SECONDARY STRUCTURE & FOLDING PATTERN OF CONSERVED DOMAIN IN ALPHA CRYSTALLIN

α-晶体蛋白结构域是一种蛋白质模块， 阻断小热休克蛋白寡聚体的组装 结构，并可能在它们的伴侣样作用中发挥核心作用， 功能 该结构域的严格保守性表明， 形成一个共同的结构核心，即，不变次级区域 三级结构。 本研究的目的是确定 序列特异性二级结构和折叠模式 这个结构域。 连续半胱氨酸突变体是 构建在残基60和120之间，在E.杆菌和 使用阴离子交换和凝胶过滤色谱法纯化。 的 突变体然后与巯基特异性顺磁自旋反应， 标记并通过电子顺磁共振光谱分析。 每个自旋标记的局部环境的特征在于： 确定其相对于蛋白质基质的迁移率及其 仅与顺磁试剂碰撞的可及性 可溶于水相。 这项分析导致了一项任务 沿着多肽链的结构类别。 定期 作为剩余数函数的结构类模式 用于鉴定二级结构的位置及其 地形 所研究的区域包含一个三圈α-螺旋 连接N-和C-末端结构域和两条β-链 由一个高度暴露于溶剂的环隔开。

The alpha-crystallin domain is a protein module used as a building block in the assembly of the small heat-shock protein oligomeric structure and might play a central role in their chaperone-like function. The stringent conservation of this domain suggests that it forms a common structural core, i.e., a region of invariant secondary and tertiary structure. The objective of this study is to determine the sequence-specific secondary structure and the folding pattern of this domain in alphaA-crystallin. Sequential cysteine mutants were constructed between residues 60 and 120, expressed in E. coli and purified using anion exchange and gel filtration chromatography. The mutants were then reacted with a sulfhydryl specific paramagnetic spin label and analyzed by Electron Paramagnetic Resonance spectroscopy. The local environment of each spin label was characterized by determining its mobility with respect to the protein matrix and its accessibility to collisions with a paramagnetic reagent exclusively soluble in the aqueous phase. This analysis resulted in an assignment of the structural class along the polypeptide chain. Periodic patterns in the structural classes as a function of the residue number were used to identify the location of secondary structures and their topography. The investigated region contains a three-turn alpha-helix connecting the N- and C-terminal domains and two beta-strands separated by a highly solvent-exposed loop.