Hodel A, Kautz R A, Adelman D M, Fox R O
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Protein Sci. 1994 Apr;3(4):549-56. doi: 10.1002/pro.5560030403.
We examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116-Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent-exposed, flexible loop of residues 112-117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side-chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112-117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.
我们研究了蛋白质环的受限末端所施加的构象限制在确定应变环构象中的作用。葡萄球菌核酸酶A的Lys 116 - Pro 117肽键存在于顺式和反式异构体之间的平衡中。折叠后的蛋白质更倾向于占比90%的应变顺式异构体。该肽键包含在112 - 117位残基的溶剂暴露柔性环中，其末端由Val 111和Asn 118固定。Asn 118通过2个侧链氢键受到限制。我们通过将Asn 118替换为天冬氨酸、丙氨酸和甘氨酸来研究这种限制的重要性。我们发现去除Asn 118中观察到的1个或更多氢键会使反式构型比顺式构型更稳定。通过降低pH值使N118D的Asp 118侧链质子化，Asp 118的氢键性质接近核酸酶A中Asn 118的氢键性质，并且顺式构型相对于反式构型得到稳定。这些数据表明环末端的刚性固定在建立应变顺式构象中很重要。核酸酶A中112 - 117位残基的片段为研究确定多肽构象的基本原理提供了一个有前景的模型系统。此类研究可能有助于蛋白质分子的合理设计或重新设计。
