Minimalist protein design:: A β-hairpin peptide that binds ssDNA
Minimalist protein design:: A β-hairpin peptide that binds ssDNA
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DOI：
10.1021/ja045002o
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发表时间：
2005-01-12
影响因子：
15
通讯作者：
Waters, ML
中科院分区：
文献类型：
--
作者：
Butterfield, SM;Cooper, WJ;Waters, ML
在β折叠的一面上有两个Trp和两个Lys残基的28个残基的β发夹二聚体（WKWK）2在低微摩尔浓度下与单链寡核苷酸形成复合物。二聚体的每个β-发夹含有对角取向的交叉链Trp-Trp对，其先前已显示产生用于插入芳族客体如腺嘌呤的裂缝（J. Am. Chem.Soc.2003，125，9580）。β-发夹二聚体在水中结合5-残基ssDNA序列5 '-AAAAA-3'、5 '-TTTTT-3'和5 '-CCCCC-3'，解离常数在12−30 μM范围内。观察到与序列5 '-AAAAA-3'结合的弱能量偏好，这被认为是由Trp和腺嘌呤碱基之间更强的堆积相互作用引起的。通过NMR证实了5 '-AAAAA-3'与肽的Lys和Trp残基的相互作用，并且证明了1：1缔合。在接近生理的离子强度和pH值下，11个残基的ssDNA序列的识别发生在3 μM的解离常数下，表明β-发夹二聚体与ssDNA的结合强度与许多天然存在的蛋白质一样强。11-残基寡核苷酸与肽二聚体相互作用的盐依赖性表明，Trp−核碱基堆积相互作用对识别的贡献约为−4 kcal/mol，这比含有Trp的非结构化肽中非离子相互作用的贡献大得多。此外，ssDNA的识别表明相对于相应的双链体降低了盐依赖性，导致在高盐条件下对ssDNA的选择性。肽（WKWK）2是OB折叠（寡核苷酸/寡糖结合）蛋白的相关模拟物，其结合β折叠表面上的ssDNA。
A 28-residue β-hairpin dimer(WKWK)2with two Trp and two Lys residues on one face of each β-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each β-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc.2003,125, 9580). The β-hairpin dimer binds 5-residue ssDNA sequences 5‘-AAAAA-3‘, 5‘-TTTTT-3‘, and 5‘-CCCCC-3‘ in water with dissociation constants in the range of 12−30 μM. A weak energetic preference for binding to sequence 5‘-AAAAA-3‘ was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5‘-AAAAA-3‘ with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 μM under near-physiological ionic strength and pH, demonstrating that the β-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp−nucleobase stacking interactions contribute about −4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide(WKWK)2is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a β-sheet.