Experimental/Computational Study of Protein Aggregation

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，将研究原纤维的分子机制和结构特征以及淀粉样蛋白纤维形成的早期事件。我们将使用两个小蛋白，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，但不同的折叠机制，来描述蛋白质聚集和纤维形成的关键序列，结构和稳定性决定因素。利用这些蛋白质进行互补实验和模拟，以检验突变蛋白质序列对成核事件、聚集倾向、聚集和折叠动力学的影响，以及折叠中间体在聚集中的作用。Blanch小组将利用蛋白质L的广泛突变文库进行实验，以确定在一定时间尺度上纤维形成的动力学，使用表面等离子体共振和动态光散射来确定短期和中期时间尺度上的蛋白质相互作用，以及荧光各向异性和硫黄素T结合来监测长时间内纤维形成的动力学。部分折叠中间体对天然折叠与聚集的划分也可以被检查并与序列相关。我们的实验工作将以模拟为指导，旨在阐明控制聚合事件的顺序和结构因素。我们将使用在Head-Gordon实验室开发的蛋白质L和G的粗粒度蛋白质模型。这些模型是高度可处理的，并且提供了完整的热力学和动力学表征，不仅折叠热力学和动力学，而且从涉及多链的模拟中完成聚集的景观表征。一旦通过实验验证，模拟将提供快速筛选序列，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导方针，以减少给定蛋白质的聚集倾向，并使用蛋白质g的广泛突变来测试这些指导方针的可转移性。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质L相比相同突变的稳定性和聚集动力学。这为本文提出的联合实验/理论项目的可行性提供了证据。

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.