Agarwalla S, Gokhale R S, Santi D V, Balaram P
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Protein Sci. 1996 Feb;5(2):270-7. doi: 10.1002/pro.5560050211.
Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.
胸苷酸合成酶（TS）是一种二聚体酶，在尿素浓度为3.3 - 5M时会形成大的可溶性聚集体，这一浓度远低于通过荧光和尺寸排阻色谱法确定的完全变性所需的浓度。与野生型酶不同，TS的一种工程突变体（T155C/E188C/C244T），即TSMox，其中两个亚基通过155 - 188'和188 - 155'残基之间的二硫键交联，不会表现出这种行为。在突变蛋白中二硫键还原后，聚集行为得以恢复，这表明界面片段参与了可溶性缔合物种的形成。分子间二硫键交联已被用作一种探针来研究更大的非天然聚集体的形成。这些研究表明，通过二聚体界面区域的多肽粘性斑块形成大的多聚体物种，该斑块在部分展开时会暴露出来。对相对脆弱的蛋白质 - 蛋白质界面进行共价增强可能是使多聚体蛋白中非天然结构聚集最小化的一种有用策略。
