Palme S, Jaenicke R, Slingsby C
Department of Crystallography, Birkbeck College, London, United Kingdom.
Protein Sci. 1998 Mar;7(3):611-8. doi: 10.1002/pro.5560070310.
GammaB-crystallin consists of two domains each comprising two "Greek key" motifs. Both domains fold independently, and domain interactions contribute significantly to the stability of the C-terminal domain. In a previous study (Palme S et al., 1996, Protein Sci 6:1529-1636) it was shown that Phe56 from the N-terminal domain, a residue involved in forming a hydrophobic core at the domain interface, effects the interaction of the two domains, and therefore, the stability of the C-terminal domain. Ala or Asp at position 56 drastically decreased the stability of the C-terminal domain, whereas Trp had a more moderate effect. In this article we present the X-ray structures of these interface mutants and correlate them with the stability data. The mutations do not effect the overall structure of the molecule. No structural changes are observed in the vicinity of the replaced residue, suggesting that the local structure is too rigid to allow compensations for the amino acid replacements. In the mutants gammaB-F56A and -F56D, a solvent-filled groove accessible to the bulk solvent is created by the replacement of the bulky Phe side chain. In gammaB-F56W, the pyrrole moiety of the indole ring replaces the phenyl side chain of the wild type. With the exception of gammaB-F56W, there is a good correlation between the hydrophobicity of the amino acid at position 56 according to the octanol scale and the stability of the C-terminal domain. In gammaB-F56W, the C-terminal domain is less stable than estimated from the hydrophobicity, presumably because the ring nitrogen (Nepsilon1) has no partner to form hydrogen bonds. The data suggest that the packing of hydrophobic residues in the interface core is important for domain interactions and the stability of gammaB-crystallin. Apparently, for protein stability, the same principles apply for hydrophobic cores within domains and at domain interfaces.
γB-晶状体蛋白由两个结构域组成，每个结构域包含两个“希腊钥匙”基序。两个结构域独立折叠，且结构域间的相互作用对C端结构域的稳定性有显著贡献。在之前的一项研究（Palme S等人，1996年，《蛋白质科学》6:1529 - 1636）中表明，来自N端结构域的Phe56（该残基参与在结构域界面形成疏水核心）影响两个结构域的相互作用，进而影响C端结构域的稳定性。56位的丙氨酸或天冬氨酸会大幅降低C端结构域的稳定性，而色氨酸的影响则较为适中。在本文中，我们展示了这些界面突变体的X射线结构，并将它们与稳定性数据相关联。这些突变不影响分子的整体结构。在被取代残基附近未观察到结构变化，这表明局部结构过于刚性，无法对氨基酸替换进行补偿。在突变体γB - F56A和 - F56D中，由于大体积苯丙氨酸侧链被取代，形成了一个可被大量溶剂接触的充满溶剂的凹槽。在γB - F56W中，吲哚环的吡咯部分取代了野生型的苯侧链。除了γB - F56W外，根据正辛醇标度，56位氨基酸的疏水性与C端结构域的稳定性之间存在良好的相关性。在γB - F56W中，C端结构域的稳定性低于根据疏水性估计的值，推测是因为环氮（Nε1）没有形成氢键的伙伴。数据表明，界面核心中疏水残基的堆积对于γB -晶状体蛋白的结构域相互作用和稳定性很重要。显然，对于蛋白质稳定性而言，相同的原则适用于结构域内和结构域界面的疏水核心。
