Importance of α–helix N–capping motif in stabilization of ββα fold

FSD‐1 (full sequence design 1) is a protein folded in a ββα 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 (S¹³RSDH¹⁷) 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 α‐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 N₃ in S¹³NEKE¹⁷ 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 N₁ Glu 15→Ser mutation also produced a considerable effect (1.0 kcal • mole⁻¹ destabilization), again emphasizing the significance of electrostatic interactions in α‐helix stabilization.