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，用于构建 阻断小分子热休克蛋白低聚体的组装 结构，并可能在其类似伴侣的结构中发挥核心作用 功能。这一领域的严格保护表明，它 形成一个共同的结构核心，即一个不变的次级区域 和三级结构。这项研究的目的是确定 序列特异的二级结构和折叠模式 αA中的这个结构域--晶体蛋白。序列半胱氨酸突变体是 构建在残基60和120之间，在大肠杆菌中表达和 采用阴离子交换和凝胶过滤层析进行纯化。这个 然后，突变体与巯基特定的顺磁自旋反应 用电子顺磁共振波谱进行标记和分析。 每个自旋标记物的局部环境被表征为 测定其相对于蛋白质基质的迁移率 只与顺磁性试剂发生碰撞的可及性 可溶于水相。这一分析导致了一项任务 沿着多肽链的结构类。周期性的 作为剩余数函数的结构类中的模式 用于确定二级结构的位置和它们的 地形。研究区域包含一个三个转角的α-螺旋。 连接N-末端和C-末端结构域和两条β-链 由高度暴露于溶剂的环路隔开。

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.