title: 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.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, UK.
authors.forename: J L
authors.initials: JL
authors.lastname: Neira
authors.forename: B
authors.initials: B
authors.lastname: Davis
authors.forename: A G
authors.initials: AG
authors.lastname: Ladurner
authors.forename: A M
authors.initials: AM
authors.lastname: Buckle
authors.forename: G de P
authors.initials: Gde P
authors.lastname: Gay
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D003460:Crystallization; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012996:Solutions
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D012996:Solutions; C093336:chymotrypsin inhibitor 2; D002918:Chymotrypsin