Visualizing chaperone-assisted protein folding


Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Crystallographic data and ensemble selection.
Figure 2
Figure 3: Spy–Im76–45 ensemble, arranged by r.m.s. deviation (r.m.s.d.) from the native state of Im76–45.
Figure 4: Contact maps of the Spy–Im76–45 complex.
Figure 5: Spy conformation changes upon substrate binding.
Figure 6: Flexibility of the Spy linker region and the effects of Super Spy mutants.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

Change history

  • 15 June 2016

    In the version of this article initially published online, the name of the program funding the author Shu Quan was incorrect. The correct name should be the Shanghai Pujiang Program. The error has been corrected for the print, PDF and HTML versions of this article.


  1. 1

    Keskin, O., Gursoy, A., Ma, B. & Nussinov, R. Principles of protein-protein interactions: what are the preferred ways for proteins to interact? Chem. Rev. 108, 1225–1244 (2008).

  2. 2

    Fraser, J.S. et al. Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl. Acad. Sci. USA 108, 16247–16252 (2011).

  3. 3

    Kay, L.E. New views of functionally dynamic proteins by solution NMR spectroscopy. J. Mol. Biol. 428 2 Pt A, 323–331 (2016).

  4. 4

    Salmon, L. & Blackledge, M. Investigating protein conformational energy landscapes and atomic resolution dynamics from NMR dipolar couplings: a review. Rep. Prog. Phys. 78, 126601 (2015).

  5. 5

    Blackledge, M.J. et al. Conformational backbone dynamics of the cyclic decapeptide antamanide: application of a new multiconformational search algorithm based on NMR data. Biochemistry 32, 10960–10974 (1993).

  6. 6

    Guerry, P. et al. Mapping the population of protein conformational energy sub-states from NMR dipolar couplings. Angew. Chem. Int. Ed. Engl. 52, 3181–3185 (2013).

  7. 7

    Jewett, A.I. & Shea, J.E. Reconciling theories of chaperonin accelerated folding with experimental evidence. Cell. Mol. Life Sci. 67, 255–276 (2010).

  8. 8

    Mashaghi, A. et al. Reshaping of the conformational search of a protein by the chaperone trigger factor. Nature 500, 98–101 (2013).

  9. 9

    Buckle, A.M., Zahn, R. & Fersht, A.R. A structural model for GroEL-polypeptide recognition. Proc. Natl. Acad. Sci. USA 94, 3571–3575 (1997).

  10. 10

    Martinez-Hackert, E. & Hendrickson, W.A. Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone. Cell 138, 923–934 (2009).

  11. 11

    Saio, T., Guan, X., Rossi, P., Economou, A. & Kalodimos, C.G. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344, 1250494 (2014).

  12. 12

    Joachimiak, L.A., Walzthoeni, T., Liu, C.W., Aebersold, R. & Frydman, J. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell 159, 1042–1055 (2014).

  13. 13

    Chen, D.H. et al. Visualizing GroEL/ES in the act of encapsulating a folding protein. Cell 153, 1354–1365 (2013).

  14. 14

    Karagöz, G.E. et al. Hsp90-Tau complex reveals molecular basis for specificity in chaperone action. Cell 156, 963–974 (2014).

  15. 15

    Dekker, C. et al. The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. EMBO J. 30, 3078–3090 (2011).

  16. 16

    Muñoz, I.G. et al. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin. Nat. Struct. Mol. Biol. 18, 14–19 (2011).

  17. 17

    Elad, N. et al. Topologies of a substrate protein bound to the chaperonin GroEL. Mol. Cell 26, 415–426 (2007).

  18. 18

    Albert, A. et al. Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase. J. Biol. Chem. 285, 6371–6376 (2010).

  19. 19

    Quan, S. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat. Struct. Mol. Biol. 18, 262–269 (2011).

  20. 20

    Friel, C.T., Smith, D.A., Vendruscolo, M., Gsponer, J. & Radford, S.E. The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints. Nat. Struct. Mol. Biol. 16, 318–324 (2009).

  21. 21

    Figueiredo, A.M., Whittaker, S.B., Knowling, S.E., Radford, S.E. & Moore, G.R. Conformational dynamics is more important than helical propensity for the folding of the all α-helical protein Im7. Protein Sci. 22, 1722–1738 (2013).

  22. 22

    Stull, F., Koldewey, P., Humes, J.R., Radford, S.E. & Bardwell, J.C. Substrate protein folds while it is bound to the ATP-independent chaperone Spy. Nat. Struct. Mol. Biol. 23, 53–58 (2016).

  23. 23

    Kwon, E., Kim, D.Y., Gross, C.A., Gross, J.D. & Kim, K.K. The crystal structure Escherichia coli Spy. Protein Sci. 19, 2252–2259 (2010).

  24. 24

    Quan, S. et al. Super Spy variants implicate flexibility in chaperone action. eLife 3, e01584 (2014).

  25. 25

    Creamer, L.K., Richardson, T. & Parry, D.A. Secondary structure of bovine alpha s1- and beta-casein in solution. Arch. Biochem. Biophys. 211, 689–696 (1981).

  26. 26

    Chak, K.F., Safo, M.K., Ku, W.Y., Hsieh, S.Y. & Yuan, H.S. The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface. Proc. Natl. Acad. Sci. USA 93, 6437–6442 (1996).

  27. 27

    Pashley, C.L. et al. Conformational properties of the unfolded state of Im7 in nondenaturing conditions. J. Mol. Biol. 416, 300–318 (2012).

  28. 28

    Burling, F.T., Weis, W.I., Flaherty, K.M. & Brünger, A.T. Direct observation of protein solvation and discrete disorder with experimental crystallographic phases. Science 271, 72–77 (1996).

  29. 29

    van den Bedem, H., Dhanik, A., Latombe, J.C. & Deacon, A.M. Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers. Acta Crystallogr. D Biol. Crystallogr. 65, 1107–1117 (2009).

  30. 30

    Brennan, S. & Cowan, P.L. A suite of programs for calculating X-ray absorption, reflection, and diffraction performance for a variety of materials at arbitrary wavelengths. Rev. Sci. Instrum. 63, 850–853 (1992).

  31. 31

    Karanicolas, J. & Brooks, C.L. III. The origins of asymmetry in the folding transition states of protein L and protein G. Protein Sci. 11, 2351–2361 (2002).

  32. 32

    Jewett, A.I. & Shea, J.E. Folding on the chaperone: yield enhancement through loose binding. J. Mol. Biol. 363, 945–957 (2006).

  33. 33

    Bardwell, J.C. & Jakob, U. Conditional disorder in chaperone action. Trends Biochem. Sci. 37, 517–525 (2012).

  34. 34

    Quan, S., Hiniker, A., Collet, J.F. & Bardwell, J.C. Isolation of bacteria envelope proteins. Methods Mol. Biol. 966, 359–366 (2013).

  35. 35

    Fischer, M., Shoichet, B.K. & Fraser, J.S. One crystal, two temperatures: cryocooling penalties alter ligand binding to transient protein sites. ChemBioChem 16, 1560–1564 (2015).

  36. 36

    Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

  37. 37

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

  38. 38

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  39. 39

    Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  40. 40

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  41. 41

    Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  42. 42

    Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

  43. 43

    Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

Download references


The authors would like to thank J. Smith, D. Akey, U. Jakob, D. Smith, Z. Wawrzak, and F. Stull for critical comments and suggestions. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). This work was funded by an NRSA National Institutes of Health (NIH) grant GM108298 (L.S.A.), a Boehringer Ingelheim Fonds fellowship (P.K.), a National Natural Science Foundation of China (NSFC) grant 31400664 (S.Q.), the Shanghai Pujiang Program (S.Q.), NIH grant GM102829 (J.C.A.B.), NIH grant GM107233 (C.L.B.), NIH grant 1P01 GM063210 (P.V.), the Phenix Industrial Consortium and the US Department of Energy Contract No. DE-AC02-05CH11231 (P.V.) and NSF grant CHE1506273 (C.L.B.). J.C.A.B. is supported as a Howard Hughes Medical Institute Investigator.

Author information

The overall concept was conceived by S.H. and J.C.A.B. Experiments were designed by S.H., S.Q., J.C.A.B., R.C.T., H.v.d.B., and P.K. Experiments were performed by S.H., S.Q., P.K., R.M., and L.W. Analyses and computational modeling were designed by C.L.B., L.S., P.V.A., L.S.A., H.v.d.B., and S.H. Computational analysis was carried out by Q.X., S.H., L.S., L.S.A., P.V.A., P.K., and R.M. The manuscript was written primarily by S.H. and J.C.A.B., with assistance from L.S., L.S.A., and all other authors.

Correspondence to Scott Horowitz or James C A Bardwell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Truncating Spy’s unstructured tails does not dramatically decrease chaperone activity in vivo or in vitro.

(a) Overexpression of Spy increases the steady state level of the unstable Im7 mutant (Im7 L53A I54A) detected via SDS-PAGE and Coomassie stain (above) or by anti-His tag antibody (below). (b) Truncating Spy’s unstructured tails does not dramatically alter chaperone activity in vitro, as determined by the aldolase refolding assay. In vitro, Spy is able to provide minor aid to aldolase refolding capacity, compared to the major folding aid provided to Im7 in vivo. Error bars plotted are s.e.m. using 3-5 replicates from separate experiments.

Supplementary Figure 2 Detection of substrates in Spy crystals.

Substrates are Im76–45 (a), casein 148-177 (b), and Im7 L18A L19A L37A (c), respectively. Lanes W1-W5: washes of crystals in crystallization solution; lane D: dissolved crystals. The lack of protein in (b) for W2 relative to W3 for casein 148-177 and in (c) for Im7 L18A L19A L37A for W2 relative to W1 was due to some crystal breakage in the latter wash drops. WT Im7 crystals were too fragile to survive the washing process.

Supplementary Figure 3 Im76–45 structural properties.

(a) Comparison of disordered Im76-45 helical propensity in simulation and experiment. Per-residue helicity profiles obtained from coarse-grained simulations (FF1-FF3) and NMR chemical shifts (Loïc Salmon, Logan S. Ahlstrom, Scott Horowitz, Alex Dickson, Charles L. Brooks III, and James C. A. Bardwell, unpublished data: Exp, black curve). The unmodified Im76-45 force field (FF1, red curve) yields an ensemble that overestimates the helicity. Applying an adjustment to the pseudo-dihedral potential (FF2, cyan curve) improves the agreement of the helicity profile with experiment. Rescaling the non-bonded interactions in addition to this adjustment results in a helicity distribution that closely matches the experimental profile (FF3, orange curve). (b) Native structure of WT Im7. Residues 6-45 are highlighted in orange.

Supplementary Figure 4 Pseudocrystal environments for Spy-Im76–45 simulations.

(a) and (b) The two pseudo-crystal environments for Spy Im76-45 simulations. Use of two environments allows all multi-Spy interactions Im76-45 would encounter within the crystal. Both environments comprise four crystallographic Spy dimers (blue, red, yellow, and magenta) and a single peptide (orange).

Supplementary Figure 5 Simulated data validation test of READ selection protocol.

(a) Flowchart of simulated data validation test. (b) Three target ensembles (cyan) overlaid with selected ensembles (magenta). RMSDs of target to selected ensembles: 5.7 Å (top left), 6.5 Å (middle right), and 3.7 Å (bottom left).

Supplementary Figure 6 Flowchart of pool corruption validation test for READ selection protocol.

Supplementary Figure 7 Bootstrapping validation test of READ selection protocol.

37% of data replaced with repeat data in 200 separate selections was used to calculate mean and standard error contact maps. Note the change in intensity scale for the standard error map of the selections. The contact profile of the ensembles from the bootstrapped selections has low error and displays a different binding profile than the initial MD pool.

Supplementary Figure 8 Additional binding and competition assays.

(a) Binding of Spy 29-124 to Im7 L18A L19A L37A H40W measured via ITC. This Im7 mutant was subsequently used for competition assays with Im76-45 and Spy 29-124. (b) Casein 148-177 competes with Im7 6-45 for Spy 29-124 binding. Due to binding between Im76-45 and casein 148-177, as identified by analytical ultracentrifugation experiments of the two proteins (c), we were unable to accurately quantitate competition between Im76-45 and casein 148-177 (b). The dissociation constant of casein 148-177 to Spy 29-124 was determined by fluorescence (d) with a 1:1 stoichiometry of casein 148-177 to Spy 29-124 (e). Also, see Fig. 5c for competition of Im76-45 with Im7 L18A L19A L37A H40W. Error bars depict s.d. of n=3 technical replicates.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Note (PDF 2016 kb)

Supplementary Data Set 1

6.5 kev anomalous difference maps of pI-Phe substituted Im76–45 (PDF 6013 kb)

Movie of six-member selected ensemble of Spy:Im76–45

Ensemble members ordered by RMSD from least to most nativelike. Individual snapshots of the six-membered ensemble shown in Fig. 3. (MP4 4262 kb)

Morph of apo Spy (PDB 3O39) to bound Spy

Depicts the twisting motion upon substrate binding (MP4 660 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Horowitz, S., Salmon, L., Koldewey, P. et al. Visualizing chaperone-assisted protein folding. Nat Struct Mol Biol 23, 691–697 (2016).

Download citation

Further reading