Kinetic role of helix caps in protein folding is context-dependent

Biochemistry. 2004 Apr 6;43(13):3814-23. doi: 10.1021/bi035683k.

Abstract

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / genetics
  • Amino Acid Motifs / genetics
  • Amino Acid Substitution / genetics
  • Arginine / genetics
  • Aspartic Acid / genetics
  • Bacteriophage lambda / chemistry
  • Bacteriophage lambda / genetics
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • Glutamine / genetics
  • Glycine / genetics
  • Kinetics
  • Lysine / genetics
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Protein Folding*
  • Protein Structure, Secondary* / genetics
  • Repressor Proteins / chemistry*
  • Repressor Proteins / genetics
  • Thermodynamics
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins

Substances

  • DNA-Binding Proteins
  • Peptide Fragments
  • Repressor Proteins
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins
  • phage repressor proteins
  • Glutamine
  • Aspartic Acid
  • Arginine
  • Lysine
  • Alanine
  • Glycine