Education & Certifications

  • B.S., Harvey Mudd College, Chemistry (1992)
  • Ph.D., UC Berkeley, Biophysical Chemistry (1998)

All Publications

  • Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis. ACS chemical biology Kuo, J., Lynch, S. R., Liu, C. W., Xiao, X., Khosla, C. 2016; 11 (9): 2636-2641


    Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode "orphan" assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a "split, stuttering" module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.

    View details for DOI 10.1021/acschembio.6b00489

    View details for PubMedID 27384917

  • Self-assembly and sequence length dependence on nanofibrils of polyglutamine peptides NEUROPEPTIDES Inayathullah, M., Tan, A., Jeyaraj, R., Lam, J., Cho, N., Liu, C. W., Manoukian, M. A., Ashkan, K., Mahmoudi, M., Rajadas, J. 2016; 57: 71-83


    Huntington's disease (HD) is recognized as a currently incurable, inherited neurodegenerative disorder caused by the accumulation of misfolded polyglutamine (polyQ) peptide aggregates in neuronal cells. Yet, the mechanism by which newly formed polyQ chains interact and assemble into toxic oligomeric structures remains a critical, unresolved issue. In order to shed further light on the matter, our group elected to investigate the folding of polyQ peptides - examining glutamine repeat lengths ranging from 3 to 44 residues. To characterize these aggregates we employed a diverse array of technologies, including: nuclear magnetic resonance; circular dichroism; Fourier transform infrared spectroscopy; fluorescence resonance energy transfer (FRET), and atomic force microscopy. The data we obtained suggest that an increase in the number of glutamine repeats above 14 residues results in disordered loop structures, with different repeat lengths demonstrating unique folding characteristics. This differential folding manifests in the formation of distinct nano-sized fibrils, and on this basis, we postulate the idea of 14 polyQ repeats representing a critical loop length for neurotoxicity - a property that we hope may prove amenable to future therapeutic intervention. Furthermore, FRET measurements on aged assemblages indicate an increase in the end-to-end distance of the peptide with time, most probably due to the intermixing of individual peptide strands within the nanofibril. Further insight into this apparent time-dependent reorganization of aggregated polyQ peptides may influence future disease modeling of polyQ-related proteinopathies, in addition to directing novel clinical innovations.

    View details for DOI 10.1016/j.npep.2016.01.011

    View details for Web of Science ID 000378193100010

    View details for PubMedID 26874369

  • Revealing an outward-facing open conformational state in a CLC Cl-/H+ exchange transporter ELIFE Khantwal, C. M., Abraham, S. J., Han, W., Jiang, T., Chavan, T. S., Cheng, R. C., Elvington, S. M., Liu, C. W., Mathews, I. I., Steins, R. A., Mchaourab, H. S., Tajkhorshid, E., Maduke, M. 2016; 5


    CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.

    View details for DOI 10.7554/eLife.11189

    View details for Web of Science ID 000373794800001

    View details for PubMedID 26799336

  • The short-term transport and transformation of phosphorus species in a saturated soil following poultry manure amendment and leaching GEODERMA Giles, C. D., Cade-Menun, B. J., Liu, C. W., Hill, J. E. 2015; 257: 134-141
  • Determination of Hydrogen Bond Structure in Water versus Aprotic Environments To Test the Relationship Between Length and Stability JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sigala, P. A., Ruben, E. A., Liu, C. W., Piccoli, P. M., Hohenstein, E. G., Martinez, T. J., Schultz, A. J., Herschlag, D. 2015; 137 (17): 5730-5740


    Hydrogen bonds profoundly influence the architecture and activity of biological macromolecules. Deep appreciation of hydrogen bond contributions to biomolecular function thus requires a detailed understanding of hydrogen bond structure and energetics and the relationship between these properties. Hydrogen bond formation energies (ΔGf) are enormously more favorable in aprotic solvents than in water, and two classes of contributing factors have been proposed to explain this energetic difference, focusing respectively on the isolated and hydrogen-bonded species: (I) water stabilizes the dissociated donor and acceptor groups much better than aprotic solvents, thereby reducing the driving force for hydrogen bond formation; and (II) water lengthens hydrogen bonds compared to aprotic environments, thereby decreasing the potential energy within the hydrogen bond. Each model has been proposed to provide a dominant contribution to ΔGf, but incisive tests that distinguish the importance of these contributions are lacking. Here we directly test the structural basis of model II. Neutron crystallography, NMR spectroscopy, and quantum mechanical calculations demonstrate that O-H···O hydrogen bonds in crystals, chloroform, acetone, and water have nearly identical lengths and very similar potential energy surfaces despite ΔGf differences >8 kcal/mol across these solvents. These results rule out a substantial contribution from solvent-dependent differences in hydrogen bond structure and potential energy after association (model II) and thus support the conclusion that differences in hydrogen bond ΔGf are predominantly determined by solvent interactions with the dissociated groups (model I). These findings advance our understanding of universal hydrogen-bonding interactions and have important implications for biology and engineering.

    View details for DOI 10.1021/ja512980h

    View details for Web of Science ID 000354338500017

    View details for PubMedID 25871450

  • C-13 NMR detects conformational change in the 100-kD membrane transporter ClC-ec1 JOURNAL OF BIOMOLECULAR NMR Abraham, S. J., Cheng, R. C., Chew, T. A., Khantwal, C. M., Liu, C. W., Gong, S., Nakamoto, R. K., Maduke, M. 2015; 61 (3-4): 209-226


    CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).

    View details for DOI 10.1007/s10858-015-9898-7

    View details for Web of Science ID 000352711900004

    View details for PubMedID 25631353

  • Stratification of Phosphorus Forms from Long-Term Conservation Tillage and Poultry Litter Application SOIL SCIENCE SOCIETY OF AMERICA JOURNAL Cade-Menun, B. J., He, Z., Zhang, H., Endale, D. M., Schomberg, H. H., Liu, C. W. 2015; 79 (2): 504-516
  • The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell Joachimiak, L. A., Walzthoeni, T., Liu, C. W., Aebersold, R., Frydman, J. 2014; 159 (5): 1042-1055


    The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

    View details for DOI 10.1016/j.cell.2014.10.042

    View details for PubMedID 25416944

  • Molecular simulation and experimental characterization of the nanoporous structures of coal and gas shale INTERNATIONAL JOURNAL OF COAL GEOLOGY Firouzi, M., Rupp, E. C., Liu, C. W., Wilcox, J. 2014; 121: 123-128
  • Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy of Soils from 2005 to 2013: A Review of Sample Preparation and Experimental Parameters SOIL SCIENCE SOCIETY OF AMERICA JOURNAL Cade-Menun, B., Liu, C. W. 2014; 78 (1): 19-37
  • Complementary Phosphorus Speciation in Agricultural Soils by Sequential Fractionation, Solution P-31 Nuclear Magnetic Resonance, and Phosphorus K-edge X-ray Absorption Near-Edge Structure Spectroscopy JOURNAL OF ENVIRONMENTAL QUALITY Liu, J., Yang, J., Cade-Menun, B. J., Liang, X., Hu, Y., Liu, C. W., Zhao, Y., Li, L., Shi, J. 2013; 42 (6): 1763-1770
  • Phosphorus Speciation in Riparian Soils: A Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy and Enzyme Hydrolysis Study SOIL SCIENCE SOCIETY OF AMERICA JOURNAL Young, E. O., Ross, D. S., Cade-Menun, B. J., Liu, C. W. 2013; 77 (5): 1636-1647
  • The Dynamic Process of beta(2)-Adrenergic Receptor Activation CELL Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Fung, J. J., Bokoch, M. P., Thian, F. S., Kobilka, T. S., Shaw, D. E., Mueller, L., Prosser, R. S., Kobilka, B. K. 2013; 152 (3): 532-542


    G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins.

    View details for DOI 10.1016/j.cell.2013.01.008

    View details for Web of Science ID 000314362800022

  • Ligand-switchable Substrates for a Ubiquitin-Proteasome System JOURNAL OF BIOLOGICAL CHEMISTRY Egeler, E. L., Urner, L. M., Rakhit, R., Liu, C. W., Wandless, T. J. 2011; 286 (36): 31328-31336


    Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.

    View details for DOI 10.1074/jbc.M111.264101

    View details for Web of Science ID 000294487500028

    View details for PubMedID 21768107

  • Small-molecule displacement of a cryptic degron causes conditional protein degradation NATURE CHEMICAL BIOLOGY Bonger, K. M., Chen, L., Liu, C. W., Wandless, T. J. 2011; 7 (8): 531-537


    The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

    View details for DOI 10.1038/NCHEMBIO.598

    View details for Web of Science ID 000292825400014

    View details for PubMedID 21725303

  • Rationally Designed Turn Promoting Mutation in the Amyloid-beta Peptide Sequence Stabilizes Oligomers in Solution PLOS ONE Rajadas, J., Liu, C. W., Novick, P., Kelley, N. W., Inayathullah, M., LeMieux, M. C., Pande, V. S. 2011; 6 (7)


    Enhanced production of a 42-residue beta amyloid peptide (A?(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the A?(42) peptide (A?(42)Nle35p37) that appear to organize A?(42) into stable oligomers. 2D NMR on the A?(42)Nle35p37 peptide revealed the occurrence of two ?-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the A?(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.

    View details for DOI 10.1371/journal.pone.0021776

    View details for Web of Science ID 000293097300006

    View details for PubMedID 21799748

  • Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase PROTEIN SCIENCE Charkoudian, L. K., Liu, C. W., Capone, S., Kapur, S., Cane, D. E., Togni, A., Seebach, D., Khosla, C. 2011; 20 (7): 1244-1255


    The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF?-S-ACP, was developed as a ¹?F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

    View details for DOI 10.1002/pro.652

    View details for Web of Science ID 000292257600017

    View details for PubMedID 21563224

  • Phosphorus Forms and Chemistry in the Soil Profile under Long-Term Conservation Tillage: A Phosphorus-31 Nuclear Magnetic Resonance Study JOURNAL OF ENVIRONMENTAL QUALITY Cade-Menun, B. J., Carter, M. R., James, D. C., Liu, C. W. 2010; 39 (5): 1647-1656


    In many regions, conservation tillage has replaced conventional tilling practices to reduce soil erosion, improve water conservation, and increase soil organic matter. However, tillage can have marked effects on soil properties, specifically nutrient redistribution or stratification in the soil profile. The objective of this research was to examine soil phosphorus (P) forms and concentrations in a long-term study comparing conservation tillage (direct drilling, "No Till") and conventional tillage (moldboard plowing to 20 cm depth, "Till") established on a fine sandy loam (Orthic Humo-Ferric Podzol) in Prince Edward Island, Canada. No significant differences in total carbon (C), total nitrogen (N), total P, or total organic P concentrations were detected between the tillage systems at any depth in the 0- to 60-cm depth range analyzed. However, analysis with phosphorus-31 nuclear magnetic resonance spectroscopy showed differences in P forms in the plow layer. In particular, the concentration of orthophosphate was significantly higher under No Till than Till at 5 to 10 cm, but the reverse was true at 10 to 20 cm. Mehlich 3-extractable P was also significantly higher in No Till at 5 to 10 cm and significantly higher in Till at 20 to 30 cm. This P stratification appears to be caused by a lack of mixing of applied fertilizer in No Till because the same trends were observed for pH and Mehlich 3-extractable Ca (significantly higher in the Till treatment at 20 to 30 cm), reflecting mixing of applied lime. The P saturation ratio was significantly higher under No Till at 0 to 5 cm and exceeded the recommended limits, suggesting that P stratification under No Till had increased the potential for P loss in runoff from these sites.

    View details for DOI 10.2134/jeq2009.0491

    View details for Web of Science ID 000281575600012

    View details for PubMedID 21043270

  • The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation JOURNAL OF BIOLOGICAL CHEMISTRY Vivona, S., Liu, C. W., Strop, P., Rossi, V., Filippini, F., Brunger, A. T. 2010; 285 (23): 17965-17973


    SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

    View details for DOI 10.1074/jbc.M110.120972

    View details for Web of Science ID 000278133400072

    View details for PubMedID 20378544

  • Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor NATURE Bokoch, M. P., Zou, Y., Rasmussen, S. G., Liu, C. W., Nygaard, R., Rosenbaum, D. M., Fung, J. J., Choi, H., Thian, F. S., Kobilka, T. S., Puglisi, J. D., Weis, W. I., Pardo, L., Prosser, R. S., Mueller, L., Kobilka, B. K. 2010; 463 (7277): 108-U121


    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

    View details for DOI 10.1038/nature08650

    View details for Web of Science ID 000273344900040

    View details for PubMedID 20054398

  • Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR EMBO JOURNAL Elvington, S. M., Liu, C. W., Maduke, M. C. 2009; 28 (20): 3090-3102


    The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.

    View details for DOI 10.1038/emboj.2009.259

    View details for Web of Science ID 000271008200004

    View details for PubMedID 19745816

  • THE DIVERSITY OF NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY BIOPHYSICS AND THE CHALLENGES OF EMERGING THREATS Liu, C. W., Alekseyev, V. Y., Allwardt, J. R., Bankovich, A. J., Cade-Menun, B. J., Davis, R. W., Du, L., Garcia, K. C., Herschlag, D., Khosla, C., Kraut, D. A., Li, Q., Null, B., Puglisi, J. D., Sigala, P. A., Stebbins, J. F., Varani, L. 2009: 65-81
  • Aggregation and conformational studies on a pentapeptide derivative BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS Sambasivam, D., Liu, C. W., Jayaraman, M., Malar, E. J., Rajadas, J. 2008; 1784 (11): 1659-1667


    Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.

    View details for DOI 10.1016/j.bbapap.2008.07.015

    View details for Web of Science ID 000261019000022

    View details for PubMedID 18775521

  • High-Resolution, In Vivo Magnetic Resonance Imaging of Drosophila at 18.8 Tesla PLOS ONE Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. 2008; 3 (7)


    High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.

    View details for DOI 10.1371/journal.pone.0002817

    View details for Web of Science ID 000264304300036

    View details for PubMedID 18665264

  • PKR: A NMR perspective PROGRESS IN NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY Lindhout, D. A., Mckenna, S. A., Aitken, C. E., Liu, C. W., Puglisi, J. D. 2007; 51 (3): 199-215
  • Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase PROTEIN SCIENCE Alekseyev, V. Y., Liu, C. W., Cane, D. E., Puglisi, J. D., Khosla, C. 2007; 16 (10): 2093-2107


    Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.

    View details for DOI 10.1110/ps.073011407

    View details for Web of Science ID 000249692400001

    View details for PubMedID 17893358

  • Solution mapping of T cell receptor docking footprints on peptide-MHC PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Varani, L., Bankovich, A. J., Liu, C. W., Colf, L. A., Jones, L. L., Kranz, D. M., Puglisi, J. D., Garcia, K. C. 2007; 104 (32): 13080-13085


    T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a "recognition code" between conserved elements of the TCR and MHC genes. Although approximately 17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2Ld-QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.

    View details for DOI 10.1073/pnas.0703702104

    View details for Web of Science ID 000248650300024

    View details for PubMedID 17670943

  • Molecular framework for the activation of RNA-dependent protein kinase JOURNAL OF BIOLOGICAL CHEMISTRY Mckenna, S. A., Lindhout, D. A., Kim, I., Liu, C. W., Gelev, V. M., Wagner, G., Puglisi, J. D. 2007; 282 (15): 11474-11486


    The RNA-dependent protein kinase (PKR) plays an integral role in the antiviral response to cellular infection. PKR contains three distinct domains consisting of two conserved N-terminal double-stranded RNA (dsRNA)-binding domains, a C-terminal Ser-Thr kinase domain, and a central 80-residue linker. Despite rich structural and biochemical data, a detailed mechanistic explanation of PKR activation remains unclear. Here we provide a framework for understanding dsRNA-dependent activation of PKR using nuclear magnetic resonance spectroscopy, dynamic light scattering, gel filtration, and autophosphorylation kinetics. In the latent state, PKR exists as an extended monomer, with an increase in self-affinity upon dsRNA association. Subsequent phosphorylation leads to efficient release of dsRNA followed by a greater increase in self-affinity. Activated PKR displays extensive conformational perturbations within the kinase domain. We propose an updated model for PKR activation in which the communication between RNA binding, central linker, and kinase domains is critical in the propagation of the activation signal and for PKR dimerization.

    View details for DOI 10.1074/jbc.M700301200

    View details for Web of Science ID 000245941500069

    View details for PubMedID 17284445

  • Methods and rationale for high-resolution magnetic resonance imaging (MRI) of Drosophila , using an 18.8 Tesla NMR spectrometer Drosophila Information Service Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. 2007; 90: 131-137
  • Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs JOURNAL OF MOLECULAR BIOLOGY Mckenna, S. A., Kim, I., Liu, C. W., Puglisi, J. D. 2006; 358 (5): 1270-1285


    Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain and a C-terminal kinase domain. Upon binding double-stranded RNA (dsRNA), PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2alpha (eIF-2alpha). Phosphorylation of eIF-2alpha results in attenuation of protein translation by the ribosome in either a general or an mRNA-specific manner. Therefore, the interaction between PKR and dsRNAs represents a crucial host cell defense mechanism against viral infection. Viruses can circumvent PKR function by transcription of virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We present here a biophysical characterization of the interactions between human PKR and two viral inhibitor RNAs, EBER(I) (from Epstein-Barr virus) and VA(I) (from human adenovirus). Autophosphorylation assays confirmed that both EBER(I) and VA(I) are inhibitors of PKR activation, and profiled the kinetics of the inhibition. Binding affinities of dsRNAs to PKR double-stranded RNA-binding domains (dsRBDs) were determined by isothermal titration calorimetry and gel electrophoresis. A single stem-loop domain from each inhibitory RNA mediates the interaction with both dsRBDs of PKR. The binding sites on inhibitor RNAs and the dsRBDs of PKR have been mapped by NMR chemical shift perturbation experiments, which indicate that inhibitors of PKR employ similar surfaces of interaction as activators. Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition.

    View details for DOI 10.1016/j.jmb.2006.03.003

    View details for Web of Science ID 000237689600008

    View details for PubMedID 16580685

  • Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR JOURNAL OF MOLECULAR BIOLOGY Kim, I., Liu, C. W., Puglisi, J. D. 2006; 358 (2): 430-442


    PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.

    View details for DOI 10.1016/j.jmb.2006.01.099

    View details for Web of Science ID 000237149600009

    View details for PubMedID 16516925

  • Testing electrostatic complementarity in enzyme catalysis: Hydrogen bonding in the ketosteroid isomerase oxyanion hole PLOS BIOLOGY Kraut, D. A., Sigala, P. A., Pybus, B., Liu, C. W., Ringe, D., Petsko, G. A., Herschlag, D. 2006; 4 (4): 501-519


    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02 A/pK(a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

    View details for DOI 10.1371/journal.pbio.0040099

    View details for Web of Science ID 000237066500005

    View details for PubMedID 16602823

  • High-level bacterial secretion of single-chain alpha beta T-cell receptors JOURNAL OF IMMUNOLOGICAL METHODS Maynard, J., Adams, E. J., Krogsgaard, M., Petersson, K., Liu, C. W., Garcia, K. C. 2005; 306 (1-2): 51-67


    While numerous antibody-antigen systems have been structurally characterized, studies of structurally analogous T-cell receptor MHC systems have lagged behind largely due to the lack of a general TCR expression system. Efforts to develop bacterial systems have resulted in low yields (< 0.5 mg/l) of active material which is prone to proteolysis and aggregation. Here we report a strategy to secrete folded, soluble single chain T-cell receptors (scTCR) in the Escherichia coli periplasm using three representative alphabeta TCRs (172.10, 1934.4/c19 and 2B4). Shake flask yields between 0.5 and 30 mg/l active, purified material were attained for all TCRs studied and found to depend on the introduction of solubility-increasing amino acid substitutions, skp chaperone co-expression and C-terminal fusion to a human kappa constant domain in the context of a tightly regulated expression vector. This system will greatly enable crystallographic, thermodynamic and other biophysical analyses of TCRs which require large quantities of homogeneous material.

    View details for DOI 10.1016/j.jim.2005.07.022

    View details for Web of Science ID 000234174500005

    View details for PubMedID 16198365

  • Characterization of the FKBP.rapamycin.FRB ternary complex. Journal of the American Chemical Society Banaszynski, L. A., Liu, C. W., Wandless, T. J. 2005; 127 (13): 4715-4721


    Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

    View details for PubMedID 15796538

  • Using NMR To Study Large RNAs: Case Study of the HCV IRES Structure, Dynamics and Function of Biological Macromolecules and Assemblies Kim, I., Lukavsky, P. J., Otto, G., Liu, C., Puglisi, J. D. IOS Press. 2005: 75–89
  • Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein BIOCHEMISTRY Li, Q., Khosla, C., Puglisi, J. D., Liu, C. W. 2003; 42 (16): 4648-4657


    During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.

    View details for DOI 10.1021/bi0274120

    View details for Web of Science ID 000182460700006

    View details for PubMedID 12705828

  • Soil and litter phosphorus-31 nuclear magnetic resonance spectroscopy: Extractants, metals, and phosphorus relaxation times Cade-Menun, B. J., Liu, C. W., Nunlist, R., McColl, J. G. AMER SOC AGRONOMY. 2002: 457–65


    Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy is an excellent tool with which to study soil organic P, allowing quantitative, comparative analysis of P forms. However, for 31P NMR to be tative, all peaks must be completely visible, and in their correct relative proportions. There must be no line broadening, and adequate delay times must be used to avoid saturation of peaks. The objective of this study was to examine the effects of extractants on delay times and peak saturation. Two samples (a forest litter and a mineral soil sample) and three extractants (0.25 M NaOH, NaOH plus Chelex (Bio-Rad Laboratories, Hercules, CA), and NaOH plus EDTA) were used to determine the differences in the concentration of P and cations solubilized by each extractant, and to measure spin-lattice (T1) relaxation times of P peaks in each extract. For both soil and litter, NaOH-Chelex extracted the lowest concentrations of P. For the litter sample, T1 values were short for all extractants due to the high Fe concentration remaining after extraction. For the soil sample, there were noticeable differences among the extractants. The NaOH-Chelex sample had less Fe and Mn remaining in solution after extraction than the other extractants, and the longest delay times used in the study, 6.4 s, were not long enough for quantitative analysis. Delay times of 1.5 to 2 s for the NaOH and NaOH-EDTA were adequate. Line broadening was highest in the NaOH extracts, which had the highest concentration of Fe. On the basis of these results, recommendations for future analyses of soil and litter samples by solution 31P NMR spectroscopy include: careful selection of an extractant; measurement of paramagnetic ions extracted with P; use of appropriate delay times and the minimum number of scans; and measurement of T1 values whenever possible.

    View details for Web of Science ID 000174330600011

    View details for PubMedID 11931434

  • Cyclic and linear oligocarbamate ligands for human thrombin BIOORGANIC & MEDICINAL CHEMISTRY Cho, C. Y., Liu, C. W., Wemmer, D. E., Schultz, P. G. 1999; 7 (6): 1171-1179


    Several classes of compounds have been tested as potential inhibitors of the serine protease thrombin, an important regulator of blood coagulation cascades. We describe here the discovery of a new class of thrombin inhibitors based on an unnatural carbamate biopolymer. Oligocarbamate thrombin inhibitors were identified through the screening of diverse cyclic trimer, cyclic tetramer, and linear tetramer libraries using the one bead, one peptide method. Whereas the cyclic trimer oligocarbamate ligands bound thrombin with modest affinity, a cyclic tetramer oligocarbamate inhibited thrombin with an apparent Ki of 31 nM. Linear oligocarbamate tetramers bound thrombin with inhibition constants in the 100-nM range. These nonpeptidic, oligomeric molecules may provide the basis for further drug development and studies of thrombin ligand interactions.

    View details for Web of Science ID 000081084000023

    View details for PubMedID 10428389

  • Biochemical and biophysical characterization of the trimerization domain from the heat shock transcription factor BIOCHEMISTRY Peteranderl, R., Rabenstein, M., Shin, Y. K., Liu, C. W., Wemmer, D. E., King, D. S., Nelson, H. C. 1999; 38 (12): 3559-3569


    Previously, we had characterized a 91 amino acid fragment of the heat shock transcription factor from the yeast Kluyveromyces lactis and had shown it to be highly alpha-helical and sufficient for formation of homotrimers [Peteranderl, R., and Nelson, H. C. M. (1992) Biochemistry 31, 12272-12276]. Based on those data, as well as the presence of hydrophobic heptad repeats, we postulated that the trimerization domain contains a three-stranded coiled-coil and that it might resemble the trimerization domain found in influenza hemagglutinin. Here, we further characterize the trimerization domain and show that the minimal domain needs 71 residues to remain trimeric and highly alpha-helical. 19F NMR spectroscopy suggests that the structure contains three parallel strands that are in register along the long axis of the coiled-coil. Electron paramagnetic resonance spectroscopy studies show that the C-termini of the subunits are in close proximity; this is in contrast to the topology of the hemaglutinin trimerization domain where the C-termini form buttressing helices. Analytical ultracentrifugation also confirms that the structure is elongated and unlikely to have buttressing helices. Additional experiments suggest that the trimerization domain has at least two subdomains. The first subdomain has the potential to form trimers independently, though not as stably as the complete domain. The second subdomain is quite helical, forms large oligomers, and appears to provide stability to the complete domain. Our current model for the heat shock transcription factor trimerization domain is a highly elongated coiled-coil structure, with a potential break in the coiled-coil region located between the two subdomains.

    View details for Web of Science ID 000079510700012

    View details for PubMedID 10090742

  • Mechanistic Studies of an Antibody-Catalyzed Pericyclic Rearrangement Journal of the American Chemical Society Driggers, E. M., Cho, H. S., Liu, C. W., Katzka, C. P., Braisted, A. C., Ulrich, H. D., Wemmer, D. E., Schultz, P. G. 1998; 120 (9): 1945–1958

    View details for DOI 10.1021/ja962933u

  • Synthesis and Screening of Linear and Cyclic Oligocarbamate Libraries. Discovery of High Affinity Ligands for GPIIb/IIIa Journal of the American Chemical Society Cho, C. Y., Youngquist, R. S., Paikoff, S. J., Beresini, M. H., Hebert, A. R., Berleau, L. T., Liu, C. W., Wemmer, D. E., Keough, T., Schultz, P. G. 1998; 120 (31): 7706–7718

    View details for DOI 10.1021/ja9734399

  • Structure of the Michaelis Complex of an Efficient Antibody Acyl Transferase Determined by Transferred Nuclear Overhauser Enhancement Spectroscopy Journal of the American Chemical Society Driggers, E. M., Liu, C. W., Wemmer, D. E., Schultz, P. G. 1998; 120 (29): 7395–7396

    View details for DOI 10.1021/ja9813313

  • A Template for Stabilization of a Peptide α-Helix:  Synthesis and Evaluation of Conformational Effects by Circular Dichroism and NMR Journal of the American Chemical Society Austin, R. E., Maplestone, R. A., Sefler, A. M., Liu, K., Hruzewicz, W. N., Liu, C. W., Cho, H. S., Wemmer, D. E., Bartlett, P. A. 1997; 119 (28): 6461-6472

    View details for DOI 10.1021/ja964231a

  • Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy PROTEIN SCIENCE Cho, H. S., Liu, C. W., Damberger, F. F., Pelton, J. G., Nelson, H. C., Wemmer, D. E. 1996; 5 (2): 262-269


    The structure and dynamics of the N-terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA-binding domain as a structural reference, we show that the protein backbone of the N-terminal activation domain undergoes rapid, large-amplitude motions and is therefore unstructured. Difference CD data also show that the N-terminal activation domain remains random-coil, even in the presence of DNA. Implications for a "polypeptide lasso" model of transcriptional activation are discussed.

    View details for Web of Science ID A1996TU72400010

    View details for PubMedID 8745404



    The solution structure of the 92 residue (11 kDa) winged helix-turn-helix DNA-binding domain from the kluyveromyces lactis heat shock factor was refined using a total of 932 NOE, 35 phi, 25 chi 1, 5 chi 2 and 44 hydrogen bond restraints. The overall root-mean-square deviation for structured regions was 0.75(+/- 0.15) A. The three-helix bundle and four-stranded beta-sheet are well defined with rmsd of 0.53(+/- 0.10) A and 0.60(+/- 0.17) A, respectively. Helix H2 is underwound and bent near Pro45. The angle between helix H2 and the proposed recognition helix H3 is 96(+/- 6) degrees. Detailed comparisons are made with the X-ray structure of this protein as well as other structural studies on HSF. Overall, the results are consistent with the earlier studies. Differences are related to protein-protein interactions in the crystal and dynamics in solution. Backbone dynamics was investigated via 15N relaxation. The average R1, R2 and NOE values for residues in segments of secondary structure were 1.9(+/- 0.9) s-1, 7.8(+/- 0.9) s-1 and 0.81(+/- 0.05), respectively. The correlation time based on these data was 5.6(+/- 0.4) ns. Motional order parameters were calculated by fitting the relaxation data to one of three models. Low-order parameters were found for residues that comprise the turn between helices H2 and H3 (residues Lys49 to Phe53), and most strikingly, the 16 residue wing (residues Val68 to Arg83). These data are consistent with the lack of long-range NOEs identified in these regions. The data provide a basis for comparison with results of the protein-DNA complex. The relationship between structure and function is discussed.

    View details for Web of Science ID A1995TJ63200016

    View details for PubMedID 7500344



    The UV-VIS absorption data of four bacteriorhodopsin (BR) analogs formed from azulene-retinals of varying polyene chain length show that the one-bond-shortened to one-bond-lengthened analogs possess comparable opsin shift values to that of BR. A two-bond-shortened analog exhibited a much smaller opsin shift. These data, combined with those reported for the C-22 retinal analog (Tokunaga et al., 1977, Biophys. J. 19, 191-198) were analyzed by molecular modelling and computer graphics in terms of a model where conformational flexibility of the appended butyl is the controlling factor in determining ease of pigment formation and protein/substrate interaction.

    View details for Web of Science ID A1991GD93400018

    View details for PubMedID 1796116