Discerning the structure and energy of multiple transition states in protein folding using psi-analysis.

We quantify the degree to which folding occurs along a complex landscape with structurally distinct pathways using psi-analysis in combination with a protein engineering method that identifies native, non-covalent polypeptide interactions and their relative populations at the rate-limiting step. By probing the proximity of two specific partners, this method is extremely well-suited for comparison to theoretical simulations. Using ubiquitin as a model system, we detect individual pathways with site-resolved resolution, demonstrating that the protein folds through a native-like transition state ensemble with a common nucleus that contains heterogeneous features on its periphery. The consensus transition state topology has part of the major helix docked against four properly aligned beta-strands. However, structural heterogeneity exists in the transition state ensemble, wherein peripheral regions are differentially populated according to their relative stability. Pathway diversity reflects the variable order of formation of these peripheral elements, which radiate outward from the common nucleus. These results, which show only moderate agreement with traditional mutational phi-analysis, provide an extraordinarily detailed and quantitative description of protein folding.

[1]  L Serrano,et al.  Development of the multiple sequence approximation within the AGADIR model of alpha-helix formation: comparison with Zimm-Bragg and Lifson-Roig formalisms. , 1997, Biopolymers.

[2]  Lorna J. Smith,et al.  Understanding protein folding via free-energy surfaces from theory and experiment. , 2000, Trends in biochemical sciences.

[3]  T. Sosnick,et al.  Distinguishing between two-state and three-state models for ubiquitin folding. , 2000 .

[4]  E I Shakhnovich,et al.  Specific nucleus as the transition state for protein folding: evidence from the lattice model. , 1994, Biochemistry.

[5]  Hue Sun Chan,et al.  Contact order dependent protein folding rates: Kinetic consequences of a cooperative interplay between favorable nonlocal interactions and local conformational preferences , 2003, Proteins.

[6]  Hongyi Zhou,et al.  Folding rate prediction using total contact distance. , 2002, Biophysical journal.

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

[8]  M. S. Briggs,et al.  Hydrogen exchange in native and alcohol forms of ubiquitin. , 1992, Biochemistry.

[9]  D. Thirumalai,et al.  Fishing for folding nuclei in lattice models and proteins. , 1998, Folding & design.

[10]  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.

[11]  T. Sosnick,et al.  Molecular collapse: The rate‐limiting step in two‐state cytochrome c folding , 1996, Proteins.

[12]  K. Kuwajima,et al.  The molten globule state as a clue for understanding the folding and cooperativity of globular‐protein structure , 1989, Proteins.

[13]  A. Horovitz,et al.  φ value analysis of heterogeneity in pathways of allosteric transitions: Evidence for parallel pathways of ATP-induced conformational changes in a GroEL ring , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Khorasanizadeh,et al.  Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues , 1996, Nature Structural Biology.

[15]  The role of a β‐bulge in the folding of the β‐hairpin structure in ubiquitin , 2001 .

[16]  D Baker,et al.  The single helix in protein L is largely disrupted at the rate-limiting step in folding. , 1998, Journal of molecular biology.

[17]  Alan R Davidson,et al.  Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state. , 2002, Journal of molecular biology.

[18]  A. Kentsis,et al.  Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Ivet Bahar,et al.  Transition states and the meaning of Φ-values in protein folding kinetics , 2001, Nature Structural Biology.

[20]  R. Sauer,et al.  Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects , 2002, Nature Structural Biology.

[21]  L. Serrano,et al.  Obligatory steps in protein folding and the conformational diversity of the transition state , 1998, Nature Structural &Molecular Biology.

[22]  E I Shakhnovich,et al.  Folding nucleus: specific or multiple? Insights from lattice models and experiments. , 1998, Folding & design.

[23]  M. Proctor,et al.  Structural changes in the transition state of protein folding: alternative interpretations of curved chevron plots. , 1999, Biochemistry.

[24]  A. Fersht,et al.  The changing nature of the protein folding transition state: implications for the shape of the free-energy profile for folding. , 1998, Journal of molecular biology.

[25]  David Baker,et al.  Experiment and theory highlight role of native state topology in SH3 folding , 1999, Nature Structural Biology.

[26]  Matthews Cr.  Effect of point mutations on the folding of globular proteins. , 1987 .

[27]  Haipeng Gong,et al.  Local secondary structure content predicts folding rates for simple, two-state proteins. , 2003, Journal of molecular biology.

[28]  C. Bugg,et al.  Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin. , 1987, The Journal of biological chemistry.

[29]  K. Nagorny,et al.  Cluster-Bildung in Cu—Fe-Mischkristallen und Bestimmung der Löslichkeit von Eisen in Kupfer im Temperaturbereich von 450—1000°C , 1977 .

[30]  D Baker,et al.  Long-range order in the src SH3 folding transition state. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[31]  T. Sosnick,et al.  Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding. , 2002, Journal of molecular biology.

[32]  Valerie Daggett,et al.  Molecular dynamics simulations of hydrophobic collapse of ubiquitin , 1998, Protein science : a publication of the Protein Society.

[33]  K. Dill,et al.  From Levinthal to pathways to funnels , 1997, Nature Structural Biology.

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

[35]  P G Wolynes,et al.  Protein folding mechanisms and the multidimensional folding funnel , 1998, Proteins.

[36]  T. Sosnick,et al.  Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil , 2001, Nature Structural Biology.

[37]  Kevin W Plaxco,et al.  The topomer search model: A simple, quantitative theory of two‐state protein folding kinetics , 2003, Protein science : a publication of the Protein Society.

[38]  D. Goldenberg,et al.  φ-Values for BPTI folding intermediates and implications for transition state analysis , 2001, Nature Structural Biology.

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

[40]  Kevin W Plaxco,et al.  How the folding rate constant of simple, single-domain proteins depends on the number of native contacts , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Fersht,et al.  Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Kiefhaber,et al.  Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states. , 2003, Journal of molecular biology.

[43]  K. Kuwajima,et al.  Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin. , 1989, Journal of molecular biology.

[44]  Dudley H. Williams,et al.  Structural characterization of a mutant peptide derived from ubiquitin: Implications for protein folding , 2000, Protein science : a publication of the Protein Society.