Experimental/Computational Study of Protein Aggregation

蛋白质聚集的实验/计算研究

• 批准号: 7100363
• 负责人: Teresa L. Head-Gordon
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7100363
• 关键词: amyloid proteins; binding proteins; chemical aggregate; chemical kinetics; computational biology; computer simulation; fluorescence polarization; genetic library; light scattering; molecular dynamics; nephelometry; protein biosynthesis; protein denaturation; protein engineering; protein folding; protein protein interaction; protein structure; surface plasmon resonance; thermodynamics

DESCRIPTION (provided by applicant): This proposal is a joint experimental (Blanch) and computational (Head-Gordon) study that will examine the molecular mechanisms and the structural characteristics of protofibril and early events of amyloid fibril formation. We will employ two small proteins, the immunoglobulin-binding proteins L and G, which have low sequence identity, high structural similarity, but different folding mechanisms, to delineate the key sequence, structural, and stability determinants of protein aggregation and fibril formation. Complementary experiments and simulations using these proteins are proposed to examine the effect of mutating protein sequence on nucleation events, aggregation propensity, the kinetics of aggregation and folding, and the role of folding intermediates on aggregation. The Blanch group, using an extensive mutant library for protein L, will perform experiments to determine the kinetics of fibril formation over a range of time scales, using surface plasmon resonance and dynamic light scattering to determine protein interactions at short and intermediate time scales, and fluorescence anisotropy and thioflavin T binding to monitor the kinetics of fibril formation at longer times. The partitioning of partially-folded intermediates to native folds versus aggregation can also be examined and correlated with sequence. Our experimental efforts will be guided by simulations aimed at elucidating the sequence and structural factors that govern aggregation events. We will use coarse-grained protein models for proteins L and G developed in the Head-Gordon laboratory. These models are highly tractable, and provide complete thermodynamic and kinetic characterization of, not only folding thermodynamic and kinetics, but also complete landscape characterization of aggregation from simulations involving multiple chains. Once validated by experiment, simulations will provide a rapid screening for sequences that minimize aggregation. Using our computational results and those from other protein engineering studies, we will construct a set of guidelines for the rational design of mutations for reducing the aggregation propensity of a given protein, and test the transferability of these guidelines using a wide-range of mutants for proteins G. We have completed early studies in which experiment and simulation characterize the same mutants for stability and aggregation kinetics compared against wild-type for protein L, which provides evidence for the feasibility of the joint experimental/theoretical project proposed here. The two investigators propose a joint experimental and computational study that will examine the mechanism of protofibril and amyloid fibril formation. The model systems used are proteins G and L. The methods will include experimental characterization of the folding and aggregation pathways by surface plasmon resonance, dynamic light scattering, fluorescence anisotropy, and thioflavin T binding. A coarse-grained off-lattice simulation and atomistic molecular dynamics will also be applied, characterizing folding trajectories, kinetics, as well as thermodynamics.

描述（由申请人提供）：该提案是一项联合实验（Blanch）和计算（Head-Gordon）研究，将检查原纤维的分子机制和结构特征以及淀粉样原纤维形成的早期事件。我们将采用两种小蛋白，即免疫球蛋白结合蛋白 L 和 G，它们具有低序列同一性、高结构相似性，但折叠机制不同，来描述蛋白质聚集和原纤维形成的关键序列、结构和稳定性决定因素。建议使用这些蛋白质进行补充实验和模拟，以检查突变蛋白质序列对成核事件、聚集倾向、聚集和折叠动力学以及折叠中间体对聚集的作用的影响。 Blanch小组将使用广泛的L蛋白突变体库进行实验，以确定在一定时间尺度内原纤维形成的动力学，使用表面等离子体共振和动态光散射来确定短期和中间时间尺度上的蛋白质相互作用，以及荧光各向异性和硫代黄素 T 结合可长时间监测原纤维形成的动力学。还可以检查部分折叠中间体到天然折叠的分配与聚集并与序列相关联。我们的实验工作将以模拟为指导，旨在阐明控制聚集事件的顺序和结构因素。我们将使用 Head-Gordon 实验室开发的蛋白质 L 和 G 的粗粒度蛋白质模型。这些模型非常容易处理，并且不仅提供折叠热力学和动力学的完整热力学和动力学表征，而且还提供涉及多链的模拟中聚集的完整景观表征。一旦通过实验验证，模拟将提供对序列的快速筛选，从而最大限度地减少聚集。利用我们的计算结果和其他蛋白质工程研究的结果，我们将构建一套合理设计突变的指南，以减少给定蛋白质的聚集倾向，并使用各种突变体测试这些指南的可转移性我们已经完成了早期研究，其中实验和模拟表征了与野生型蛋白 L 相比的相同突变体的稳定性和聚集动力学，这为此处提出的联合实验/理论项目的可行性提供了证据。 两位研究人员提出了一项联合实验和计算研究，将研究原纤维和淀粉样原纤维形成的机制。使用的模型系统是蛋白质 G 和 L。这些方法将包括通过表面等离子共振、动态光散射、荧光各向异性和硫黄素 T 结合对折叠和聚集途径进行实验表征。还将应用粗粒度的离格模拟和原子分子动力学，来表征折叠轨迹、动力学以及热力学。