Probing the Structure of Transient Unfolded States with NMR Relaxation Dispersion

用核磁共振弛豫色散探测瞬态未折叠态的结构

• 批准号: 7612049
• 负责人: Derrick Walter Meinhold
• 金额: $ 5.01万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7612049
• 关键词: Active Sites; Address; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Arts; Behavior; Biological Models; Complex; Core Protein; Data; Dependence; Detection; Development; Disease; Disulfides; Enzymes; Equilibrium; Event; Exhibits; Goals; High temperature of physical object; Kinetics; Lead; Maps; Measurement; Measures; Modeling; Molecular Conformation; Mutagenesis; Mutation; Myoglobin; Parkinson Disease; Pathway interactions; Physeteridae; Plant Roots; Population; Positioning Attribute; Prealbumin; Process; Proteins; Protons; Relaxation; Research; Sampling; Site; Solutions; Staging; Structure; Superoxide Dismutase; System; Techniques; Temperature; Thermodynamics; Urea; Vertebral column; aggregation pathway; amyloid formation; human disease; improved; insight; mutant; novel; protein function; protein misfolding; public health relevance; research study

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.

描述（由申请人提供）：瞬时天然态解折叠被认为是蛋白质错误折叠的初始阶段以及随后在许多系统中聚集，从而传播成多种疾病。本研究的基本目标是利用 NMR R2 分散技术绘制脱辅基肌红蛋白的天然能量图谱，其应用目标是开发一种新的模型系统来理解瞬时蛋白质解折叠和聚集。 R2 色散已成功应用于活性位点周围酶功能的动力学研究，然而，很少有研究关注轻微扰动的自然状态下的复杂波动。 15N R2 分散体将用于了解 WT 载脂蛋白肌红蛋白在载脂蛋白 Mb 保持其天然状态结构的 pH 值范围内表现出的展开转变。将研究激发态的特性，以及展开和重折叠事件的 pH 和温度依赖性。初步结果表明，在某些条件下，自然状态和特征良好的熔球之间会发生两位点交换。来自约 90 个共振的数据将适合局部两态机制，并评估协同性和机制。将应用全局和集群拟合例程来改进交换参数的估计并识别合作结构的单元。多个温度将用于了解转变的热力学以及构象交换的温度依赖性。突变体将用于探测动态波动的 F 螺旋在天然状态能量景观中的贡献，而不可交换质子的完全置换将用于选择性地探测疏水效应对构象波动的贡献。然后，将分别使用 15N 和 13C-甲基 R2 分散技术对 WT 脱辅基肌红蛋白样品进行调整，以产生有关导致无定形（约 1M 尿素）和淀粉样蛋白（pH 9，>40°C）聚集过程的动态信息。将仔细控制样品条件，以保持样品可溶，同时促进引发聚集的动力学。将进行具体比较，以揭示诱导聚集的条件和促进短暂展开但不聚集的条件之间展开和重折叠机制的差异。公共健康相关性 为了了解许多聚集机制的初始阶段，有必要了解蛋白质解折叠的过程。由于蛋白质功能经常依赖于瞬时解折叠，因此揭示不涉及聚集的解折叠事件与导致有毒多聚体物种形成的解折叠事件之间的关键差异非常重要。