2005 – 2009 H. B. Sc. University of Toronto, Chemistry and Physics
2009 – 2013 Ph. D. University of Illinois, Chemical Physics

Honors & Awards

  • University of Toronto Excellence Award, University of Toronto (2007)
  • University of Toronto Excellence Award, University of Toronto (2008)
  • Walter Brown Fellowship, University of Illinois at Urbana-Champaign (2010-2011)
  • NSF Center for the Physics of Living Cells Teaching Fellowship, University of Illinois at Urbana-Champaign (2010-2011)
  • 2nd US-Mexico Workshop in Biological Chemistry: Protein Folding, Misfolding and Design, Travel Award, National Science Foundation (2011)
  • John C. Bailar Fellowship, University of Illinois at Urbana-Champaign (2011-2012)
  • 2012 Graduate College Travel Award, University of Illinois at Urbana-Champaign (2012)
  • Howard Hughes Medical Institute International Student Research Fellowship, Howard Hughes Medical Institute (2012-2013)
  • 2013 Josephine G. Geerdes Memorial Travel Award, University of Illinois at Urbana-Champaign (2013)
  • Dean's Postdoctoral Fellowship, Stanford University (2014-2015)
  • Helen Hay Whitney Foundation Postdoctoral Fellowship, Helen Hay Whitney Foundation (2015-2018)
  • 2016 PHYS Division Postdoctoral Research Award, American Chemical Society (2016)

Professional Education

  • Honours Bachelor of Science, University of Toronto, Chemistry and Physics (2009)
  • Doctor of Philosophy, University of Illinois at Urbana-Champaign, Chemical Physics (2013)

Stanford Advisors

Lab Affiliations

All Publications

  • Mapping fast protein folding with multiple-site fluorescent probes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Prigozhin, M. B., Chao, S., Sukenik, S., Pogorelov, T. V., Gruebele, M. 2015; 112 (26): 7966-7971


    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

    View details for DOI 10.1073/pnas.1422683112

    View details for Web of Science ID 000357079400039

    View details for PubMedID 26080403

  • Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Wirth, A. J., Liu, Y., Prigozhin, M. B., Schulten, K., Gruebele, M. 2015; 137 (22): 7152-7159


    The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for β-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.

    View details for DOI 10.1021/jacs.5b02474

    View details for Web of Science ID 000356322300039

    View details for PubMedID 25988868

  • Criteria for Selecting PEGylation Sites on Proteins for Higher Thermodynamic and Proteolytic Stability JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lawrence, P. B., Gavrilov, Y., Matthews, S. S., Langlois, M. I., Shental-Bechor, D., Greenblatt, H. M., Pandey, B. K., Smith, M. S., Paxman, R., Torgerson, C. D., Merrell, J. P., Ritz, C. C., Prigozhin, M. B., Levy, Y., Price, J. L. 2014; 136 (50): 17547-17560


    PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

    View details for DOI 10.1021/ja5095183

    View details for Web of Science ID 000346682600033

    View details for PubMedID 25409346

  • Mechanical Modeling and Computer Simulation of Protein Folding JOURNAL OF CHEMICAL EDUCATION Prigozhin, M. B., Scott, G. E., Denos, S. 2014; 91 (11): 1939-1942

    View details for DOI 10.1021/ed400719c

    View details for Web of Science ID 000345267100028

  • Observation of Complete Pressure-Jump Protein Refolding in Molecular Dynamics Simulation and Experiment JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, Y., Prigozhin, M. B., Schulten, K., Gruebele, M. 2014; 136 (11): 4265-4272


    Density is an easily adjusted variable in molecular dynamics (MD) simulations. Thus, pressure-jump (P-jump)-induced protein refolding, if it could be made fast enough, would be ideally suited for comparison with MD. Although pressure denaturation perturbs secondary structure less than temperature denaturation, protein refolding after a fast P-jump is not necessarily faster than that after a temperature jump. Recent P-jump refolding experiments on the helix bundle λ-repressor have shown evidence of a <3 μs burst phase, but also of a ~1.5 ms "slow" phase of refolding, attributed to non-native helical structure frustrating microsecond refolding. Here we show that a λ-repressor mutant is nonetheless capable of refolding in a single explicit solvent MD trajectory in about 19 μs, indicating that the burst phase observed in experiments on the same mutant could produce native protein. The simulation reveals that after about 18.5 μs of conformational sampling, the productive structural rearrangement to the native state does not occur in a single swift step but is spread out over a brief series of helix and loop rearrangements that take about 0.9 μs. Our results support the molecular time scale inferred for λ-repressor from near-downhill folding experiments, where transition-state population can be seen experimentally, and also agrees with the transition-state transit time observed in slower folding proteins by single-molecule spectroscopy.

    View details for DOI 10.1021/ja412639u

    View details for Web of Science ID 000333435500028

    View details for PubMedID 24437525

  • Misplaced helix slows down ultrafast pressure-jump protein folding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Prigozhin, M. B., Liu, Y., Wirth, A. J., Kapoor, S., Winter, R., Schulten, K., Gruebele, M. 2013; 110 (20): 8087-8092


    Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.

    View details for DOI 10.1073/pnas.1219163110

    View details for Web of Science ID 000319803500032

    View details for PubMedID 23620522

  • Impact of Site-Specific PEGylation on the Conformational Stability and Folding Rate of the Pin WW Domain Depends Strongly on PEG Oligomer Length BIOCONJUGATE CHEMISTRY Pandey, B. K., Smith, M. S., Torgerson, C., Lawrence, P. B., Matthews, S. S., Watkins, E., Groves, M. L., Prigozhin, M. B., Price, J. L. 2013; 24 (5): 796-802


    Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.

    View details for DOI 10.1021/bc3006122

    View details for Web of Science ID 000319250300005

    View details for PubMedID 23578107

  • Microsecond folding experiments and simulations: a match is made PHYSICAL CHEMISTRY CHEMICAL PHYSICS Prigozhin, M. B., Gruebele, M. 2013; 15 (10): 3372-3388


    For the past two decades, protein folding experiments have been speeding up from the second or millisecond time scale to the microsecond time scale, and full-atom simulations have been extended from the nanosecond to the microsecond and even millisecond time scale. Where the two meet, it is now possible to compare results directly, allowing force fields to be validated and refined, and allowing experimental data to be interpreted in atomistic detail. In this perspective we compare recent experiments and simulations on the microsecond time scale, pointing out the progress that has been made in determining native structures from physics-based simulations, refining experiments and simulations to provide more quantitative underlying mechanisms, and tackling the problems of multiple reaction coordinates, downhill folding, and complex underlying structure of unfolded or misfolded states.

    View details for DOI 10.1039/c3cp43992e

    View details for Web of Science ID 000314846600002

    View details for PubMedID 23361200

  • Functional Importance of Short-Range Binding and Long-Range Solvent Interactions in Helical Antifreeze Peptides BIOPHYSICAL JOURNAL Ebbinghaus, S., Meister, K., Prigozhin, M. B., DeVries, A. L., Havenith, M., Dzubiella, J., Gruebele, M. 2012; 103 (2): L20-L22


    Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.

    View details for DOI 10.1016/j.bpj.2012.06.013

    View details for Web of Science ID 000306522300002

    View details for PubMedID 22853917

  • Studying IDP stability and dynamics by fast relaxation imaging in living cells. Methods in molecular biology (Clifton, N.J.) Dhar, A., Prigozhin, M., Gelman, H., Gruebele, M. 2012; 895: 101-111


    Fast relaxation imaging (FReI) temperature-tunes living cells and applies small temperature jumps to them, to monitor biomolecular stability and kinetics in vivo. The folding or aggregation state of a target protein is monitored by Förster resonance energy transfer (FRET). Intrinsically disordered proteins near the structured-unstructured boundary are particularly sensitive to their environment. We describe, using the IDP α-synuclein as an example, how FReI can be used to measure IDP stability and folding inside the cell.

    View details for DOI 10.1007/978-1-61779-927-3_8

    View details for PubMedID 22760315

  • The Fast and the Slow: Folding and Trapping of lambda(6-85) JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Prigozhin, M. B., Gruebele, M. 2011; 133 (48): 19338-19341


    Molecular dynamics simulations combining many microsecond trajectories have recently predicted that a very fast folding protein like lambda repressor fragment λ(6-85) D14A could have a slow millisecond kinetic phase. We investigated this possibility by detecting temperature-jump relaxation to 5 ms. While λ(6-85) D14A has no significant slow phase, two even more stable mutants do. A slow phase of λ(6-85) D14A does appear in mild denaturant. The experimental data and computational modeling together suggest the following hypothesis: λ(6-85) takes only microseconds to reach its native state from an extensively unfolded state, while the latter takes milliseconds to reach compact β-rich traps. λ(6-85) is not only thermodynamically but also kinetically protected from reaching such "intramolecular amyloids" while folding.

    View details for DOI 10.1021/ja209073z

    View details for Web of Science ID 000297606500021

    View details for PubMedID 22066714

  • Conformational energy gaps and scaling of conformer density in chain molecules CHEMICAL PHYSICS LETTERS Walters, P. L., Prigozhin, M. B., Takeshita, T. Y., Xu, L., Olivarez, F. M., Gruebele, M. 2011; 507 (1-3): 15-18
  • Reducing Lambda Repressor to the Core JOURNAL OF PHYSICAL CHEMISTRY B Prigozhin, M. B., Sarkar, K., Law, D., Swope, W. C., Gruebele, M., Pitera, J. 2011; 115 (9): 2090-2096


    Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.

    View details for DOI 10.1021/jp110175x

    View details for Web of Science ID 000287833000021

    View details for PubMedID 21319829

  • Porous silicon: electrochemical microstructuring, photoluminescence and covalent modification International Conference on Application of Photonics Technology (Photonics North 2008) Prigozhin, M. B., Shiwsankar, P., Algar, W. R., Krull, U. J. SPIE-INT SOC OPTICAL ENGINEERING. 2008

    View details for DOI 10.1117/12.807175

    View details for Web of Science ID 000261333300045