Experimental/Computational Study of Protein Aggregation

描述（由申请人提供）：本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蛋白原纤维形成的早期事件。我们将采用两个小的蛋白质，免疫球蛋白结合蛋白L和G，这具有低序列同一性，高结构相似性，但不同的折叠机制，描绘的关键序列，结构和稳定性的蛋白质聚集和原纤维形成的决定因素。互补的实验和模拟，使用这些蛋白质的突变蛋白质序列上的成核事件，聚集倾向，聚集和折叠的动力学，以及折叠中间体聚集的作用，提出了检查的效果。Blanch小组使用广泛的蛋白L突变库，将进行实验以确定在一系列时间尺度上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时间和中等时间尺度下的蛋白质相互作用，以及荧光各向异性和硫磺素T结合来监测长时间内原纤维形成的动力学。还可以检查部分折叠的中间体向天然折叠相对于聚集的分配并与序列相关。我们的实验工作将指导旨在阐明的序列和结构因素，治理聚集事件的模拟。我们将使用粗粒度的蛋白质模型的蛋白质L和G在头戈登实验室开发。这些模型是非常容易处理的，并提供完整的热力学和动力学表征，不仅折叠热力学和动力学，但也完整的景观特征的聚集模拟涉及多个链。一旦通过实验验证，模拟将提供一个快速筛选的序列，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，我们将构建一套指导原则，用于合理设计突变以降低给定蛋白质的聚集倾向，并使用广泛的蛋白质G突变体来测试这些指导原则的可转移性。我们已经完成了早期的研究，其中实验和模拟表征相同的突变体的稳定性和聚集动力学相比，野生型的蛋白L，这提供了证据的可行性，联合实验/理论项目在这里提出。 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。这些方法将包括实验表征的折叠和聚集途径的表面等离子体共振，动态光散射，荧光各向异性，和硫磺素T结合。一个粗粒度的非格子模拟和原子分子动力学也将被应用，表征折叠轨迹，动力学，以及热力学。

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.