How is the function of biomolecules in living systems related to their atomic structure?

Professor Doniach's research group uses scattering of synchotron X-rays from electron storage rings at SLAC and at the Argonne National Laboratory to study changes in the conformation of molecules as their solvent environments are changed. The research also involves computer simulations of the dynamics and energetic of the resulting changes.

recent Advances in the biology of DNA have shown that a very large part of the genome in eukaryotes codes for small RNA molecules that appear to be centralto the way the genes (coding for proteins) are put together. Doniach's group is currently studying structural changes that occur when some small functional RNA's turn on and off gene expression (riboswitches) without needing to involve protein transcription factors. Understanding RNA control mechanisms is central to our ability to intervene in biological functions such as generation of biofuels by bacteria or of intervention when cells start to go cancerous.

The Doniach group's bio-simulation work involves new ways to represent changes in molecular structure, in which the entire trajectory for a change of conformation is represented in a large number of CPU's where each time slice of the trajectory is managed by one of the CPU's. In this way, a representationof changes involving thousands of degrees of freedom may be obtained at atomic reslution. This method has recently been applied to look at protein misfolding. Another project involves using a highly simplified normal mode representationto represent large scale conformational changes in molecular motor molecules and DNS polymerase.

The group is also working on ways to improve the methods of computing the statistical mechanics of counter-ion shielding of the very large Coulomb forces endangered by the phosphate backbones of DNA and RNA. Software has been developed that modifies the solving of the Poisson Boltzmann equation to include the effects of finite ion size. Further modifications are being worked in to include effects of ion-on correlations.

Current Area of Focus:
- Membrane Proteins

Academic Appointments

Administrative Appointments

  • Visiting Fellow, Los Alamos National Laboratory (1987 - 1991)
  • Professor, of Physics and Applied Physics, Stanford University (1979 - Present)
  • JSPS Visiting Professor, University of Tokyo (1978 - Present)
  • Professor Associe; 1975-76, 1978, 1982, University of Paris, France (1975 - 1982)
  • Director, Stanford Synchrotron Radiation Laboratory (1973 - 1978)
  • Lecturer, Imperial College (1967 - 1969)
  • Reader in Physics, Imperial College (1967 - 1969)
  • Lecturer, Queen Mary College (1960 - 1964)
  • ICI Fellow, University of Liverpool (1958 - 1960)

Professional Education

  • Ph.D., University of Liverpool, England, Physics (1958)
  • B.A., Cambridge University, England, Physics (1954)

Current Research and Scholarly Interests

Study of changes in conformation of proteins and RNA using x-ray scattering

2015-16 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • The linac coherent light source single particle imaging road map STRUCTURAL DYNAMICS Aquila, A., Barty, A., Bostedt, C., Boutet, S., Carini, G., Deponte, D., DRELL, P., Doniach, S., Downing, K. H., Earnest, T., Elmlund, H., Elser, V., Guehr, M., Hajdu, J., Hastings, J., Hau-Riege, S. P., Huang, Z., Lattman, E. E., Maia, F. R., Marchesini, S., Ourmazd, A., Pellegrini, C., Santra, R., Schlichting, I., Schroer, C., Spence, J. C., Vartanyants, I. A., Wakatsuki, S., Weis, W. I., Williams, G. J. 2015; 2 (4)

    View details for DOI 10.1063/1.4918726

    View details for Web of Science ID 000360649200003

  • Tuning Micelle Dimensions and Properties with Binary Surfactant Mixtures LANGMUIR Oliver, R. C., Lipfert, J., Fox, D. A., Lo, R. H., Kim, J. J., Doniach, S., Columbus, L. 2014; 30 (44): 13353-13361


    Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.

    View details for DOI 10.1021/la503458n

    View details for Web of Science ID 000344905100027

    View details for PubMedID 25312254

  • Observation of correlated X-ray scattering at atomic resolution PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Mendez, D., Lane, T. J., Sung, J., Sellberg, J., Levard, C., Watkins, H., Cohen, A. E., Soltis, M., Sutton, S., Spudich, J., Pande, V., Ratner, D., Doniach, S. 2014; 369 (1647)
  • Understanding Nucleic Acid-Ion Interactions ANNUAL REVIEW OF BIOCHEMISTRY, VOL 83 Lipfert, J., Doniach, S., Das, R., Herschlag, D. 2014; 83: 813-841


    Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

    View details for DOI 10.1146/annurev-biochem-060409-092720

    View details for Web of Science ID 000348432500031

    View details for PubMedID 24606136

  • Dependence of Micelle Size and Shape on Detergent Alkyl Chain Length and Head Group PLOS ONE Oliver, R. C., Lipfert, J., Fox, D. A., Lo, R. H., Doniach, S., Columbus, L. 2013; 8 (5)


    Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

    View details for DOI 10.1371/journal.pone.0062488

    View details for Web of Science ID 000319055600027

    View details for PubMedID 23667481

  • 'Hidden' states are pervasive in RNA folding: detection and dissection through mutate-and-map experiments Das, R., Doniach, S., Ali, M., Cordero, P., VanLang, C. FEDERATION AMER SOC EXP BIOL. 2013
  • Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hong, S., Toro, E., Mortensen, K. I., de la Rosa, M. A., Doniach, S., Shapiro, L., Spakowitz, A. J., McAdams, H. H. 2013; 110 (5): 1674-1679


    We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 μm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

    View details for DOI 10.1073/pnas.1220824110

    View details for Web of Science ID 000314558100027

    View details for PubMedID 23319648

  • Salt dependence of the radius of gyration and flexibility of single-stranded DNA in solution probed by small-angle x-ray scattering PHYSICAL REVIEW E Sim, A. Y., Lipfert, J., Herschlag, D., Doniach, S. 2012; 86 (2)


    Short single-stranded nucleic acids are ubiquitous in biological processes; understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small-angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

    View details for DOI 10.1103/PhysRevE.86.021901

    View details for Web of Science ID 000307277300008

    View details for PubMedID 23005779

  • Electrostatics of Nucleic Acid Folding under Conformational Constraint JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Anthony, P. C., Sim, A. Y., Chu, V. B., Doniach, S., Block, S. M., Herschlag, D. 2012; 134 (10): 4607-4614


    RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg(2+) stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson-Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg(2+) titrations. This finding is consistent with previous work, suggesting that Poisson-Boltzmann and other mean-field theories fail for higher valency cations where ion-ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.

    View details for DOI 10.1021/ja208466h

    View details for Web of Science ID 000301990600036

    View details for PubMedID 22369617

  • AquaSAXS: a web server for computation and fitting of SAXS profiles with non-uniformally hydrated atomic models NUCLEIC ACIDS RESEARCH Poitevin, F., Orland, H., Doniach, S., Koehl, P., Delarue, M. 2011; 39: W184-W189


    Small Angle X-ray Scattering (SAXS) techniques are becoming more and more useful for structural biologists and biochemists, thanks to better access to dedicated synchrotron beamlines, better detectors and the relative easiness of sample preparation. The ability to compute the theoretical SAXS profile of a given structural model, and to compare this profile with the measured scattering intensity, yields crucial structural informations about the macromolecule under study and/or its complexes in solution. An important contribution to the profile, besides the macromolecule itself and its solvent-excluded volume, is the excess density due to the hydration layer. AquaSAXS takes advantage of recently developed methods, such as AquaSol, that give the equilibrium solvent density map around macromolecules, to compute an accurate SAXS/WAXS profile of a given structure and to compare it to the experimental one. Here, we describe the interface architecture and capabilities of the AquaSAXS web server (

    View details for DOI 10.1093/nar/gkr430

    View details for Web of Science ID 000292325300031

    View details for PubMedID 21665925

  • RNA Structure, Function, and (Thermo-) Dynamics: A SAXS and Single-Molecule Perspective Lipfert, J., Herschlag, D., Doniach, S., Dekker, N. H. CELL PRESS. 2011: 1-2
  • ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit NATURE STRUCTURAL & MOLECULAR BIOLOGY Jaru-Ampornpan, P., Shen, K., Lam, V. Q., Ali, M., Doniach, S., Jia, T. Z., Shan, S. 2010; 17 (6): 696-U64


    Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA(+)) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.

    View details for DOI 10.1038/nsmb.1836

    View details for Web of Science ID 000278393400013

    View details for PubMedID 20424608

  • Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding RNA-A PUBLICATION OF THE RNA SOCIETY Lipfert, J., Sim, A. Y., Herschlag, D., Doniach, S. 2010; 16 (4): 708-719


    Riboswitches are gene-regulating RNAs that are usually found in the 5'-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg(2+), Ca(2+), and Mn(2+) facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

    View details for DOI 10.1261/rna.1985110

    View details for Web of Science ID 000275951000006

    View details for PubMedID 20194520

  • The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure JOURNAL OF MOLECULAR BIOLOGY Ali, M., Lipfert, J., Seifert, S., Herschlag, D., Doniach, S. 2010; 396 (1): 153-165


    Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.

    View details for DOI 10.1016/j.jmb.2009.11.030

    View details for Web of Science ID 000274766500013

    View details for PubMedID 19925806

  • Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K alpha-Helix BIOPHYSICAL JOURNAL Sivaramakrishnan, S., Sung, J., Ali, M., Doniach, S., Flyvbjerg, H., Spudich, J. A. 2009; 97 (11): 2993-2999


    A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K alpha-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K alpha-helix behaves as a wormlike chain with a persistence length of approximately 15 nm or approximately 28 turns of alpha-helix. The ER/K alpha-helix length in proteins varies from 3 to 60 nm, with a median length of approximately 5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.

    View details for DOI 10.1016/j.bpj.2009.09.009

    View details for Web of Science ID 000272274500017

    View details for PubMedID 19948129

  • Do conformational biases of simple helical junctions influence RNA folding stability and specificity? RNA-A PUBLICATION OF THE RNA SOCIETY Chu, V. B., Lipfert, J., Bai, Y., Pande, V. S., Doniach, S., Herschlag, D. 2009; 15 (12): 2195-2205


    Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

    View details for DOI 10.1261/rna.1747509

    View details for Web of Science ID 000272169000011

    View details for PubMedID 19850914

  • Mixing and Matching Detergents for Membrane Protein NMR Structure Determination JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Columbus, L., Lipfert, J., Jambunathan, K., Fox, D. A., Sim, A. Y., Doniach, S., Lesley, S. A. 2009; 131 (21): 7320-7326


    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

    View details for DOI 10.1021/ja808776j

    View details for Web of Science ID 000266484900031

    View details for PubMedID 19425578

  • Riboswitch conformations revealed by small-angle X-ray scattering. Methods in molecular biology (Clifton, N.J.) Lipfert, J., Herschlag, D., Doniach, S. 2009; 540: 141-159


    Riboswitches are functional RNA molecules that control gene expression through conformational changes in response to small-molecule ligand binding. In addition, riboswitch 3D structure, like that of other RNA molecules, is dependent on cation-RNA interactions as the RNA backbone is highly negatively charged. Here, we show how small-angle X-ray scattering (SAXS) can be used to probe RNA conformations as a function of ligand and ion concentration. In a recent study of a glycine-binding tandem aptamer from Vibrio cholerae, we have used SAXS data and thermodynamic modeling to investigate how Mg(2+)-dependent folding and glycine binding are energetically coupled. In addition, we have employed ab initio shape reconstruction algorithms to obtain low-resolution models of the riboswitch structure from SAXS data under different solution conditions.

    View details for DOI 10.1007/978-1-59745-558-9_11

    View details for PubMedID 19381558



    Small-angle X-ray scattering (SAXS) is emerging as an important technique to characterize the structure of RNA molecules. While lower in resolution than X-ray crystallography or NMR spectroscopy, SAXS has the great advantage to have virtually no molecular weight limitations and does not require crystallization. In addition, SAXS can be readily applied under a large range of solution conditions, allowing to monitor RNA folding, ligand binding, and to characterize partially folded intermediates. Here, we review how the development of SAXS as a structural technique is driven by advances in computer algorithms that allow to reconstruct low-resolution electron density maps ab initio from scattering profiles. In addition, we delineate how these low-resolution models can be used in free energy electrostatics calculations. Finally, we discuss how one can exploit the hierarchical nature of RNA folding by combining the low resolution, global information provided by SAXS with local information on RNA structure, from either experiments or state-of-the-art RNA structure prediction algorithms, to further increase the resolution and quality of models obtained from SAXS.

    View details for DOI 10.1016/S0076-6879(09)69011-X

    View details for Web of Science ID 000272797000011

    View details for PubMedID 20946792

  • A repulsive field: advances in the electrostatics of the ion atmosphere CURRENT OPINION IN CHEMICAL BIOLOGY Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2008; 12 (6): 619-625


    The large electrostatic repulsion arising from the negatively charged backbone of RNA molecules presents a large barrier to folding. Solution counterions assist in the folding process by screening this electrostatic repulsion. While early research interpreted the effect of these counterions in terms of an empirical ligand-binding model, theories based on physical models have supplanted them and revised our view of the roles that ions play in folding. Instead of specific ion-binding sites, most ions in solution interact inside an 'ion atmosphere'--a fluctuating cloud of nonspecifically associated ions surrounding any charged molecule. Recent advances in experiments have begun the task of characterizing the ion atmosphere, yielding valuable data that have revealed deficiencies in Poisson-Boltzmann theory, the most widely used theory of the ion atmosphere. The continued development of experiments will help guide the development of improved theories, with the ultimate goal of understanding RNA folding and function and nucleic acid/protein interactions from a quantitative perspective.

    View details for DOI 10.1016/j.cbpa.2008.10.010

    View details for Web of Science ID 000262541300004

    View details for PubMedID 19081286

  • Critical assessment of nucleic acid electrostatics via experimental and computational investigation of an unfolded state ensemble JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Chu, V. B., Lipfert, J., Pande, V. S., Herschlag, D., Doniach, S. 2008; 130 (37): 12334-12341


    Electrostatic forces, acting between helices and modulated by the presence of the ion atmosphere, are key determinants in the energetic balance that governs RNA folding. Previous studies have employed Poisson-Boltzmann (PB) theory to compute the energetic contribution of these forces in RNA folding. However, the complex interaction of these electrostatic forces with RNA features such as tertiary contact formation, specific ion-binding, and complex interhelical junctions present in prior studies precluded a rigorous evaluation of PB theory, especially in physiologically important Mg(2+) solutions. To critically assess PB theory, we developed a model system that isolates these electrostatic forces. The model system, composed of two DNA duplexes tethered by a polyethylene glycol junction, is an analog for the unfolded state of canonical helix-junction-helix motifs found in virtually all structured RNAs. This model system lacks the complicating features that have precluded a critical assessment of PB in prior studies, ensuring that interhelical electrostatic forces dominate the behavior of the system. The system's simplicity allows PB predictions to be directly compared with small-angle X-ray scattering experiments over a range of monovalent and divalent ion concentrations. These comparisons indicate that PB is a reasonable description of the underlying electrostatic energies for monovalent ions, but large deviations are observed for divalent ions. The validation of PB for monovalent solutions allows analysis of the change in the conformational ensemble of this simple motif as salt concentration is changed. Addition of ions allows the motif to sample more compact microstates, increasing its conformational entropy. The increase of conformational entropy presents an additional barrier to folding by stabilizing the unfolded state. Neglecting this effect will adversely impact the accuracy of folding analyses and models.

    View details for DOI 10.1021/ja800854u

    View details for Web of Science ID 000259139900046

    View details for PubMedID 18722445

  • The complete VS ribozyme in solution studied by small-angle X-ray scattering STRUCTURE Lipfert, J., Ouellet, J., Norman, D. G., Doniach, S., Lilley, D. M. 2008; 16 (9): 1357-1367


    We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.

    View details for DOI 10.1016/j.str.2008.07.007

    View details for Web of Science ID 000259164500011

    View details for PubMedID 18786398

  • Dynamic charge interactions create surprising rigidity in the ER/K alpha-helical protein motif PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sivaramakrishnan, S., Spink, B. J., Sim, A. Y., Doniach, S., Spudich, J. A. 2008; 105 (36): 13356-13361


    Protein alpha-helices are ubiquitous secondary structural elements, seldom considered to be stable without tertiary contacts. However, amino acid sequences in proteins that are based on alternating repeats of four glutamic acid (E) residues and four positively charged residues, a combination of arginine (R) and lysine (K), have been shown to form stable alpha-helices in a few proteins, in the absence of tertiary interactions. Here, we find that this ER/K motif is more prevalent than previously reported, being represented in proteins of diverse function from archaea to humans. By using molecular dynamics (MD) simulations, we characterize a dynamic pattern of side-chain interactions that extends along the backbone of ER/K alpha-helices. A simplified model predicts that side-chain interactions alone contribute substantial bending rigidity (0.5 pN/nm) to ER/K alpha-helices. Results of small-angle x-ray scattering (SAXS) and single-molecule optical-trap analyses are consistent with the high bending rigidity predicted by our model. Thus, the ER/K alpha-helix is an isolated secondary structural element that can efficiently span long distances in proteins, making it a promising tool in designing synthetic proteins. We propose that the significant rigidity of the ER/K alpha-helix can help regulate protein function, as a force transducer between protein subdomains.

    View details for DOI 10.1073/pnas.0806256105

    View details for Web of Science ID 000259251700034

    View details for PubMedID 18768817

  • Long single alpha-helical tail domains bridge the gap between structure and function of myosin VI NATURE STRUCTURAL & MOLECULAR BIOLOGY Spink, B. J., Sivaramakrishnan, S., Lipfert, J., Doniach, S., Spudich, J. A. 2008; 15 (6): 591-597


    Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take approximately 36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single alpha-helix of approximately 10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the approximately 36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

    View details for DOI 10.1038/nsmb.1429

    View details for Web of Science ID 000256388900013

    View details for PubMedID 18511944

  • Quantitative and comprehensive decomposition of the ion atmosphere around nucleic acids JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Greenfeld, M., Travers, K. J., Chu, V. B., Lipfert, J., Doniach, S., Herschlag, D. 2007; 129 (48): 14981-14988


    The ion atmosphere around nucleic acids critically affects biological and physical processes such as chromosome packing, RNA folding, and molecular recognition. However, the dynamic nature of the ion atmosphere renders it difficult to characterize. The basic thermodynamic description of this atmosphere, a full accounting of the type and number of associated ions, has remained elusive. Here we provide the first complete accounting of the ion atmosphere, using buffer equilibration and atomic emission spectroscopy (BE-AES) to accurately quantitate the cation association and anion depletion. We have examined the influence of ion size and charge on ion occupancy around simple, well-defined DNA molecules. The relative affinity of monovalent and divalent cations correlates inversely with their size. Divalent cations associate preferentially over monovalent cations; e.g., with Na+ in 4-fold excess of Mg2+ (20 vs 5 mM), the ion atmosphere nevertheless has 3-fold more Mg2+ than Na+. Further, the dicationic polyamine putrescine2+ does not compete effectively for association relative to divalent metal ions, presumably because of its lower charge density. These and other BE-AES results can be used to evaluate and guide the improvement of electrostatic treatments. As a first step, we compare the BE-AES results to predictions from the widely used nonlinear Poisson Boltzmann (NLPB) theory and assess the applicability and precision of this theory. In the future, BE-AES in conjunction with improved theoretical models, can be applied to complex binding and folding equilibria of nucleic acids and their complexes, to parse the electrostatic contribution from the overall thermodynamics of important biological processes.

    View details for DOI 10.1021/ja075020g

    View details for Web of Science ID 000251293500034

    View details for PubMedID 17990882

  • Evaluation of ion binding to DNA duplexes using a size-modified Poisson-Boltzmann theory BIOPHYSICAL JOURNAL Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2007; 93 (9): 3202-3209


    Poisson-Boltzmann (PB) theory is among the most widely applied electrostatic theories in biological and chemical science. Despite its reasonable success in explaining a wide variety of phenomena, it fails to incorporate two basic physical effects, ion size and ion-ion correlations, into its theoretical treatment. Recent experimental work has shown significant deviations from PB theory in competitive monovalent and divalent ion binding to a DNA duplex. The experimental data for monovalent binding are consistent with a hypothesis that attributes these deviations to counterion size. To model the observed differences, we have generalized an existing size-modified Poisson-Boltzmann (SMPB) theory and developed a new numerical implementation that solves the generalized theory around complex, atomistic representations of biological molecules. The results of our analysis show that good agreement to data at monovalent ion concentrations up to approximately 150 mM can be attained by adjusting the ion-size parameters in the new size-modified theory. SMPB calculations employing calibrated ion-size parameters predict experimental observations for other nucleic acid structures and salt conditions, demonstrating that the theory is predictive. We are, however, unable to model the observed deviations in the divalent competition data with a theory that only accounts for size but neglects ion-ion correlations, highlighting the need for theoretical descriptions that further incorporate ion-ion correlations. The accompanying numerical solver has been released publicly, providing the general scientific community the ability to compute SMPB solutions around a variety of different biological structures with only modest computational resources.

    View details for DOI 10.1529/biophysj.106.099168

    View details for Web of Science ID 000250199300024

    View details for PubMedID 17604318

  • Size and shape of detergent micelles determined by small-angle x-ray scattering JOURNAL OF PHYSICAL CHEMISTRY B Lipfert, J., Columbus, L., Chu, V. B., Lesley, S. A., Doniach, S. 2007; 111 (43): 12427-12438


    We present a systematic analysis of the aggregation number and shape of micelles formed by nine detergents commonly used in the study of membrane proteins. Small-angle X-ray scattering measurements are reported for glucosides with 8 and 9 alkyl carbons (OG/NG), maltosides and phosphocholines with 10 and 12 alkyl carbons (DM/DDM and FC-10/FC-12), 1,2-dihexanoyl-sn-glycero-phosphocholine (DHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). The SAXS intensities are well described by two-component ellipsoid models, with a dense outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core. These models provide an intermediate resolution view of micelle size and shape. In addition, we show that Guinier analysis of the forward scattering intensity can be used to obtain an independent and model-free measurement of the micelle aggregation number and radius of gyration. This approach has the advantage of being easily generalizable to protein-detergent complexes, where simple geometric models are inapplicable. Furthermore, we have discovered that the position of the second maximum in the scattering intensity provides a direct measurement of the characteristic head group-head group spacing across the micelle core. Our results for the micellar aggregation numbers and dimensions agree favorably with literature values as far as they are available. We de novo determine the shape of FC-10, FC-12, DM, LPPG, and CHAPS micelles and the aggregation numbers of FC-10 and OG to be ca. 50 and 250, respectively. Combined, these data provide a comprehensive view of the determinants of micelle formation and serve as a starting point to correlate detergent properties with detergent-protein interactions.

    View details for DOI 10.1021/jp073016l

    View details for Web of Science ID 000250556600013

    View details for PubMedID 17924686

  • MinActionPath: maximum likelihood trajectory for large-scale structural transitions in a coarse-grained locally harmonic energy landscape NUCLEIC ACIDS RESEARCH Franklin, J., Koehl, P., Doniach, S., Delarue, M. 2007; 35: W477-W482


    The non-linear problem of simulating the structural transition between two known forms of a macromolecule still remains a challenge in structural biology. The problem is usually addressed in an approximate way using 'morphing' techniques, which are linear interpolations of either the Cartesian or the internal coordinates between the initial and end states, followed by energy minimization. Here we describe a web tool that implements a new method to calculate the most probable trajectory that is exact for harmonic potentials; as an illustration of the method, the classical Calpha-based Elastic Network Model (ENM) is used both for the initial and the final states but other variants of the ENM are also possible. The Langevin equation under this potential is solved analytically using the Onsager and Machlup action minimization formalism on each side of the transition, thus replacing the original non-linear problem by a pair of linear differential equations joined by a non-linear boundary matching condition. The crossover between the two multidimensional energy curves around each state is found numerically using an iterative approach, producing the most probable trajectory and fully characterizing the transition state and its energy. Jobs calculating such trajectories can be submitted on-line at:

    View details for DOI 10.1093/nar/gkm342

    View details for Web of Science ID 000255311500090

    View details for PubMedID 17545201

  • Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Zheng, W., Liao, J., Brooks, B. R., Doniach, S. 2007; 67 (4): 886-896


    Hepatitis C virus NS3 helicase is an enzyme that unwinds double-stranded polynucleotides in an ATP-dependent reaction. It provides a promising target for small molecule therapeutic agents against hepatitis C. Design of such drugs requires a thorough understanding of the dynamical nature of the mechanochemical functioning of the helicase. Despite recent progress, the detailed mechanism of the coupling between ATPase activity and helicase activity remains unclear. Based on an elastic network model (ENM), we apply two computational analysis tools to probe the dynamical mechanism underlying the allosteric coupling between ATP binding and polynucleotide binding in this enzyme. The correlation analysis identifies a network of hot-spot residues that dynamically couple the ATP-binding site and the polynucleotide-binding site. Several of these key residues have been found by mutational experiments as functionally important, while our analysis also reveals previously unexplored hot-spot residues that are potential targets for future mutational studies. The conformational changes between different crystal structures of NS3 helicase are found to be dominated by the lowest frequency mode solved from the ENM. This mode corresponds to a hinge motion of the highly flexible domain 2. This motion simultaneously modulates the opening/closing of the domains 1-2 cleft where ATP binds, and the domains 2-3 cleft where the polynucleotide binds. Additionally, a small twisting motion of domain 1, observed in both mode 1 and the computed ATP binding induced conformational change, fine-tunes the binding affinity of the domains 1-3 interface for the polynucleotide. The combination of these motions facilitates the translocation of a single-stranded polynucleotide in an inchworm-like manner.

    View details for DOI 10.1002/prot.21326

    View details for Web of Science ID 000246415700009

    View details for PubMedID 17373706

  • Low-resolution models for nucleic acids from small-angle X-ray scattering with applications to electrostatic modeling Lipfert, J., Chu, V. B., Bai, Y., Herschlag, D., Doniach, S. WILEY-BLACKWELL. 2007: S229-S234
  • Analysis of small-angle X-ray scattering data of protein-detergent complexes by singular value decomposition JOURNAL OF APPLIED CRYSTALLOGRAPHY Lipfert, J., Columbus, L., Chu, V. B., Doniach, S. 2007; 40: S235-S239
  • Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae JOURNAL OF MOLECULAR BIOLOGY Lipfert, J., Das, R., Chu, V. B., Kudaravalli, M., Boyd, N., Herschlag, D., Doniach, S. 2007; 365 (5): 1393-1406


    Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.

    View details for DOI 10.1016/j.jmb.2006.10.022

    View details for Web of Science ID 000243749600013

    View details for PubMedID 17118400

  • Quantitative and comprehensive decomposition of the ion atmosphere around nucleic acids Bai, Y., Travers, K., Chu, V. B., Lipfert, J., Doniach, S., Herschlag, D. CELL PRESS. 2007: 46A-46A
  • Small-angle X-ray scattering from RNA, proteins, and protein complexes ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Lipfert, J., Doniach, S. 2007; 36: 307-327


    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

    View details for DOI 10.1146/annurev.biophys.36.040306.132655

    View details for Web of Science ID 000247773000015

    View details for PubMedID 17284163

  • Towards the mechanism of dynamic couplings and translocation in Hepatitis C virus NS3 helicase using elastic network model Zheng, W., Liao, J., Brooks, B. R., Doniach, S. CELL PRESS. 2007: 177A-178A
  • Modeling RNA low resolution structure and thermodynamics from small-angle X-ray scattering Lipfert, J., Chu, V. B., Bai, Y., Ouellet, J., Lilley, D. M., Herschlag, D., Doniach, S. CELL PRESS. 2007: 417A-417A
  • Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination PROTEIN SCIENCE Columbus, L., Lipfert, J., Klock, H., Millett, I., Doniach, S., Lesley, S. A. 2006; 15 (5): 961-975


    Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.

    View details for DOI 10.1110/ps.05184706

    View details for Web of Science ID 000237237800002

    View details for PubMedID 16597824

  • Dynamic bond constraints in protein Langevin dynamics JOURNAL OF CHEMICAL PHYSICS Franklin, J., Doniach, S. 2006; 124 (15)


    Bond constraint algorithms for molecular dynamics typically take, as the target constraint lengths, the values of the equilibrium bond lengths defined in the potential. In Langevin form, the equations of motion are temperature dependent, which gives the average value for the individual bond lengths a temperature dependence. In addition to this, locally constant force fields can shift the local equilibrium bond lengths. To restore the average bond lengths in constrained integration to their unconstrained values, we suggest changing the constraint length used by popular constraint methods such as RATTLE [H. C. Andersen, J. Comput. Phys. 52, 23 (1983)] at each step. This allows us to more accurately capture the equilibrium bond length changes (with respect to the potential) due to the local equilibration and temperature effects. In addition, the approximations to the unconstrained nonbonded energies are closer using the dynamic constraint method than a traditional fixed constraint algorithm. The mechanism for finding the new constrained lengths involves one extra calculation of the bonded components of the force, and therefore adds O(N) time to the constraint algorithm. Since most molecular dynamics calculations are dominated by the O(N2) nonbonded forces, this new method does not take significantly more time than a fixed constraint algorithm.

    View details for DOI 10.1063/1.2178325

    View details for Web of Science ID 000236969500049

    View details for PubMedID 16674259

  • Sample holder for small-angle x-ray scattering static and flow cell measurements REVIEW OF SCIENTIFIC INSTRUMENTS Lipfert, J., Millett, I. S., Seifert, S., Doniach, S. 2006; 77 (4)

    View details for DOI 10.1063/1.2194484

    View details for Web of Science ID 000237136500066

  • How large is an alpha-helix? Studies of the radii of gyration of helical peptides by small-angle X-ray scattering and molecular dynamics JOURNAL OF MOLECULAR BIOLOGY Zagrovic, B., Jayachandran, G., Millett, I. S., Doniach, S., Pande, V. S. 2005; 353 (2): 232-241


    Using synchrotron radiation and the small-angle X-ray scattering technique we have measured the radii of gyration of a series of alanine-based alpha-helix-forming peptides of the composition Ace-(AAKAA)(n)-GY-NH(2), n=2-7, in aqueous solvent at 10(+/-1) degrees C. In contrast to other techniques typically used to study alpha-helices in isolation (such as nuclear magnetic resonance and circular dichroism), small-angle X-ray scattering reports on the global structure of a molecule and, as such, provides complementary information to these other, more sequence-local measuring techniques. The radii of gyration that we measure are, except for the 12-mer, lower than the radii of gyration of ideal alpha-helices or helices with frayed ends of the equivalent sequence-length. For example, the measured radius of gyration of the 37-mer is 14.2(+/-0.6)A, which is to be compared with the radius of gyration of an ideal 37-mer alpha-helix of 17.6A. Attempts are made to analyze the origin of this discrepancy in terms of the analytical Zimm-Bragg-Nagai (ZBN) theory, as well as distributed computing explicit solvent molecular dynamics simulations using two variants of the AMBER force-field. The ZBN theory, which treats helices as cylinders connected by random walk segments, predicts markedly larger radii of gyration than those measured. This is true even when the persistence length of the random walk parts is taken to be extremely short (about one residue). Similarly, the molecular dynamics simulations, at the level of sampling available to us, give inaccurate values of the radii of gyration of the molecules (by overestimating them by around 25% for longer peptides) and/or their helical content. We conclude that even at the short sequences examined here (< or =37 amino acid residues), these alpha-helical peptides behave as fluctuating semi-broken rods rather than straight cylinders with frayed ends.

    View details for DOI 10.1016/j.jmb.2005.08.053

    View details for Web of Science ID 000232505600003

    View details for PubMedID 16171817

  • Adaptive time stepping in biomolecular dynamics JOURNAL OF CHEMICAL PHYSICS Franklin, J., Doniach, S. 2005; 123 (12)


    We present an adaptive time stepping scheme based on the extrapolative method of Barth and Schlick [LN, J. Chem. Phys. 109, 1633 (1998)] to numerically integrate the Langevin equation with a molecular-dynamics potential. This approach allows us to use (on average) a time step for the strong nonbonded force integration corresponding to half the period of the fastest bond oscillation, without compromising the slow degrees of freedom in the problem. We show with simple examples how the dynamic step size stabilizes integration operators, and discuss some of the limitations of such stability. The method introduced uses a slightly more accurate inner integrator than LN to accommodate the larger steps. The adaptive time step approach reproduces temporal features of the bovine pancreatic trypsin inhibitor (BPTI) test system (similar to the one used in the original introduction of LN) compared to short-time integrators, but with energies that are shifted with respect to both LN, and traditional stochastic versions of Verlet. Although the introduction of longer steps has the effect of systematically heating the bonded components of the potential, the temporal fluctuations of the slow degrees of freedom are reproduced accurately. The purpose of this paper is to display a mechanism by which the resonance traditionally associated with using time steps corresponding to half the period of oscillations in molecular dynamics can be avoided. This has theoretical utility in terms of designing numerical integration schemes--the key point is that by factoring a propagator so that time steps are not constant one can recover stability with an overall (average) time step at a resonance frequency. There are, of course, limitations to this approach associated with the complicated, nonlinear nature of the molecular-dynamics (MD) potential (i.e., it is not as straightforward as the linear test problem we use to motivate the method). While the basic notion remains in the full Newtonian problem, it is easier to see the effects when damping is considered to be physical--that is, we do not view our method as a perturbation of Newtonian dynamics, we associate the damping with the environment, for example, a water bath (with gamma approximately 90 ps(-1)) [Zagrovic and Pande, J. Comp. Chem. 24, 1432 (2003)]. All stochastic approaches to MD are stabilized by large physical damping, but here, we are really using it only to show that the resonance frequency can be obtained. Another simplifying assumption used in this paper is "heavy" hydrogen (we take the hydrogen mass to be 10 amu)--the view here is that we are interested primarily in the slowest degrees of freedom, and this approach has effects similar to bond freezing and united atom treatments of hydrogen. So from the point of view of biomolecular applications, the method described here is best suited to studies in which water is not explicit (so that damping in the problem can really be viewed as environmental interaction), and the interest is in slow dynamics where the effects of hydrogen are neglectable. There are a number of parameters in the LN method and the one derived here, and we cannot in a short paper address all adjustments, so our primary goal as a first pass is to show that stability can be recovered for a set of numerically forced (and hence artificial) bond oscillations, and compare stability to fixed-step methods.

    View details for DOI 10.1063/1.1997137

    View details for Web of Science ID 000232206500067

    View details for PubMedID 16392529

  • Unusual compactness of a polyproline type II structure PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zagrovic, B., Lipfert, J., Sorin, E. J., Millettt, I. S., van Gunsteren, W. F., Doniach, S., Pande, V. S. 2005; 102 (33): 11698-11703


    Polyproline type II (PPII) helix has emerged recently as the dominant paradigm for describing the conformation of unfolded polypeptides. However, most experimental observables used to characterize unfolded proteins typically provide only short-range, sequence-local structural information that is both time- and ensemble-averaged, giving limited detail about the long-range structure of the chain. Here, we report a study of a long-range property: the radius of gyration of an alanine-based peptide, Ace-(diaminobutyric acid)2-(Ala)7-(ornithine)2-NH2. This molecule has previously been studied as a model for the unfolded state of proteins under folding conditions and is believed to adopt a PPII fold based on short-range techniques such as NMR and CD. By using synchrotron radiation and small-angle x-ray scattering, we have determined the radius of gyration of this peptide to be 7.4 +/- 0.5 angstroms, which is significantly less than the value expected from an ideal PPII helix in solution (13.1 angstroms). To further study this contradiction, we have used molecular dynamics simulations using six variants of the AMBER force field and the GROMOS 53A6 force field. However, in all cases, the simulated ensembles underestimate the PPII content while overestimating the experimental radius of gyration. The conformational model that we propose, based on our small angle x-ray scattering results and what is known about this molecule from before, is that of a very flexible, fluctuating structure that on the level of individual residues explores a wide basin around the ideal PPII geometry but is never, or only rarely, in the ideal extended PPII helical conformation.

    View details for DOI 10.1073/pnas.0409693102

    View details for Web of Science ID 000231317000025

    View details for PubMedID 16085707

  • Protein misfolding and amyloid formation for the peptide GNNQQNY from yeast prion protein Sup35: Simulation by reaction path annealing JOURNAL OF MOLECULAR BIOLOGY Lipfert, J., Franklin, J., Wu, F., Doniach, S. 2005; 349 (3): 648-658


    We study the early steps of amyloid formation of the seven residue peptide GNNQQNY from yeast prion-like protein Sup35 by simulating the random coil to beta-sheet and alpha-helix to beta-sheet transition both in the absence and presence of a cross-beta amyloid nucleus. The simulation method at atomic resolution employs a new implementation of a Langevin dynamics "reaction path annealing" algorithm. The results indicate that the presence of amyloid-like cross-beta-sheet strands both facilitates the transition into the cross-beta conformation and substantially lowers the free energy barrier for this transition. This model systems allows us to investigate the energetic and kinetic details of this transition, which is consistent with an auto-catalyzed, nucleation-like mechanism for the formation of beta-amyloid. In particular, we find that electrostatic interactions of peptide backbone dipoles contribute significantly to the stability of the beta-amyloid state. Furthermore, we find water exclusion and interactions of polar side-chains to be driving forces of amyloid formation: the cross-beta conformation is stabilized by burial of polar side-chains and inter-residue hydrogen bonds in the presence of an amyloid-like "seed". These findings are in support of a "dry, polar zipper model" of amyloid formation.

    View details for DOI 10.1016/j.jmb.2005.03.083

    View details for Web of Science ID 000229401700017

    View details for PubMedID 15896350

  • Fold recognition aided by constraints from small angle X-ray scattering data PROTEIN ENGINEERING DESIGN & SELECTION Zheng, W. J., Doniach, S. 2005; 18 (5): 209-219


    We performed a systematic exploration of the use of structural information derived from small angle X-ray scattering (SAXS) measurements to improve fold recognition. SAXS data provide the Fourier transform of the histogram of atomic pair distances (pair distribution function) for a given protein and hence can serve as a structural constraint on methods used to determine the native conformational fold of the protein. Here we used it to construct a similarity-based fitness score with which to evaluate candidate structures generated by a threading procedure. In order to combine the SAXS scores with the standard energy scores and other 1D profile-based scores used in threading, we made use both of a linear regression method and of a neural network-based technique to obtain optimal combined fitness scores and applied them to the ranking of candidate structures. Our results show that the use of SAXS data with gapless threading significantly improves the performance of fold recognition.

    View details for DOI 10.1093/protein/gzi026

    View details for Web of Science ID 000229699600001

    View details for PubMedID 15845555

  • Network of dynamically important residues in the open/closed transition in polymerases is strongly conserved STRUCTURE Zheng, W. J., Brooks, B. R., Doniach, S., Thirumalai, D. 2005; 13 (4): 565-577


    The open/closed transition in polymerases is a crucial event in DNA replication and transcription. We hypothesize that the residues that transmit the signal for the open/closed transition are also strongly conserved. To identify the dynamically relevant residues, we use an elastic network model of polymerases and probe the residue-specific response to a local perturbation. In a variety of DNA/RNA polymerases, a network of residues spanning the fingers and palm domains is involved in the open/closed transition. The similarity in the network of residues responsible for large-scale domain movements supports the notion of a common induced-fit mechanism in the polymerase families for the formation of a closed ternary complex. Multiple sequence alignment shows that many of these residues are also strongly conserved. Residues with the largest sensitivity to local perturbations include those that are not so obviously involved in the polymerase catalysis. Our results suggest that mutations of the mechanical "hot spots" can compromise the efficiency of the enzyme.

    View details for DOI 10.1016/j.str.2005.01.017

    View details for Web of Science ID 000228685900011

    View details for PubMedID 15837195

  • Effects of nitration on the structure and aggregation of alpha-synuclein MOLECULAR BRAIN RESEARCH Uversky, V. N., Yamin, G., Munishkina, L. A., Karymov, M. A., Millett, I. S., Doniach, S., Lyubchenko, Y. L., Fink, A. L. 2005; 134 (1): 84-102


    Substantial evidence suggests that the aggregation of the presynaptic protein alpha-synuclein is a key step in the etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying alpha-synuclein aggregation remain unknown, oxidative stress has been implicated in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant alpha-synuclein to fibrillate in vitro. The properties of nitrated alpha-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was monomeric. alpha-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of non-modified alpha-synuclein at neutral pH. Potential implications of these findings to the etiology of Parkinson's disease are discussed.

    View details for DOI 10.1016/j.molbrainres.2004.11.014

    View details for Web of Science ID 000228321100010

    View details for PubMedID 15790533

  • Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bai, Y., Das, R., Millett, I. S., Herschlag, D., Doniach, S. 2005; 102 (4): 1035-1040


    Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na+), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT/bp. Addition of high concentrations of Na+ (1.2 M), Mg2+ (0.6 M), and spermidine3+ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated.

    View details for DOI 10.1073/pnas.0404448102

    View details for Web of Science ID 000226617900016

    View details for PubMedID 15647360

  • Principles of RNA compaction: Insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations JOURNAL OF MOLECULAR BIOLOGY Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D., Chance, M. R. 2004; 343 (5): 1195-1206


    Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.

    View details for DOI 10.1016/j.jmb.2004.08.080

    View details for Web of Science ID 000224838800004

    View details for PubMedID 15491606

  • Random-coil behavior and the dimensions of chemically unfolded proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kohn, J. E., Millett, I. S., JACOB, J., Zagrovic, B., Dillon, T. M., Cingel, N., Dothager, R. S., Seifert, S., Thiyagarajan, P., Sosnick, T. R., Hasan, M. Z., Pande, V. S., Ruczinski, I., Doniach, S., Plaxco, K. W. 2004; 101 (34): 12491-12496


    Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

    View details for DOI 10.1073/pnas.0403643101

    View details for Web of Science ID 000223596200019

    View details for PubMedID 15314214

  • Stimulation of insulin fibrillation by urea-induced intermediates JOURNAL OF BIOLOGICAL CHEMISTRY Ahmad, A., Millett, I. S., Doniach, S., Uversky, V. N., Fink, A. L. 2004; 279 (15): 14999-15013


    Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased alpha-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.

    View details for DOI 10.1074/jbc.M313134200

    View details for Web of Science ID 000220594700064

    View details for PubMedID 14736893

  • Protein misfolding and aggregation in a 7 residue peptide from the yeast prion protein Sup35 - Simulation by reaction path annealing Lipfert, J., Wu, F., Franklin, J., Doniach, S. CELL PRESS. 2004: 416A-416A
  • Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Permyakov, S. E., Millett, I. S., Doniach, S., Permyakov, E. A., Uversky, V. N. 2003; 53 (4): 855-862


    The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.

    View details for DOI 10.1002/prot.10481

    View details for Web of Science ID 000186976700008

    View details for PubMedID 14635127

  • A comparative study of motor-protein motions by using a simple elastic-network model PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zheng, W. J., Doniach, S. 2003; 100 (23): 13253-13258


    In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996) Phys. Rev. Lett. 77, 1905-1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the "nucleotide-binding-induced power-stroke" scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities.

    View details for DOI 10.1073/pnas.2235686100

    View details for Web of Science ID 000186573700027

    View details for PubMedID 14585932

  • Partially folded intermediates in insulin fibrillation BIOCHEMISTRY Ahmad, A., Millett, I. S., Doniach, S., Uversky, V. N., Fink, A. L. 2003; 42 (39): 11404-11416


    Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.

    View details for DOI 10.1021/bi034868o

    View details for Web of Science ID 000185724100002

    View details for PubMedID 14516191

  • The fastest global events in RNA folding: Electrostatic relaxation and tertiary collapse of the tetrahymena ribozyme JOURNAL OF MOLECULAR BIOLOGY Das, R., Kwok, L. W., Millett, I. S., Bai, Y., Mills, T. T., JACOB, J., Maskel, G. S., Seifert, S., Mochrie, S. G., Thiyagarajan, P., Doniach, S., Pollack, L., Herschlag, D. 2003; 332 (2): 311-319


    Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

    View details for DOI 10.1016/S0022-2836(03)00854-4

    View details for Web of Science ID 000185306700002

    View details for PubMedID 12948483

  • Nuclear localization of alpha-synuclein and its interaction with histones BIOCHEMISTRY Goers, J., Manning-Bog, A. B., McCormack, A. L., Millett, I. S., Doniach, S., Di Monte, D. A., Uversky, V. N., Fink, A. L. 2003; 42 (28): 8465-8471


    The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.

    View details for DOI 10.1021/bi0341152

    View details for Web of Science ID 000184249000010

    View details for PubMedID 12859192

  • Counterion distribution around DNA probed by solution X-ray scattering PHYSICAL REVIEW LETTERS Das, R., Mills, T. T., Kwok, L. W., Maskel, G. S., Millett, I. S., Doniach, S., Finkelstein, K. D., Herschlag, D., Pollack, L. 2003; 90 (18)


    Counterion atmospheres condensed onto charged biopolymers strongly affect their physical properties and biological functions, but have been difficult to quantify experimentally. Here, monovalent and divalent counterion atmospheres around DNA double helices in solution are probed using small-angle x-ray scattering techniques. Modulation of the ion scattering factors by anomalous (resonant) x-ray scattering and by interchanging ion identities yields direct measurements of the scattering signal due to the spatial correlation of surrounding ions to the DNA. The quality of the data permit, for the first time, quantitative tests of extended counterion distributions calculated from atomic-scale models of biologically relevant molecules.

    View details for DOI 10.1103/PhysRevLett.90.188103

    View details for Web of Science ID 000182823900054

    View details for PubMedID 12786045

  • Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis CELL Meyer, A. S., Gillespie, J. R., Walther, D., Millet, I. S., Doniach, S., Frydman, J. 2003; 113 (3): 369-381


    Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

    View details for Web of Science ID 000182640800011

    View details for PubMedID 12732144

  • Three-dimensional flux states as a model for the pseudogap phase of transition metal oxides PHYSICAL REVIEW B Schroeter, D. F., Doniach, S. 2002; 66 (7)
  • Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation - Evidence for an off-pathway oligomer at acidic pH JOURNAL OF BIOLOGICAL CHEMISTRY Souillac, P. O., Uversky, V. N., Millett, I. S., Khurana, R., Doniach, S., Fink, A. L. 2002; 277 (15): 12666-12679


    Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

    View details for DOI 10.1074/jbc.M109229200

    View details for Web of Science ID 000175036300024

    View details for PubMedID 11815604

  • Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH JOURNAL OF BIOLOGICAL CHEMISTRY Souillac, P. O., Uversky, V. N., Millett, I. S., Khurana, R., Doniach, S., Fink, A. L. 2002; 277 (15): 12657-12665


    Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

    View details for DOI 10.1074/jbc.M109230200

    View details for Web of Science ID 000175036300023

    View details for PubMedID 11815605

  • Biophysical properties of the synucleins and their propensities to fibrillate - Inhibition of alpha-synuclein assembly by beta- and gamma-synucleins JOURNAL OF BIOLOGICAL CHEMISTRY Uversky, V. N., Li, J., Souillac, P., Millett, I. S., Doniach, S., Jakes, R., Goedert, M., Fink, A. L. 2002; 277 (14): 11970-11978


    The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous alpha-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, beta- and gamma-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. beta-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas alpha- and gamma-synucleins were slightly more compact and structured. gamma-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to alpha-synuclein, beta- And gamma-synucleins inhibit alpha-synuclein fibril formation. Complete inhibition of alpha-synuclein fibrillation was observed at 4:1 molar excess of beta- and gamma-synucleins. No significant incorporation of beta-synuclein into the fibrils was detected. The lack of fibrils formed by beta-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.

    View details for DOI 10.1074/jbc.M109541200

    View details for Web of Science ID 000174846400049

    View details for PubMedID 11812782

  • Rapid compaction during RNA folding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Millettt, I. S., Tate, M. W., Kwok, L. W., Nakatani, B., Gruner, S. M., Mochrie, S. G., Pande, V., Doniach, S., Herschlag, D., Pollack, L. 2002; 99 (7): 4266-4271


    We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

    View details for DOI 10.1073/pnas.072589599

    View details for Web of Science ID 000174856000027

    View details for PubMedID 11929997

  • Protein structure prediction constrained by solution X-ray scattering data and structural homology identification JOURNAL OF MOLECULAR BIOLOGY Zheng, W. J., Doniach, S. 2002; 316 (1): 173-187


    Here we perform a systematic exploration of the use of distance constraints derived from small angle X-ray scattering (SAXS) measurements to filter candidate protein structures for the purpose of protein structure prediction. This is an intrinsically more complex task than that of applying distance constraints derived from NMR data where the identity of the pair of amino acid residues subject to a given distance constraint is known. SAXS, on the other hand, yields a histogram of pair distances (pair distribution function), but the identities of the pairs contributing to a given bin of the histogram are not known. Our study is based on an extension of the Levitt-Hinds coarse grained approach to ab initio protein structure prediction to generate a candidate set of C(alpha) backbones. In spite of the lack of specific residue information inherent in the SAXS data, our study shows that the implementation of a SAXS filter is capable of effectively purifying the set of native structure candidates and thus provides a substantial improvement in the reliability of protein structure prediction. We test the quality of our predicted C(alpha) backbones by doing structural homology searches against the Dali domain library, and find that the results are very encouraging. In spite of the lack of local structural details and limited modeling accuracy at the C(alpha) backbone level, we find that useful information about fold classification can be extracted from this procedure. This approach thus provides a way to use a SAXS data based structure prediction algorithm to generate potential structural homologies in cases where lack of sequence homology prevents identification of candidate folds for a given protein. Thus our approach has the potential to help in determination of the biological function of a protein based on structural homology instead of sequence homology.

    View details for DOI 10.1006/jmbi.2001.5324

    View details for Web of Science ID 000174025900014

    View details for PubMedID 11829511

  • Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques JOURNAL OF MOLECULAR BIOLOGY Choy, W. Y., Mulder, F. A., Crowhurst, K. A., Muhandiram, D. R., Millett, I. S., Doniach, S., Forman-Kay, J. D., Kay, L. E. 2002; 316 (1): 101-112


    The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride.

    View details for DOI 10.1006/jmbi.2001.5328

    View details for Web of Science ID 000174025900009

    View details for PubMedID 11829506

  • Exploring the folding landscape of a structured RNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Zhuang, X. W., Babcock, H. P., Millett, I. S., Doniach, S., Chu, S., Herschlag, D. 2002; 99 (1): 155-160


    Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

    View details for Web of Science ID 000173233300031

    View details for PubMedID 11756689

  • Equilibrium collapse and the kinetic 'foldability' of proteins BIOCHEMISTRY Millet, I. S., Townsley, L. E., Chiti, F., Doniach, S., Plaxco, K. W. 2002; 41 (1): 321-325


    An important element of protein folding theory has been the identification of equilibrium parameters that might uniquely distinguish rapidly folding polypeptide sequences from those that fold slowly. One such parameter, termed sigma, is a dimensionless, equilibrium measure of the coincidence of chain compaction and folding that is predicted to be an important determinant of relative folding kinetics. To test this prediction and improve our understanding of the putative relationship between nonspecific compaction of the unfolded state and protein folding kinetics, we have used small-angle X-ray scattering and circular dichroism spectroscopy to measure the sigma of five well-characterized proteins. Consistent with theoretical predictions, we find that near-perfect coincidence of the unfolded state contraction and folding (sigma approximately 0) is associated with the rapid kinetics of these naturally occurring proteins. We do not, however, observe any significant correlation between sigma and either the relative folding rates of these proteins or the presence or absence of well-populated kinetic intermediates. Thus, while sigma approximately 0 may be a necessary condition to ensure rapid folding, differences in sigma do not account for the wide range of rates and mechanisms with which naturally occurring proteins fold.

    View details for DOI 10.1021/bi015695a

    View details for Web of Science ID 000173216500036

    View details for PubMedID 11772031

  • Toward a taxonomy of the denatured state: Small angle scattering studies of unfolded proteins UNFOLDED PROTEINS Millet, I. S., Doniach, S., Plaxco, K. W. 2002; 62: 241-262

    View details for Web of Science ID 000179263800007

    View details for PubMedID 12418105

  • Changes in biomolecular conformation seen by small angle X-ray scattering CHEMICAL REVIEWS Doniach, S. 2001; 101 (6): 1763-1778

    View details for DOI 10.1021/cr990071k

    View details for Web of Science ID 000169375600008

    View details for PubMedID 11709998

  • Simulation of protein folding by reaction path annealing JOURNAL OF CHEMICAL PHYSICS Eastman, P., Gronbech-Jensen, N., Doniach, S. 2001; 114 (8): 3823-3841
  • Exploring the RNA folding landscape, one molecule at a time. Herschlag, D., Russell, R., Zhuang, X. W., Bartley, L., Babcock, H. P., Miller, I. S., Doniach, S., Chu, S. AMER CHEMICAL SOC. 2000: U82-U82
  • Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein JOURNAL OF MOLECULAR BIOLOGY Bada, M., Walther, D., Arcangioli, B., Doniach, S., Delarue, M. 2000; 300 (3): 563-574


    Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.

    View details for DOI 10.1006/jmbi.2000.3854

    View details for Web of Science ID 000088223600012

    View details for PubMedID 10884352

  • Small angle X-ray scattering reveals a compact intermediate in RNA folding NATURE STRUCTURAL BIOLOGY Russell, R., Millett, I. S., Doniach, S., Herschlag, D. 2000; 7 (5): 367-370


    We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

    View details for Web of Science ID 000086908800009

    View details for PubMedID 10802731

  • Fourth-generation X-ray sources: some possible applications to biology JOURNAL OF SYNCHROTRON RADIATION Doniach, S. 2000; 7: 116-120


    The term 'fourth generation X-ray sources' has come to mean X-ray free-electron lasers which use multi-GeV electron beams from linear accelerators to generate X-rays by self-amplified stimulated emission when fired into long undulators. Properties of the radiation expected from such sources are reviewed briefly and two possible applications of the resulting pulsed highly collimated X-radiation to problems in biology are discussed: use of X-ray photon correlation spectroscopy to measure time correlations of atoms in protein crystals, and use of Mössbauer radiation extracted from the photon beams by resonant Bragg diffraction from (57)Fe-containing crystals, for MAD phasing of very large unit-cell biomolecular crystals and possibly for photon echo measurements.

    View details for Web of Science ID 000087250300003

    View details for PubMedID 16609184

  • Reconstruction of low-resolution three-dimensional density maps from one-dimensional small-angle X-ray solution scattering data for biomolecules JOURNAL OF APPLIED CRYSTALLOGRAPHY Walther, D., Cohen, F. E., Doniach, S. 2000; 33: 350-363
  • Zn2+-mediated structure formation and compaction of the "Natively unfolded" human prothymosin alpha BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Uversky, V. N., Gillespie, J. R., Millett, I. S., Khodyakova, A. V., Vasilenko, R. N., Vasiliev, A. M., Rodionov, I. L., Kozlovskaya, G. D., Dolgikh, D. A., Fink, A. L., Doniach, S., Permyakov, E. A., Abramov, V. M. 2000; 267 (2): 663-668


    Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

    View details for Web of Science ID 000085013600034

    View details for PubMedID 10631119

  • Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering BIOCHEMISTRY Segel, D. J., ELIEZER, D., Uversky, V., Fink, A. L., Hodgson, K. O., Doniach, S. 1999; 38 (46): 15352-15359


    The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.

    View details for Web of Science ID 000083899400034

    View details for PubMedID 10563821

  • Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH BIOCHEMISTRY Uversky, V. N., Gillespie, J. R., Millett, I. S., Khodyakova, A. V., Vasiliev, A. M., Chernovskaya, T. V., Vasilenko, R. N., Kozovskaya, G. D., Dolgikh, D. A., Fink, A. L., Doniach, S., Abramov, V. M. 1999; 38 (45): 15009-15016


    Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

    View details for Web of Science ID 000083726100030

    View details for PubMedID 10555983

  • Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Segel, D. J., Bachmann, A., Hofrichter, J., Hodgson, K. O., Doniach, S., Kiefhaber, T. 1999; 288 (3): 489-499


    We have used synchrotron radiation, together with stopped-flow and continuous-flow mixing techniques to monitor refolding of lysozyme at pH 5.2. From data measured at times which range from 14 ms to two seconds, we can monitor changes in the size, the shape and the pair distribution function of the polypeptide chain during the folding process. Comparison of the results with the properties of native and GdmCl-unfolded lysozyme shows that a major chain collapse occurs in the dead-time of mixing. During this process about 50 % of the change in radius of gyration between the unfolded protein and the native state occurs and the polypeptide chain adopts a globular shape. Time-resolved fluorescence spectra of this collapsed state suggest that the hydrophobic side-chains are still highly solvent accessible. A subsequently formed intermediate with helical structure in the alpha-domain is nearly identical in size and shape with native lysozyme and has a solvent-inaccessible hydrophobic core. Despite its native-like properties, this intermediate is only slightly more stable (DeltaG0=-4 kJ/mol) than the collapsed state and still much less stable than native lysozyme (DeltaDeltaG0=36 kJ/mol) at 20 degrees C.

    View details for Web of Science ID 000080204600014

    View details for PubMedID 10329156

  • Protein dynamics simulations from nanoseconds to microseconds CURRENT OPINION IN STRUCTURAL BIOLOGY Doniach, S., Eastman, P. 1999; 9 (2): 157-163


    There have been a number of advances in atomic resolution simulations of biomolecules during the past few years. These have arisen partly from improvements to computer power and partly from algorithmic improvements. There have also been advances in measuring time-dependent fluctuations in proteins using NMR spectroscopy, revealing the importance of fluctuations in the microsecond to millisecond time range. Progress has also been made in measuring how far the simulations are able to represent the accessible phase space that is available to the protein in its native state, in solution, at room temperature. Another area of development is the simulation of protein unfolding at atomic resolution.

    View details for Web of Science ID 000085219800002

    View details for PubMedID 10322213

  • Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability PROTEIN SCIENCE Uversky, V. N., Karnoup, A. S., Khurana, R., Segel, D. J., Doniach, S., Fink, A. L. 1999; 8 (1): 161-173


    Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.

    View details for Web of Science ID 000077956900017

    View details for PubMedID 10210194

  • Protein denaturation: A small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c BIOCHEMISTRY Segel, D. J., Fink, A. L., Hodgson, K. O., Doniach, S. 1998; 37 (36): 12443-12451


    Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (Rg) shows a cooperative transition with increasing denaturant with a similar Cm to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.

    View details for Web of Science ID 000075909800009

    View details for PubMedID 9730816

  • Anion-induced folding of Staphylococcal nuclease: Characterization of multiple equilibrium partially folded intermediates JOURNAL OF MOLECULAR BIOLOGY Uversky, V. N., Karnoup, A. S., Segel, D. J., Seshadri, S., Doniach, S., Fink, A. L. 1998; 278 (4): 879-894


    The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three different partially folded intermediates (A states). The three intermediates lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of secondary structure, size, stability and globularity. The least structured conformation, A1, is stabilized by chloride (600 mM) or sulfate (100 mM). It is about 50% folded (based on circular dichroism and small angle X-ray scattering (SAXS) data). The next most structured intermediate, A2, is induced by trifluoroacetate (300 mM) and has approximately 70% native-like secondary structure. The most structured intermediate, A3, is stabilized by trichloroacetate (50 mM) and has native-like secondary structure content and is almost as compact as the native state. The stability toward urea denaturation increases with increasing structure of the intermediates. Moreover, ureainduced unfolding studies show that these partially folded species are separated from each other, and from the unfolded state, by significant free energy barriers, suggesting that they are distinct conformational states. Kratky plots, based on the SAXS data, indicate that the two more structured intermediates have significant globularity (i.e. a tightly packed core), whereas the less structured intermediate has very little globularity. These observations support a model of protein folding in which certain conformations are of particularly low free energy and hence populated under conditions which differentially destabilize the native state. These partially folded intermediates probably consist of ensembles of substates with a common core of native-like secondary structure, which is responsible for their stability. Consequently, it is likely that the intermediates observed here represent the equilibrium counterparts of transient kinetic intermediates.

    View details for Web of Science ID 000073774000014

    View details for PubMedID 9614949

  • Association-induced folding of globular proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uversky, V. N., Segel, D. J., Doniach, S., Fink, A. L. 1998; 95 (10): 5480-5483


    It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.

    View details for Web of Science ID 000073595500013

    View details for PubMedID 9576907

  • Multiple time step diffusive Langevin dynamics for proteins PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Eastman, P., Doniach, S. 1998; 30 (3): 215-227


    We present an algorithm for simulating the long time scale dynamics of proteins and other macromolecules. Our method applies the concept of multiple time step integration to the diffusive Langevin equation, in which short time scale dynamics are replaced by friction and noise. The macromolecular force field is represented at atomic resolution. Slow motions are modeled by constrained Langevin dynamics with very large time steps, while faster degrees of freedom are kept in local thermal equilibrium. In the limit of a sufficiently large molecule, our algorithm is shown to reduce the CPU time required by two orders of magnitude. We test the algorithm on two systems, alanine dipeptide and bovine pancreatic trypsin inhibitor (BPTI), and find that it accurately calculates a variety of equilibrium and dynamical properties. In the case of BPTI, the CPU time required is reduced by nearly a factor of 60 compared to a conventional, unconstrained Langevin simulation using the same force field.

    View details for Web of Science ID 000072331600001

    View details for PubMedID 9517537

  • Kinetics of lysozyme refolding: Structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Chen, L. L., Wildegger, G., Kiefhaber, T., Hodgson, K. O., Doniach, S. 1998; 276 (1): 225-237


    We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.

    View details for Web of Science ID 000072310200016

    View details for PubMedID 9514723

  • Simulations of the thermodynamic properties of a short polyalanine peptide using potentials of mean force PHYSICA A-STATISTICAL MECHANICS AND ITS APPLICATIONS Pellegrini, M., GRONBECHJENSEN, N., Doniach, S. 1997; 239 (1-3): 244-254
  • A lysozyme folding intermediate revealed by solution X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Chen, L. L., Hodgson, K. O., Doniach, S. 1996; 261 (5): 658-671


    Equilibrium unfolding of hen egg lysozyme as a function of urea concentration at pH 2.9 has been studied by solution X-ray scattering. Differences in the unfolding transition are observed as monitored by the radius of gyration Rg, and by far and near UV CD (circular dichroism) at 222 nm and 298 nm, respectively. This suggests the existence of a third unfolding species, in addition to the native and the unfolded states. A singular value decomposition (SVD) analysis was made of the scattering curves at different urea concentrations. This analysis shows clear evidence of a third basis component in the X-ray scattering curves, thus supporting the results of the Rg and CD measurements. The denaturant binding model was employed to estimate the thermodynamic parameters of denaturation for the intermediate and unfolded states. Use of these parameters to refine the SVD analysis allows us to reconstruct a scattering profile for the pure intermediate state. Simplified partially folded models, based on the crystal structure of hen lysozyme, support a working model for the intermediate, whose structure may be correlated with that of the kinetic intermediate found in the refolding pathway studied by Dobson and coworkers.

    View details for Web of Science ID A1996VF29600007

    View details for PubMedID 8800214



    Freshly-prepared blue membranes from Halobacterium halobium, previously reported to be disordered, are shown to have a distinct crystal lattice structure, slightly different from the native form. The lattice of the blue form is disrupted irreversibly when dehydrated. The disorder process was observed using time-resolved small-angle X-ray diffraction and analyzed by radial autocorrelation functions. The diffraction peaks of the in-plane lattice first sharpen and increase due to improved membrane orientation, then the trimer lattice becomes disordered and the unit cell dimension decreases by 1.8 A. In contrast, dehydration of purple membranes does not disorder the lattice, and the unit cell dimension shrinks by only 1.0 A. Comparisons of radial autocorrelation functions for the blue membrane during drying show drastic loss of inter-trimer, long-range correlation while the intra-trimer, short-range correlations remain more or less unchanged. This suggests that the deionized protein trimers can maintain their overall structure during the dehydration, even though the lattice dimension decreases appreciably and the two-dimensional crystallinity is disrupted.

    View details for Web of Science ID A1994NK09700003

    View details for PubMedID 8167134



    Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

    View details for Web of Science ID A1994MV63100028

    View details for PubMedID 8106367



    The nitrogenase enzyme complex, consisting of the molybdenum-iron protein and the iron protein, plays a critical role in the biological reduction of dinitrogen to ammonia (nitrogen fixation). The nitrogen-fixing site within the molybdenum-iron protein is an iron-molybdenum-sulfur cofactor (FeMoco) of roughly 1000-2000 Dalton mass. Structural aspects of FeMoco have been determined by spectroscopic and more recently by crystallographic studies. In order to determine the radius of gyration (Rg) of isolated FeMoco, we have performed small-angle x-ray scattering studies of FeMoco in N-methylformamide solution, in the absence of the molybdenum-iron protein. Model compounds of known structure have also been examined in similar solvents, N,N-dimethylformamide and acetonitrile, as controls and for calibration purposes. The Rg values obtained for the models are in good agreement with calculations based upon their respective crystal structures. However, the Rg obtained for FeMoco clearly indicates that the cofactor is not monomeric in solution, but rather aggregated and possibly polydisperse. Further, Rg values were also measured after addition of thiol, dithionite, and thiol and dithionite, to the FeMoco samples. The results indicate, surprisingly, that oxidation state and putative thiol coordination have no detectable effect on the aggregation behavior of FeMoco in solution, as determined by these measurements.

    View details for Web of Science ID A1993MA28800050

    View details for PubMedID 8407930



    Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.

    View details for Web of Science ID A1993LU23300038

    View details for PubMedID 8218914



    A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives8) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form.

    View details for Web of Science ID A1993KT96700009

    View details for PubMedID 8460113



    X-ray crystallographic studies of troponin C (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S.T., and Roychowdhury, P. (1985a) Science 227, 945-948) have revealed a novel protein structure consisting of two globular domains, each containing two Ca2+-binding sites, connected via a nine-turn alpha-helix, three turns of which are fully exposed to solvent. Since the crystals were grown at pH approximately 5, it is of interest to determine whether this structure is applicable to the protein in solution under physiological conditions. We have used small-angle x-ray scattering to examine the solution structure of troponin C at pH 6.8 and the effect of Ca2+ on the structure. The scattering data are consistent with an elongated structure in solution with a radius of gyration of approximately 23.0 A, which is quite comparable to that computed for the crystal structure. The experimental scattering profile and the scattering profile computed from the crystal structure coordinates do, however, exhibit differences at the 40-A level. A weak Ca2+-facilitated dimerization of troponin C was observed. The data rule out large Ca2+-induced structural changes, indicating rather that the molecule with Ca2+ bound is only slightly more compact than the Ca2+-free molecule.

    View details for Web of Science ID A1988M662000018

    View details for PubMedID 3346242

  • POLARIZED X-RAY ABSORPTION NEAR-EDGE STRUCTURE OF HIGHLY OXIDIZED CHROMIUM PORPHYRINS INORGANIC CHEMISTRY PENNERHAHN, J. E., Benfatto, M., Hedman, B., Takahashi, T., Doniach, S., Groves, J. T., Hodgson, K. O. 1986; 25 (13): 2255-2259

    View details for Web of Science ID A1976CN37400031

    View details for PubMedID 993517