Probing the Structure of Transient Unfolded States with NMR Relaxation Dispersion

描述（由申请人提供）：瞬时天然状态展开与许多系统中蛋白质错误折叠和随后聚集的初始阶段有关，这些系统传播为多种疾病。本研究的基本目标是利用核磁共振R2分散技术绘制载脂蛋白肌红蛋白的天然状态能量图，其应用目标是开发一种新的模型系统来理解瞬态蛋白质的展开和聚集。R2分散已成功地应用于酶活性位点周围的动力学研究，然而，很少有研究关注于轻微扰动的天然状态下的复杂波动。15nr2分散体将被应用于理解WT载脂蛋白肌红蛋白在一定pH值范围内所表现出的展开转变，在这种pH值范围内，apoMb保持其天然状态结构。将研究激发态的同一性，以及展开和再折叠事件对pH和温度的依赖。初步结果表明，在某些条件下，原生态和表征良好的熔融球之间发生了双向交换。来自~90个共振的数据将适合于局部双态机制，并评估了协同性和机制。将应用全局和聚类拟合例程来改进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波动的f -螺旋对原生态能量格局的贡献，而非交换质子的完全渗透将被用来选择性地探测疏水效应对构象波动的贡献。然后使用15N和13c -甲基r2分散技术分别对WT载脂蛋白肌红蛋白样品进行调整，以产生有关导致无定形（在~1M尿素下）和淀粉样蛋白（在pH 9， bbb40¿C）聚集过程的动力学信息。样品条件将被仔细控制，以保持可溶性样品，同时促进启动聚合的动力学。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聚集的条件之间展开和再折叠机制的差异。为了了解许多聚集机制的初始阶段，有必要了解蛋白质展开的过程。由于蛋白质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物种形成的展开事件之间的关键差异非常重要。

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.