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Center for Protein Folding Machinery

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Executive Summary

Naturally occurring cellular nanomachines called chaperones fold many critical cellular proteins in all human and animal cells [2, 3]. Different classes of chaperones work together to form elaborate cooperative networks and also ensure that potentially damaging misfolded polypeptides are cleared from the cell. Such misfolded proteins would otherwise cause a cascade of cellular damage and ultimately lead to globalized cell death. Our center envisions using chaperones as important targets for therapeutic interventions or diagnostic analyses in several human diseases related to protein misfolding. In particular, chaperonins represent a particularly intriguing class of chaperones because they possess a unique ability to fold some proteins that are not folded by more simple chaperones. We are focusing on the type II molecular chaperonins, namely, the eukaryotic chaperonin, TRiC/CCT and the archaebacterial chaperonin, Mm-cpn. We aim to understand the folding properties of a broad array of eukaryotic proteins that will establish an essential foundation for designing and optimizing proteins with novel functions.


Human cells contain many structurally and functionally distinct classes of chaperones of varying size and complexity. These range from those that only bind misfolded polypeptides and prevent their aggregation, to those that recognize specific classes of folding intermediates and promote their folding to the proper native state in an energy dependent manner [4]. Hundreds of enzymes depend on fully-functioning chaperones. As our population grows older, an increasing socio-economic burden stems from a class of diseases resulting from protein misfolding and protein aggregation. Millions of Americans suffer from the most common of these: Alzheimer's disease, Parkinson's disease, and Huntington's disease. In addition to such neurodegenerative diseases, folding defects play important roles in stroke, various types of cancer, and cataract formation. Protein chains that are required for healthy cell and organ function can misfold and ultimately aggregate into toxic fibers and large complexes in all these diseases [1].

An intriguing class of oligomeric, double-ring, high molecular weight chaperones, called chaperonins, possess a unique ability to fold some proteins that otherwise could not be folded by simpler chaperone systems. The specific protein folding nanomachine that our NDC has chosen is called the type II molecular chaperonin, which includes the eukaryotic chaperonin, TRiC/CCT, and the archaebacterial chaperonin, Mm-cpn.

The human chaperonin (TRiC) is essential for de novo folding of approximately 10 percent of newly synthesized proteins. TRiC acts on proteins of high biomedical relevance such as cytoskeletal components (actin and tubulin), cell cycle regulators (cyclin E and Cdc20), and many regulatory proteins containing a beta-propeller domain. Notably, many of these TRiC substrates cannot be folded by other prokaryotic or eukaryotic chaperones. TRiC and Mm-cpn differ in complexity. Although each consists of two eight-membered rings with a total molecular mass close to 1 MDa, eight different proteins form the ring in TRiC. In contrast, the archaebacterial chaperonin Mm-cpn contains eight copies of a single protein. These barrel-shaped chaperonins open and close their central chamber in response to ATP-binding and hydrolysis. We believe that re-engineering chaperonins could present a promising and cell-friendly bio-delivery container for drugs and nano-devices for therapeutic, diagnostic or industrial purposes. Describing the conformational dynamics of this process is important for understanding and controlling protein folding inside living cells.

NDC Goals

The overarching goal of our NDC is to develop chaperonin-based nanomachines to manipulate protein folding pathways for therapeutic and biomedical applications. A quantitative description of the motions of the chaperonin in a sequence of biochemical events during the protein folding process will provide the necessary specifications to permit a rational design of new chaperonins or their substrates. This will open up new therapeutic avenues, such as:

  • New anti-tumor therapies by designing small molecule adaptors to enable cellular chaperonins to correct folding defects in tumor suppressor proteins
  • New anti-amyloid therapies by selecting small molecules which increase the affinity of cellular chaperonins to prevent polyglutamine aggregation in Huntington's and related amyloid diseases
  • Modifying the chaperonin machinery to more effectively fold pharmaceutically important target proteins that currently cannot fold into their native state in the laboratory.

Figure 1 - The NDC is organized with synergistic interactions across disciplines and laboratories in seven institutions.

Our NDC has 17 established investigators from seven institutions. We take an integrated approach to study these chaperonins by combining the most suitable physical, chemical, computational and engineering methods for measurements and analysis, including cryo-electron microscopy and tomography, single-molecule spectroscopy and imaging, restrained protein structure modeling, protein dynamics simulation, in silico protein folding in the chaperonin environment, and protein engineering design. We continue developing these methodologies to suit the studies of this relatively large and structurally dynamic cellular nanomachine both in vitro and in vivo, and we are certain that these technologies will have wide ranging impact for the study of many large cellular nanomachines.

Operationally, we aim to establish a highly inter-dependent working relationship among all the investigators who specialize in different experimental and computational methodologies (Figure 1). All of our research priorities are driven by the goal of producing translational outcomes. We recognize that it is necessary to work together cohesively and to co-develop research strategies in tackling the fundamentally important and technically challenging problem of manipulating chaperonins. We believe that our team has created a novel research pathway that is optimal for studying cellular nanomachines in general and will lead to more rapid development of medical applications. Furthermore, an important aspect of our center is our constant attention to integrating the results from various methodologies to stimulate collaborations among the participating scientists in our NDC. For instance, there is ongoing interaction between the experimental and computational investigators at various stages of the project. In addition, our team is exploring a design engineering approach to describe how the chaperonin works as a nano-device in their folding cycle and how an engineered chaperonin or substrate can be developed. Consequently, our studies will create approaches and strategies with translational outcomes.

NDC accomplishments

Structural designs of Mm-cpn and TRiC and their conformational dynamics in the folding cycle (Investigators: Chiu, Ludtke, Levitt, Sali, Adams, King, Frydman)

Chaperonin architectural design is the most fundamental requirement required for any engineering approach to designing a chaperonin or substrate with new functionalities. We have undertaken this approach using cryo-EM, cryo-EM based modeling and x-ray crystallography to determine the chaperonin design principles. We now have established for the first time a high resolution structural mechanism used by the Mm-cpn to transition from open to closed conformation triggered by the ATP hydrolysis, which is needed to initiate protein folding in the chaperonin. This study leads us to hypothesize which residues of Mm-cpn may play pivotal roles in allowing such large molecular motion while maintaining the integrity of the complex, and how we will approach redesign of the chaperonin with different functional capabilities The ABEL Trap Enables ATP Counting for Multi-Subunit Enzymes in Solution (Investigators: Moerner, Frydman)

W. E. Moerner has built a new type of instrument, the Anti-Brownian Electrokinetic trap (ABEL trap), which can trap single biomolecules in free solution without the use of laser tweezers or large beads. A single biomolecule can thereby be examined for a long time without surface attachment in an aqueous environment. Starting with the eukaryotic protein-folding machine TRiC, the Moerner group has developed a way to measure the stoichiometry of ATP binding for a single enzyme; i.e., the number of ATP molecules that are bound to the various binding sites, subunit by subunit. We expect that the ABEL trap will become one of our standard biophysical measurements in assessing the biochemical activities for the future generation of engineered chaperonins, which inevitably would depend on ATP hydrolysis. Advances in Simulation of Chaperonin Dynamics in Water (Investigator: Pande)

Protein folding in the cell often relies on the help of chaperonins. Water, which is crucial to the folding of all proteins, is confined on the nanometer scale inside chaperonins and might therefore be expected to behave differently there than it does in bulk. Vijay Pande has hypothesized that the polar residues in the interior surface of GroE sequester some of the cavity water. This drop in water concentration decreases the hydrophobicity of the substrate protein allowing easier unfolding and refolding. They tested their prediction by analyzing simulations of mutants of the GroE complex whose activities have been characterized in past experiments. This simulation points out the importance of the use of computing to fill the information gap not directly available in experiments. 

Design of Functional Loop in Chaperonin (Investigator: Kortemme)

Proteins exploit the conformational variability of loop regions to carry out diverse biological tasks. Despite significant progress in loop prediction, more accurate methods to sample and evaluate the conformational space accessible to loops are still a major bottleneck for high-resolution protein modeling. Tanja Kortemme’s team has introduced a novel robotics-inspired local loop reconstruction method in modeling loop structure. She has validated her approach with two well-studied datasets giving sub-angstrom accuracy. This approach will be deployed in her adaptor design for our chaperonins.TRiC, a Biological Nanomachine to Prevent Huntingtin Fibril Aggregation (Investigators: Frydman, Pande, Chiu, Moerner)

Over the past two years, our Center has incorporated a newly-described TRiC substrate for nanomedicine applications. Following on the Frydman lab’s finding that TRiC is a central regulator of aggregation of polyQ-expanded variants of the Huntingtin protein (htt), the Frydman, Pande, Moerner and Chiu labs have started a closely-linked collaboration to understand the molecular and theoretical basis of chaperonin inhibition of aggregation. The Frydman lab has reconstituted the TRiC-Huntingtin interaction in vitro with purified components, which makes it possible to determine how TRiC changes the conformation and dynamic properties of the Huntingtin aggregation intermediates and the structure of the aggregates themselves. The biochemical experiments in the Frydman lab have been closely linked to the modeling efforts in the Pande lab to model key aspects of the aggregation process. In addition, Chiu and Moerner are applying cryo-EM and single moleucle imaging to characterize the molecular mechanism of Huntingtin aggregation and its modulation by the chaperonin.

Identification and progress in newly identified protein misfolding diseases (Investigators: Frydman, King, Jonasch, Eissa, Mobley, Graef, Tweardy, Pande, Kortemme, Chiu)

We have initiated new collaborations with five clinician scientists. Tony Eissa’s project on refolding cystic fibrosis transmembrane conductance regulator (CFTR) has been funded through a competitive supplement. His lab is now set up to express and purify the NDB1 domain (~21kDa) of wild type CTFR and of its mutant (with the Loss of Phe508 disrupting the folding pathway of CFTR in the endoplasmic reticulum). This project benefits from previous proteomic studies from Bill Balch’s lab indicating that TRiC does interact with CFTR during its folding.

With our scale-up funds, we have also allocated resources to support David Tweardy, William Mobley, Eric Jonasch, Isabella Graef and Jonathan King. These investigators conduct clinical research in fields where manipulation of folding pathways and intermediates can have great biomedical impact. Eric Jonasch is setting up assays to test for approaches to ameliorate folding of tumor-causing VHL mutants; while Mobley and Graef are leading the investigation of aggregation-related neurodegenerative diseases, including Alzheimer’s and Huntington’s. David Tweardy investigates how the JAK/STAT pathway regulates apoptosis and cell division during stress and cancer. These and future findings result from productive interactions of these investigators with Wah Chiu, Judith Frydman, Tanja Kortemme and Vijay Pande in connection with planned biochemical, cellular and biophysical experiments. We are excited about these new translational explorations.

To facilitate progress in moving from the laboratory to the clinic, King's group has purified human TRiC from Hela cells for cryo-EM characterization and mechanistic studies, and is developing protocols for isolating TRiC and its substrates from other human cell lines, such as lymphocytes.

Training and outreach: (King, Gossard, Frydman, Chiu)

We have successfully completed in the spring term of 2009 a didactic graduate level course on chaperonin and human disease offered at the Department of Biological Science at Stanford University. This course was broadcast to other sites via VC. The instructors are drawn from the investigators of our Center. Both the on-site and remote audience have benefitted from this course. We plan to offer such course again in the winter term of 2010.

A summer undergraduate research program was active in this summer with 6 trainees placed in the labs of Chiu, Mobely, King and Frydman.



  1. Dobson, C.M., The structural basis of protein folding and its links with human disease. Philos Trans R Soc Lond B Biol Sci, 2001. 356(1406): p. 133-45.
  2. Hartl, F.U. and M. Hayer-Hartl, Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 2002. 295(5561): p. 1852-8.
  3. Frydman, J., Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem, 2001. 70: p. 603-47.
  4. Dobson, C.M., Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol, 2004. 15(1): p. 3-16.

Publications supported by the NDC award since the inception of the Center

  1. Chen, D. H., Song, J. L., Chuang, D. T., Chiu, W. Ludtke, S. J. (2006). An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate. Structure 14: 1711-22. PMID: 17098196
  2. Speiss, C., Miller, E. J., McClellan, A. J., and Frydman, J. (2006). Identification of the TRIC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins. Molecular Cell 23(1):25-37. PMID: 17018290
  3. Marsh, M. P., Chang, J. T., Booth, C. R., Liang, N. L., Schmid, M. F., and Chiu, W. (2007). Modular software platform for low-dose electron microscopy and tomography. J Microsc 228: 384-389. PMID: 18045333
  4. Reissman, S., Parnot, C., Booth, C. R., Chiu, W., and Frydman, J. (2007). Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat Struct Mol Biol, 14: 432-40. PMID: 17460696
  5. Lucent, D. V., Vishal, V., and Pande, V. S. (2007). Protein folding under confinement: A new role for solvent. Proc Natl Acad Sci U S A, 104: 10430-10434.
  6. Schroder, G., Brunger, A., and Levitt, M. (2007) Deformable elastic network model to combine initial structural information with low resolution data: application to real-space refinement. Structure, 15:1630-1641.
  7. Ludtke, S., Baker, M.L., Chen, D.H., Song, J., Chuang, D. and Chiu, W. (2008) De Novo Backbone Trace of GroEL from Single Particle Electron Cryo-Microscopy. Structure, 16: 441-8. PMID:1833421
  8. Topf, M., Lasker, K., Webb, B., Wolfson, H., Chiu, W. and Sali, A.(2008). Protein Structure Fitting and Refinement Guided by Cryo-EM Density. Structure 16: 295-307. PMID: 18275820. PMCID: PMC2409374.
  9. England, J., Park, S., and Pande, V. S. (2008) Theory for an order-driven disruption of the liquid state in water. J Chem Physics, 128:044503. PMID: 18247965
  10. Booth, C. R., Meyer, A. S., Cong, Y., Topf, M., Sali, A., Ludtke, S, J., Chiu, W. and Frydman, J. (2008). Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT. Nature Struct & Molec Biol, 15:746-53. PMID: 18536725. PMCID: PMC2546500
  11. England, J. L., Lucent, D., and Pande, V. S. (2008). A Role for Confined Water in Chaperonin Function. J Am Chem Soc, 130:11838-9. PMID: 18710231. PMCID: PMC2646679.
  12. Förster, F., Webb B., Krukenberg, K.A., Tsuruta, H., Agard, D. A., Sali, A. (2008) Integration of small angle X-ray scattering data into structural modeling of proteins and assemblies, J. Mol Biol., 382(4):1089-106. PMID: 18694757. NIHMSID: 105380.
  13. Cohen, A. E., and Moerner, W. E. (2008). Controlling Brownian motion of single protein moleculesand single fluorophores in aqueous buffer. Optics Express 16: 6941-6956. PMID: 18545398. PMCID: PMC2435051.
  14. Chen, DH, Luke, K., Zhang, J., Chiu, W., and Wittung-Stafshede, P. (2008). Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein10 as derived by cryo-EM and biophysical techniques. J Mol Biol, 381:707-17. PMID: 18588898. PMCID: PMC2612737.
  15. England, J. and Pande, V. S. (2008). Potential for modulation of the hydrophobic effect inside chaperonins. Biophysical J, 95 3391-9 PMID: 18599630. PMCID: PMC2547425.
  16. Y. Jiang, Q. Wang, A. E. Cohen, N. Douglas, J. Frydman, and W. E. Moerner (2008), Hardwarebased anti-Brownian electrokinetic trap (ABEL trap) for single molecules: Control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes, Proc. SPIE 7038, 703807. NIHMSID: 131769
  17. England, J., Lucent, D., and Pande, V. S. (2008). Rattling the cage: computational models of chaperonin-mediated protein folding. Current Opin in Struct Biol, 18:163-9. PMID: 18291636
  18. Lucent, D. Lucent, England, J., Pande, V. S. (2008). Inside the Chaperonin toolbox: Theoretical and computational models for chaperonin mechanism, Physical Biology 6:15003. PMID: 19208937
  19. Zhang, J., Nakamura, N., Shimizu, Y., Liang, N. L., Liu, X., Jakana, J., Marsh, M. P., Booth, C. R. Shinkawa, T., Nakata, M. And Chiu, W. (2009). JADAS: a customizable automated data acquisition system and its application to ice-embedded single particles. J Struct Biol, 165: 1-9. PMID: 18926912. PMCID: PMC2634810.
  20. Baker, M. L., Marsh, M. P., and Chiu, W. (2009). Cryo-EM of molecular nanomachines and cells. Nanotechnology, ed. Viola Vogel, Wiley VCH, 91-111.
  21. Kelley, N.W., Huang, X., Tamm, S., Spies, C., Frydman, J., and Pande, V.S. (2009). The predicted structure of the headpiece of the Huntingtin protein and its implications on Huntingtin aggregation. J Mol Biol 388: 919-27. PMID: 19361448. PMCID: PMC2677131
  22. Zhang, R., X. Hu, H. Khant, S.J. Ludtke, W. Chiu, M.F. Schmid, C. Frieden, and J.M. Lee (2009). Interprotofilament interactions between Alzheimer's A{beta}1-42 peptides in amyloid fibrils revealed by cryoEM. Proc Natl Acad Sci U S A. 106: 4653-4658. PMID: 19264960. PMCID: PMC2660777.
  23. Lasker, K., Topf, M., Sali, A. and Wolfson, H. J. (2009). Inferential optimization for simultaneous fitting of multiple components into a cryoEM map of their assembly. J Mol Bio. 388: 180-94. PMID: 19233204. PMCID: PMC2680734
  24. Mandell, D.J., Coutsias E.A., Kortemme T. (2009). Sub-angstrom accuracy in protein loop reconstruction by robotics-inspired conformational sampling. Nature Methods 6:551-2. PMID: 19644455.
  25. Pintilie, G., J. Zhang, W. Chiu, D. Gossard (2009). Identifying Components in 3-D Density maps of protein nanomachines by multi-scale segmentation, in: Life Science Systems and Applications Workshop, 2009. LiSSA 2009. IEEE/NIH April 9-10, pp.44-47.

NDC-related publications

  1. Krukenberg, K. A., Förster, F., Rice, L. M., Sali, A., Agard, D.A. (2008). A novel conformation of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90. Structure 16, 755-765. PMID: 18462680
  2. Cong, Y., Topf, M., Sali, A., Matsudaira, P., Dougherty, M., Chiu, W. and Schmid, M.F. (2008). Crystallographic conformers of actin in a biologically active bundle of filaments. J Mol Biol 375: 331-6. PMID: 18022194
  3. Chandramouli, P., Topf, M., Ménétret, J. F., Eswar, N., Gutell, R. R., Sali, A., Akey, C. W. (2008). Structure of the Mammalian 80S Ribosome at 8.7Å Resolution. Structure 16, 535-548. PMID: 18400176
  4. Jiang, W., Baker, M.L., Jakana, J., Weigele, P. R., King, J. and Chiu, W. (2008). Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 451: 1130-4. PMID: 18305544
  5. Serysheva, I. I., Ludtke, S. J., Baker, M. L., Cong, Y., Topf, M., Eramian, D., Sali, A., Hamilton, S.L., and Chiu, W. (2008). Subnanometer resolution cryo-EM based domain models for the cytoplasmic region of skeletal muscle RyR channel. PNAS, 105:9610-5. PMID: 18621707
  6. Schwede, T., A. Sali, B. Honig, M. Levitt, H.M. Berman, D. Jones, S.E. Brenner, S.K. Burley, R. Das, N.V. Dokholyan, R.L. Dunbrack, Jr., K. Fidelis, A. Fiser, A. Godzik, Y.J. Huang, C. Humblet, M.P. Jacobson, A. Joachimiak, S.R. Krystek, Jr., T. Kortemme, A. Kryshtafovych, G.T. Montelione, J. Moult, D. Murray, R. Sanchez, T.R. Sosnick, D.M. Standley, T. Stouch, S. Vajda, M. Vasquez, J.D. Westbrook, and I.A. Wilson, 2009. Outcome of a workshop on applications of protein models in biomedical research. Structure 17: 151-159. PMID: 19217386
  7. Cong, Y., Zhang, Q., Woolford, D., Schweikardt, T., Khant, H., Dougherty, M., Ludtke, S.J., Chiu, W., Decker H. (2009). Structural mechanism of SDS-induced enzyme activity of Scorpion Hemocyanin revealed by electron cryo-microscopy. Structure 17: 749-758. PMID: 19446530.
  8. Samson, A. O., Levitt, M. (2008). Inhibition Mechanism of the Acetylcholine Receptor by R-Neurotoxins as Revealed by Normal-Mode Dynamics. Biochemistry, 47, 4065–4070. PMCID: 115709.
  9. Samson, A. O. and M. Levitt. (2009). Protein segment finder: an online search engine for segment motifs in the PDB, Nucleic Acid Research, 37, D224-D228. PMID: 2686524.

Contact Information

Center Website: http://proteinfoldingcenter.org Exit Disclaimer

Administrative Contact

  • Lenora Trujillo (lenorat@bcm.edu)
    Phone: 713-798-2191, Fax: 713-798-8682

Key Investigators

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