title: Importance of alpha-helix N-capping motif in stabilization of betabetaalpha fold.
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.
authors.affiliation: Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Koscielska-Kasprzak
authors.forename: Tomasz
authors.initials: T
authors.lastname: Cierpicki
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D000577:Amides / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: D000577:Amides; D004268:DNA-Binding Proteins; C108466:FSD-1 protein, synthetic; D010455:Peptides; D014157:Transcription Factors