Koscielska-Kasprzak Katarzyna, Cierpicki Tomasz, Otlewski Jacek
Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
Protein Sci. 2003 Jun;12(6):1283-9. doi: 10.1110/ps.0301703.
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.
FSD-1（全序列设计1）是一种折叠成ββα基序的蛋白质，它基于Zif268的第二个锌指结构域设计，通过用疏水核心取代其金属配位位点而形成。在这项工作中，我们分析了将模板锌指的DNA识别基序（S(13)RSDH(17)）引入FSD-1序列的可能性，以便获得一个不含交联或金属辅因子的小DNA结合模块。根据圆二色性（CD）和核磁共振（NMR）标准判断，该杂合蛋白发生了去折叠。为了揭示构成α-螺旋N-封端基序的五个氨基酸各自的作用，我们分析了八个FSD-1突变体的构象和稳定性特性。我们使用Leu 18的屏蔽甲基和215 nm处的CD信号作为折叠状态的便捷测量指标。在S(13)NEKE(17)变体中，N(3)位的Glu 17→His取代通过破坏N-封端相互作用，使折叠结构含量从90%降至25%（相当于1.8千卡·摩尔⁻¹的去稳定化），并且在此处研究的单突变体中具有最显著的影响。N(cap)位的Asn 14被Arg取代显著降低了稳定性，通过破坏螺旋封端氢键和螺旋大偶极的去稳定化作用，使结构含量从90%降至40%（1.4千卡·摩尔⁻¹的去稳定化）。N(1)位的Glu 15→Ser突变也产生了相当大的影响（1.0千卡·摩尔⁻¹的去稳定化），再次强调了静电相互作用在α-螺旋稳定中的重要性。
