Probing the Structure of Transient Unfolded States with NMR Relaxation Dispersion

描述(由申请人提供)：瞬时自然状态展开被认为是蛋白质错误折叠和随后在许多系统中聚集的初始阶段，这些系统传播到多种疾病中。这项研究的基本目标是使用核磁共振R2弥散技术绘制脱脂肌红蛋白的本征能量图谱，其应用目标是开发一种新的模型系统来理解瞬时蛋白质的展开和聚集。R2分散体已经成功地应用于活性部位附近酶功能的动力学研究，然而，很少有研究集中在略微扰动的自然状态下的复杂波动。15N R2色散将被用来理解WT载脂蛋白在一定的pH值范围内表现出的展开转变，在这个范围内，apoMb保持其本征结构。将研究激发态的一致性，以及展开和重新折叠事件的pH和温度依赖性。初步结果表明，在某些条件下，在自然状态和特征良好的熔融球体之间发生了两个位置的交换。来自~90个共振的数据将符合局部两态机制，并评估协作性和机制。将应用全局和集群拟合例程来改进对交换参数的估计，并确定合作结构的单元。多个温度将被用来理解转变的热力学，以及构象交换的温度依赖性。突变体将被用来探测动态涨落的F-螺旋在本态能量格局中的贡献，而非交换质子的全重氢将被用来选择性地探索疏水效应对构象涨落的贡献。然后，使用15N和13C-甲基R2分散技术，对WT载脂肌红蛋白样品进行调整，以产生关于导致无定形(在~1M尿素)和淀粉样蛋白(在pH 9，&gt；40？C)聚集的过程的动力学信息。样品条件将被仔细控制，以保持可溶样品，同时促进启动聚集的动态。将进行具体的比较，以揭示聚集诱导条件和促进瞬时展开但不促进聚集的条件之间在展开和重新折叠机制上的差异。为了了解许多聚集机制的初始阶段与公共卫生的相关性，有必要了解蛋白质的展开过程。由于蛋白质的功能经常依赖于瞬时展开，因此揭示与聚集无关的展开事件与导致有毒多聚体形成的事件之间的关键差异是很重要的。

DESCRIPTION (provided by applicant): Transient native-state unfolding has been implicated as the initial stage of protein mis-folding and subsequent aggregation in numerous systems that propagate into a multitude of diseases. The basic goal of the proposed research is to map the native-state energy landscape of apo-myoglobin using NMR R2 dispersion techniques, with the applied goal of developing a new model system for understanding transient protein unfolding and aggregation. R2 dispersion has been successfully applied to dynamics studies of enzyme function around the active site, however, few studies have focused on the complex fluctuations accessible to a slightly perturbed native-state. 15N R2 dispersion will be applied to understand the unfolding transitions exhibited by WT apo-myoglobin at a range of pH values for which the apoMb retains its native-state structure. The identity of the excited states will be investigated, along with the pH and temperature dependence of unfolding and refolding events. Initial results suggest that under some conditions a two-site exchange occurs between the native-state, and the well characterized molten globule. Data from ~90 resonances will be fit to a local two-state mechanism, and assessed for cooperativity and mechanism. Global and cluster fitting routines will be applied to improve estimates of the exchange parameters and to identify units of cooperative structure. Multiple temperatures will be used to understand the thermodynamics of the transitions, as well as the temperature dependence of conformational exchange. Mutants will be used to probe the contribution of the dynamically fluctuating F-helix in the native-state energy landscape, while full perdeuteration of non-exchangeable protons will be used to selectively probe the contribution of the hydrophobic effect on the conformational fluctuations. WT apo-myoglobin samples will then be tuned to yield dynamics information about the processes leading to amorphous (at ~1M urea) and amyloid (at pH 9, >40¿ C) aggregation using 15N and 13C-methyl R2-dispersion techniques respectively. The sample conditions will be carefully controlled to maintain soluble samples while promoting the dynamics that initiate aggregation. Specific comparisons will be made to uncover differences in the mechanism of unfolding and refolding between aggregation-inducing conditions, and those which promote transient unfolding but no aggregation. PUBLIC HEALTH RELEVANCE In order to understand the initial stage of many aggregation mechanisms, it is necessary to understand the process of protein unfolding. Since protein function is frequently dependent on transient unfolding, it is important to uncover the key differences between unfolding events that are not implicated in aggregation, with those which result in the formation of toxic multimeric species.