An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms

In recent years, improvements in experimental techniques and enhancements in computing power have revolutionized our understanding of the mechanisms of protein folding. By combining insights gained from theory, experiment and simulation we are moving toward an atomistic view of folding landscapes. Future challenges involve exploiting the knowledge gained and methods developed to enable us to elucidate a molecular description of folding dynamics in the complex environment of the cell.

[1]  Richard I. Morimoto,et al.  Adapting Proteostasis for Disease Intervention , 2008, Science.

[2]  K. Lindorff-Larsen,et al.  Parallel protein-unfolding pathways revealed and mapped , 2003, Nature Structural Biology.

[3]  Terrence G. Oas,et al.  Backbone Dynamics of the Monomeric λ Repressor Denatured State Ensemble under Nondenaturing Conditions , 2007 .

[4]  M. Gruebele,et al.  Kinetics are probe-dependent during downhill folding of an engineered λ6–85 protein , 2005 .

[5]  R. Seckler,et al.  Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy , 2006, Proceedings of the National Academy of Sciences.

[6]  Aleksandra M. Walczak,et al.  The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures , 2008, PLoS Comput. Biol..

[7]  J. Elf,et al.  Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell , 2007, Science.

[8]  L. Gierasch,et al.  Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant , 2006, Proceedings of the National Academy of Sciences.

[9]  Gary J. Pielak,et al.  Macromolecular Crowding in the Escherichia coli Periplasm Maintains α-Synuclein Disorder , 2006 .

[10]  K. Wüthrich,et al.  Folding trajectories of human dihydrofolate reductase inside the GroEL–GroES chaperonin cavity and free in solution , 2007, Proceedings of the National Academy of Sciences.

[11]  M. Oliveberg,et al.  Malleability of protein folding pathways: a simple reason for complex behaviour. , 2007, Current opinion in structural biology.

[12]  H. Chan,et al.  Criteria for downhill protein folding: Calorimetry, chevron plot, kinetic relaxation, and single‐molecule radius of gyration in chain models with subdued degrees of cooperativity , 2006, Proteins.

[13]  Andreas Martin,et al.  Prolyl isomerization as a molecular timer in phage infection , 2005, Nature Structural &Molecular Biology.

[14]  A. Fersht,et al.  Structural Biology: Analysis of 'downhill' protein folding , 2007, Nature.

[15]  D. Baker,et al.  Contact order, transition state placement and the refolding rates of single domain proteins. , 1998, Journal of molecular biology.

[16]  D Thirumalai,et al.  Effects of crowding and confinement on the structures of the transition state ensemble in proteins. , 2007, The journal of physical chemistry. B.

[17]  C. Dobson,et al.  Determination of the transition state ensemble for the folding of ubiquitin from a combination of Phi and Psi analyses. , 2008, Journal of molecular biology.

[18]  Robert B. Best,et al.  Thermodynamics and kinetics of protein folding under confinement , 2008, Proceedings of the National Academy of Sciences.

[19]  C. Dobson,et al.  The folding of hen lysozyme involves partially structured intermediates and multiple pathways , 1992, Nature.

[20]  Stefan M. Larson,et al.  The family feud: do proteins with similar structures fold via the same pathway? , 2005, Current opinion in structural biology.

[21]  T. Kiefhaber,et al.  Origin of unusual phi-values in protein folding: evidence against specific nucleation sites. , 2003, Journal of molecular biology.

[22]  D. Baker,et al.  The Highly Cooperative Folding of Small Naturally Occurring Proteins Is Likely the Result of Natural Selection , 2007, Cell.

[23]  Michele Vendruscolo,et al.  The Mechanism of Folding of Im7 Reveals Competition between Functional and Kinetic Evolutionary Constraints , 2009, Nature Structural &Molecular Biology.

[24]  J. Onuchic,et al.  Funnels, pathways, and the energy landscape of protein folding: A synthesis , 1994, Proteins.

[25]  A. Fersht,et al.  Distinguishing between cooperative and unimodal downhill protein folding , 2007, Proceedings of the National Academy of Sciences.

[26]  José N Onuchic,et al.  Extracting function from a β-trefoil folding motif , 2008, Proceedings of the National Academy of Sciences.

[27]  M. Vendruscolo,et al.  Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins , 2008, Proceedings of the National Academy of Sciences.

[28]  A. Fersht,et al.  Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. , 1991, Biochemistry.

[29]  Dmitry M Korzhnev,et al.  Probing invisible, low-populated States of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding. , 2008, Accounts of chemical research.

[30]  Yan Zhang,et al.  Structure-function-folding relationship in a WW domain. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Robin S. Dothager,et al.  Random-coil behavior and the dimensions of chemically unfolded proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Dirar Homouz,et al.  Crowded, cell-like environment induces shape changes in aspherical protein , 2008, Proceedings of the National Academy of Sciences.

[33]  W. Eaton,et al.  Protein folding studied by single-molecule FRET. , 2008, Current opinion in structural biology.

[34]  S. Hagen Probe‐dependent and nonexponential relaxation kinetics: Unreliable signatures of downhill protein folding , 2007, Proteins.

[35]  Wilfred F van Gunsteren,et al.  Molecular simulation as an aid to experimentalists. , 2008, Current opinion in structural biology.

[36]  B. Fierz,et al.  Loop formation in unfolded polypeptide chains on the picoseconds to microseconds time scale , 2007, Proceedings of the National Academy of Sciences.

[37]  Doug Barrick,et al.  Rerouting the folding pathway of the Notch ankyrin domain by reshaping the energy landscape. , 2008, Journal of the American Chemical Society.

[38]  Eugene I Shakhnovich,et al.  Identification of the minimal protein-folding nucleus through loop-entropy perturbations. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[39]  D. Raleigh,et al.  Denatured state effects and the origin of nonclassical phi values in protein folding. , 2006, Journal of the American Chemical Society.

[40]  Sebastian Doniach,et al.  Small-angle X-ray scattering from RNA, proteins, and protein complexes. , 2007, Annual review of biophysics and biomolecular structure.

[41]  Michele Vendruscolo,et al.  Life on the edge: a link between gene expression levels and aggregation rates of human proteins. , 2007, Trends in biochemical sciences.

[42]  Changbong Hyeon,et al.  Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations , 2007, Proceedings of the National Academy of Sciences.

[43]  G. Ulrich Nienhaus,et al.  Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[44]  A. Soldatova,et al.  Protein dynamics from time resolved UV Raman spectroscopy. , 2008, Current opinion in structural biology.

[45]  Alan R Davidson,et al.  The analysis of protein folding kinetic data produced in protein engineering experiments. , 2004, Methods.

[46]  Joseph A Marsh,et al.  Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure. , 2007, Journal of molecular biology.

[47]  Bernd Bukau,et al.  The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins , 2009, Nature Structural &Molecular Biology.

[48]  Ellinor Haglund,et al.  Changes of Protein Folding Pathways by Circular Permutation , 2008, Journal of Biological Chemistry.

[49]  José N Onuchic,et al.  Backtracking on the folding landscape of the β-trefoil protein interleukin-1β? , 2008, Proceedings of the National Academy of Sciences.

[50]  Carlos Bustamante,et al.  Direct Observation of the Three-State Folding of a Single Protein Molecule , 2005, Science.

[51]  A. R. Fersht,et al.  Solution structure of a protein denatured state and folding intermediate , 2005, Nature.

[52]  A. Liljas,et al.  Crystal structure of the ribosomal protein S6 from Thermus thermophilus. , 1994, The EMBO journal.

[53]  F. Hartl,et al.  Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding , 2008, Cell.

[54]  J. Hofrichter,et al.  The protein folding 'speed limit'. , 2004, Current opinion in structural biology.

[55]  M. Textor,et al.  Probing protein-chaperone interactions with single-molecule fluorescence spectroscopy. , 2008, Angewandte Chemie.

[56]  H. Saibil Chaperone machines in action. , 2008, Current opinion in structural biology.

[57]  F. Hartl,et al.  Converging concepts of protein folding in vitro and in vivo , 2009, Nature Structural &Molecular Biology.

[58]  H Videler,et al.  A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity. , 1998, The Biochemical journal.

[59]  S. Radford,et al.  Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding. , 2006, Journal of molecular biology.

[60]  Shang-Te Danny Hsu,et al.  Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy , 2007, Proceedings of the National Academy of Sciences.

[61]  Victor Muñoz,et al.  Atom-by-atom analysis of global downhill protein folding , 2006, Nature.

[62]  Sheena E Radford,et al.  Intermediates: ubiquitous species on folding energy landscapes? , 2007, Current opinion in structural biology.

[63]  A. Fersht,et al.  The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. , 1992, Journal of molecular biology.

[64]  Michele Vendruscolo,et al.  Determination of the folding transition states of barnase by using PhiI-value-restrained simulations validated by double mutant PhiIJ-values. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[65]  E. Paci,et al.  Transition states for protein folding using molecular dynamics and experimental restraints , 2007 .

[66]  Heinz Fabian,et al.  Methods to study protein folding by stopped-flow FT-IR. , 2004, Methods.

[67]  M. Saraste,et al.  Crystal structure of the SH3 domain in human Fyn; comparison of the three‐dimensional structures of SH3 domains in tyrosine kinases and spectrin. , 1993, The EMBO journal.

[68]  S. Englander,et al.  Hydrogen exchange methods to study protein folding. , 2004, Methods.

[69]  C. Royer Probing Protein Folding and Conformational Transitions with Fluorescence , 2006 .

[70]  C. Dobson,et al.  Formation of native and non-native interactions in ensembles of denatured ACBP molecules from paramagnetic relaxation enhancement studies. , 2005, Journal of molecular biology.

[71]  S. Marqusee,et al.  Destabilization of the Escherichia coli RNase H kinetic intermediate: switching between a two-state and three-state folding mechanism. , 2004, Journal of molecular biology.

[72]  Huan‐Xiang Zhou,et al.  Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. , 2008, Annual review of biophysics.

[73]  B. Schuler,et al.  Two-state folding observed in individual protein molecules. , 2004, Journal of the American Chemical Society.

[74]  M Karplus,et al.  The Levinthal paradox: yesterday and today. , 1997, Folding & design.

[75]  M. Gruebele Comment on probe‐dependent and nonexponential relaxation kinetics: Unreliable signatures of downhill protein folding , 2008, Proteins.

[76]  Daphne H. E. W. Huberts,et al.  Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding* , 2008, Journal of Biological Chemistry.

[77]  C. Dobson Protein folding and misfolding , 2003, Nature.

[78]  Hidekazu Hiroaki,et al.  High-resolution multi-dimensional NMR spectroscopy of proteins in human cells , 2009, Nature.

[79]  Jonathan S. Weissman,et al.  A kinetic explanation for the rearrangement pathway of BPTI folding , 1995, Nature Structural Biology.

[80]  Peter J McCormick,et al.  Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins , 2004, Cell.

[81]  Alan R. Fersht,et al.  From the first protein structures to our current knowledge of protein folding: delights and scepticisms , 2008, Nature Reviews Molecular Cell Biology.

[82]  Claudia S Maier,et al.  Protein conformations, interactions, and H/D exchange. , 2005, Methods in enzymology.

[83]  M. Searle,et al.  Population of on-pathway intermediates in the folding of ubiquitin. , 2006, Journal of molecular biology.

[84]  A. Fersht,et al.  Phi-value analysis and the nature of protein-folding transition states. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[85]  B. Schuler,et al.  Unfolded protein and peptide dynamics investigated with single-molecule FRET and correlation spectroscopy from picoseconds to seconds. , 2008, The journal of physical chemistry. B.

[86]  H. Roder,et al.  Early events in protein folding explored by rapid mixing methods. , 2006, Chemical reviews.

[87]  Gerhard Wagner,et al.  Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes , 2006, Proceedings of the National Academy of Sciences.

[88]  A. Kidera,et al.  Atomically detailed description of the unfolding of alpha-lactalbumin by the combined use of experiments and simulations. , 2005, Journal of molecular biology.

[89]  V. Muñoz,et al.  Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha+beta topology that folds downhill. , 2008, Journal of the American Chemical Society.

[90]  Masaki Mishima,et al.  Protein structure determination in living cells by in-cell NMR spectroscopy , 2009, Nature.

[91]  R. Ellis Macromolecular crowding : obvious but underappreciated , 2022 .

[92]  William J. Fitzgerald,et al.  Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells , 2007, Proceedings of the National Academy of Sciences.

[93]  J. Onuchic,et al.  Theory of Protein Folding This Review Comes from a Themed Issue on Folding and Binding Edited Basic Concepts Perfect Funnel Landscapes and Common Features of Folding Mechanisms , 2022 .

[94]  K. Dill,et al.  The Protein Folding Problem , 1993 .

[95]  Zoya Ignatova,et al.  Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[96]  M. Gruebele,et al.  Kinetics are probe-dependent during downhill folding of an engineered lambda6-85 protein. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[97]  P. Schwille,et al.  Fluorescence correlation spectroscopy: novel variations of an established technique. , 2007, Annual review of biophysics and biomolecular structure.

[98]  Peter E Wright,et al.  Elucidation of the protein folding landscape by NMR. , 2005, Methods in enzymology.

[99]  Detection of a hidden folding intermediate in the focal adhesion target domain: Implications for its function and folding , 2006, Proteins.

[100]  N. C. Price,et al.  How to study proteins by circular dichroism. , 2005, Biochimica et biophysica acta.

[101]  Peter G Wolynes,et al.  Localizing frustration in native proteins and protein assemblies , 2007, Proceedings of the National Academy of Sciences.

[102]  Conggang Li,et al.  Residue-level interrogation of macromolecular crowding effects on protein stability. , 2008, Journal of the American Chemical Society.

[103]  C. Dobson,et al.  Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR , 2004, Nature.

[104]  S. Radford,et al.  Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[105]  R. B. Dyer,et al.  Ultrafast and downhill protein folding. , 2007, Current opinion in structural biology.

[106]  Alan R Lowe,et al.  Rational redesign of the folding pathway of a modular protein , 2007, Proceedings of the National Academy of Sciences.

[107]  A. Zarrine-Afsar,et al.  Φ-Value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy , 2007, Proceedings of the National Academy of Sciences.