Importance of alpha-helix N-capping motif in stabilization of betabetaalpha fold

Protein Sci. 2003 Jun;12(6):1283-9. doi: 10.1110/ps.0301703.

Abstract

FSD-1 (full sequence design 1) is a protein folded in a betabetaalpha motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S(13)RSDH(17)) into FSD-1 sequence in order to obtain a small DNA-binding module devoid of cross-link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N-capping motif of the alpha-helix, we analyzed conformational and stability properties of eight FSD-1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17-->His substitution at the N(3) in S(13)NEKE(17) variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal * mole(-1) destabilization) by disruption of N-capping interactions, and had the most significant effect among single mutants studied here. The N(cap) Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal * mole(-1) destabilization) by disruption of a helix-capping hydrogen bond and destabilization of a helix macrodipole. The N(1) Glu 15-->Ser mutation also produced a considerable effect (1.0 kcal * mole(-1) destabilization), again emphasizing the significance of electrostatic interactions in alpha-helix stabilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides / chemistry
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Circular Dichroism
  • DNA-Binding Proteins / chemistry*
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptides / chemistry
  • Peptides / genetics
  • Protein Folding*
  • Protein Structure, Secondary*
  • Thermodynamics
  • Transcription Factors / chemistry*
  • Zinc Fingers

Substances

  • Amides
  • DNA-Binding Proteins
  • FSD-1 protein, synthetic
  • Peptides
  • Transcription Factors