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Towards the complete structural characterization of a protein folding pathway: the structures of the denatured, transition and native states for the association/folding of two complementary fragments of cleaved chymotrypsin inhibitor 2. Direct evidence for a nucleation-condensation mechanism.

Neira JL, Davis B, Ladurner AG, Buckle AM, Gay Gde P, Fersht AR
Fold Des 1 189-208 (1996)
Cited: 22 times
EuropePMC logo PMID: 9079381

Abstract

Background

Single-module proteins, such as chymotrypsin inhibitor 2 (CI2), fold as a single cooperative unit. To solve its folding pathway, we must characterize, under conditions that favour folding, its denatured state, its transition state, and its final folded structure. To obtain a "denatured state' that can readily be thus characterized, we have used a trick of cleaving CI2 into two complementary fragments that associate and fold in a similar way to intact protein.

Results

Fragment CI2(1-40)-which contains the sequence of the single alpha-helix, spanning residues 12-24-and CI2(41-64), and mutants thereof, were analyzed by NMR spectroscopy, the transition state for association/folding was characterized by the protein engineering method, and the structure of the complex was solved by NMR and X-ray crystallography. Both isolated fragments are largely disordered. The transition state for association/folding is structured around a nucleus of a nearly fully formed alpha-helix, as is the transition state for the folding of intact CI2, from residues Ser12 to Leu21, Ala16, a residue from the helix whose sidechain is buried in the hydrophobic core, makes interactions with Leu49 and Ile57 in the other fragment. Ala16 makes its full interaction energy in the transition state for the association/folding reaction, just as found during the folding of the intact protein.

Conclusion

The specific contacts in the transition state from a nucleus that extends from one fragment to the next, but the nucleus is only "flickeringly' present in the denatured state. This is direct evidence for the nucleation-condensation mechanism in which the nucleus is only weakly formed in the ground state and develops in the transition state. The low conformational preferences in the denatured state are not enough to induce significant local secondary structure, but are reinforced by tertiary interactions during the rapid condensation around the nucleus.

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  1. From Levinthal to pathways to funnels. Dill KA, Chan HS. Nat Struct Biol 4 10-19 (1997)
  2. Nucleation mechanisms in protein folding. Fersht AR. Curr Opin Struct Biol 7 3-9 (1997)
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  4. Take home lessons from studies of related proteins. Nickson AA, Wensley BG, Clarke J. Curr Opin Struct Biol 23 66-74 (2013)
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  6. NMR of plant proteins. Kaas Q, Craik DJ. Prog Nucl Magn Reson Spectrosc 71 1-34 (2013)

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  1. Topological determinants of protein folding. Dokholyan NV, Li L, Ding F, Shakhnovich EI. Proc Natl Acad Sci U S A 99 8637-8641 (2002)
  2. Identification of side-chain clusters in protein structures by a graph spectral method. Kannan N, Vishveshwara S. J Mol Biol 292 441-464 (1999)
  3. Calculations on folding of segment B1 of streptococcal protein G. Sheinerman FB, Brooks CL. J Mol Biol 278 439-456 (1998)
  4. The fast protein folding problem. Gruebele M. Annu Rev Phys Chem 50 485-516 (1999)
  5. Molecular picture of folding of a small alpha/beta protein. Sheinerman FB, Brooks CL. Proc Natl Acad Sci U S A 95 1562-1567 (1998)
  6. Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat. Chen YW, Stott K, Perutz MF. Proc Natl Acad Sci U S A 96 1257-1261 (1999)
  7. Folding of a pressure-denatured model protein. Mohana-Borges R, Silva JL, Ruiz-Sanz J, de Prat-Gay G. Proc Natl Acad Sci U S A 96 7888-7893 (1999)
  8. Are residues in a protein folding nucleus evolutionarily conserved? Tseng YY, Liang J. J Mol Biol 335 869-880 (2004)
  9. Folding and stability of the three-stranded beta-sheet peptide Betanova: insights from molecular dynamics simulations. Colombo G, Roccatano D, Mark AE. Proteins 46 380-392 (2002)
  10. Deciphering the folding transition state structure and denatured state properties of nucleophosmin C-terminal domain. Scaloni F, Federici L, Brunori M, Gianni S. Proc Natl Acad Sci U S A 107 5447-5452 (2010)
  11. The rate of isomerisation of peptidyl-proline bonds as a probe for interactions in the physiological denatured state of chymotrypsin inhibitor 2. Tan YJ, Oliveberg M, Otzen DE, Fersht AR. J Mol Biol 269 611-622 (1997)
  12. Conversion of two-state to multi-state folding kinetics on fusion of two protein foldons. Inaba K, Kobayashi N, Fersht AR. J Mol Biol 302 219-233 (2000)
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  14. In silico protein fragmentation reveals the importance of critical nuclei on domain reassembly. Contreras Martínez LM, Borrero Quintana EE, Escobedo FA, DeLisa MP. Biophys J 94 1575-1588 (2008)
  15. Autonomous Sequences in Myoglobin Emerging from X-ray Structure of Holomyoglobin. Narita M, Narita M, Itsuno Y, Itsuno S. ACS Omega 4 992-999 (2019)


Related citations provided by authors (2)

  1. The Structure of the Transition State for the Association of Two Fragments of the Barley Chymotrypsin Inhibitor-2 to Generate Native-Like Protein: Implications for Mechanisms of Protein Folding. De Prat Gay G, Ruiz-Sanz J, Davis B, Fersht AR Proc. Natl. Acad. Sci. U.S.A. 91 10943- (1994)
  2. Generation of a Family of Protein Fragments for Structure-Folding Studies. 1. Folding Complementation of Two Fragments of Chymotrypsin Inhibitor-2 Formed by Cleavage at its Unique Methionine Residue. De Prat Gay G, Fersht AR Biochemistry 33 7957- (1994)

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