Hydrogen-exchange stability analysis of Bergerac-Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Proc Natl Acad Sci U S A. 2003 May 13;100(10):5730-5. doi: 10.1073/pnas.0837456100. Epub 2003 Apr 28.

Abstract

Amide hydrogendeuterium exchange rates have been determined for two mutants of alpha-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from high-energy barrier crossings. Comparison of DeltaG(ex) and m(ex) parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long beta-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.

MeSH terms

  • Amino Acid Sequence
  • Genetic Variation
  • Hydrogen
  • Hydrogen Bonding
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Protein Conformation
  • Protein Folding
  • Spectrin / chemistry*
  • Spectrin / genetics
  • Spectrin / metabolism
  • Thermodynamics
  • src Homology Domains*

Substances

  • Spectrin
  • Hydrogen