Guanidine-HCl Dependent Structural Unfolding of M-Crystallin: Fluctuating Native State Like Topologies and Intermolecular Association

Numerous experimental techniques and computational studies, proposed in recent times, have revolutionized the understanding of protein-folding paradigm. The complete understanding of protein folding and intermediates are of medical relevance, as the aggregation of misfolding proteins underlies various diseases, including some neurodegenerative disorders. Here, we describe the unfolding of M-crystallin, a βγ-crystallin homologue protein from archaea, from its native state to its denatured state using multidimensional NMR and other biophysical techniques. The protein, which was earlier characterized to be a predominantly β-sheet protein in its native state, shows different structural propensities (α and β), under different denaturing conditions. In 2 M GdmCl, the protein starts showing two distinct sets of peaks, with one arising from a partially unfolded state and the other from a completely folded state. The native secondary structural elements start disappearing as the denaturant concentration approaches 4 M. Subsequently, the protein is completely unfolded when the denaturant concentration is 6 M. The 15N relaxation data (T1/T2), heteronuclear 1H-15N Overhauser effects (nOes), NOESY data, and other biophysical data taken together indicate that the protein shows a consistent, gradual change in its structural and motional preferences with increasing GdmCl concentration.近年来提出的诸多实验技术与计算研究，极大地革新了人们对蛋白质折叠范式（protein-folding paradigm）的认知。全面阐明蛋白质折叠过程及其中间态具有重要医学意义：错误折叠蛋白质的聚集是多种疾病（包括部分神经退行性疾病）的致病基础。本研究阐述了利用多维核磁共振波谱法（multidimensional NMR）及其他生物物理技术，对来自古菌（archaea）的βγ-晶状体蛋白同源物M晶状体蛋白（M-crystallin）从天然态至变性态的解折叠过程。该蛋白此前被表征为天然态下以β折叠为主的蛋白质，但其在不同变性条件下会表现出α螺旋与β折叠两种不同的结构倾向。在2 M盐酸胍（GdmCl）环境中，该蛋白开始出现两组截然不同的峰信号：一组源自部分解折叠状态，另一组则源自完全折叠状态。随着变性剂浓度趋近4 M，该蛋白的天然二级结构元件开始逐渐消失。当变性剂浓度达到6 M时，该蛋白完全解折叠。综合15N弛豫数据（T1/T2）、异核1H-15N奥弗豪泽增强效应（nOes）、核奥弗豪泽效应谱（NOESY）及其他生物物理数据可知，随着GdmCl浓度升高，该蛋白的结构与运动偏好呈现出一致且渐进的变化过程。