Kinetic role of helix caps in protein folding is context-dependent.
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.
Kapp
Gregory T
GT
Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710, USA.
Richardson
Jane S
JS
Oas
Terrence G
TG
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D005973:Glutamine; D001224:Aspartic Acid; D001120:Arginine; D008239:Lysine; D000409:Alanine; D005998:Glycine