Experimental/Computational Study of Protein Aggregation

描述(由申请人提供)：这项建议是一项联合实验(Blanch)和计算(Head-Gordon)研究，将研究原纤维和淀粉样纤维形成的早期事件的分子机制和结构特征。我们将使用两种序列同源性低、结构相似性高但折叠机制不同的免疫球蛋白结合蛋白L和G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了互补实验和模拟，以考察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产物对聚集的影响。Blanch团队将利用广泛的L蛋白质突变体文库进行实验，以确定一系列时间尺度上的纤维形成动力学，使用表面等离子激元共振和动态光散射来确定短时间和中时间尺度上的蛋白质相互作用，并使用荧光各向异性和硫代黄素T结合来监测较长时间内的纤维形成动力学。部分折叠的中间体相对于聚集的天然折叠的分配也可以被检查并与序列相关联。我们的实验工作将由旨在阐明支配聚集事件的序列和结构因素的模拟来指导。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。这些模型非常易于处理，不仅提供了折叠热力学和动力学的完整热力学和动力学表征，而且还提供了涉及多个链的模拟的聚集的完整景观表征。一旦通过实验验证，模拟将提供对最大限度减少聚集的序列的快速筛选。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以降低特定蛋白质的聚集倾向的指南，并使用广泛的蛋白质G突变体来测试这些指南的可转移性。我们已经完成了早期的研究，其中实验和模拟表征了与野生型L相比相同的突变体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。 这两位研究人员提出了一项联合实验和计算研究，将检验原纤维和淀粉样纤维形成的机制。所使用的模型系统是蛋白质G和L。方法将包括通过表面等离子体共振、动态光散射、荧光各向异性和硫代黄素T结合来表征折叠和聚集途径的实验表征。还将应用粗粒非晶格模拟和原子分子动力学，表征折叠轨迹、动力学和热力学。

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.