Experimental/Computational Study of Protein Aggregation

蛋白质聚集的实验/计算研究

• 批准号: 7616197
• 负责人: Teresa L. Head-Gordon
• 金额: $ 26.37万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616197
• 关键词: Abate; Adopted; Alzheimer' s Disease; Amino Acid Sequence; Amyloid Fibrils; Amyloid fibers; Binding; Biological Models; Biological Process; Biotechnology; Cereals; Characteristics; Crystallography; Disease; Entropy; Event; Exhibits; Fluorescence Anisotropy; Free Energy; G-substrate; Generic Drugs; Guidelines; Head; Huntington Disease; Immunoglobulin binding proteins; Investigation; Joints; Kinetics; Knowledge; Laboratories; Libraries; Methods; Modeling; Molecular; Monitor; Morphology; Mutate; Mutation; Peptide Sequence Determination; Pharmacologic Substance; Process; Property; Protein Engineering; Proteins; Research; Research Personnel; Role; Science; Screening procedure; Structure; Surface Plasmon Resonance; Testing; Thermodynamics; Thioflavin T; Time; Work; aggregation pathway; amyloid fibril formation; computer studies; design; disulfide bond; in vivo; inhibitor/antagonist; light scattering; molecular dynamics; mutant; nanomaterials; polypeptide; protein aggregation; protein aminoacid sequence; protein folding; prototype; research study; simulation; three dimensional structure

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（头 - 盖顿）研究，该研究将检查分子机制和原纤维的结构特征以及淀粉样纤维形成的早期事件。我们将采用两种小蛋白质，即免疫球蛋白结合蛋白L和G，它们具有低序列认同，高结构相似性但不同的折叠机制，以描绘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。提出了使用这些蛋白质的补充实验和模拟，以检查突变蛋白序列对成核事件，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。 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.也可以检查部分折中间体与天然褶皱与聚集的分区，并可以与序列进行检查并相关。我们的实验工作将以旨在阐明控制聚合事件的序列和结构因素的模拟来指导。我们将使用粗粒蛋白模型来用于在头盖实验室中开发的蛋白质L和G。这些模型是高度拖动的，不仅可以折叠热力学和动力学，而且还提供了完整的热力学和动力学表征，而且还通过涉及多个链的模拟来完成聚集的景观表征。一旦通过实验验证，模拟将对最小化聚合的序列进行快速筛选。使用我们的计算结果和其他蛋白质工程研究的结果，我们将构建一组指南，用于降低特定蛋白质的聚集倾向的合理设计，并测试这些准则的转移性，使用蛋白质的广泛范围的突变体的转移性。我们已经完成了针对稳定性的蛋白质的蛋白质GINTICT和仿真的蛋白质。这里提出的联合实验/理论项目的可行性。 两名研究人员提出了一项联合实验和计算研究，该研究将检查原纤维和淀粉样纤维形成的机理。所使用的模型系统是蛋白质G和L。该方法将包括表面等离子体共振，动态光散射，荧光各向异性和硫非红素T结合对折叠和聚集途径的实验表征。还将应用粗粒颗粒的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。

项目成果

A coarse-grained alpha-carbon protein model with anisotropic hydrogen-bonding.

具有各向异性氢键的粗粒 α-碳蛋白模型。

• DOI: 10.1002/prot.21515
• 发表时间: 2008
• 期刊: Proteins
• 影响因子: 2.9
• 作者: Yap,Eng-Hui;Fawzi,NicolasLux;Head-Gordon,Teresa
• 通讯作者: Head-Gordon,Teresa

Driving forces for transmembrane alpha-helix oligomerization.

跨膜α螺旋寡聚的驱动力。

• DOI: 10.1016/j.bpj.2010.03.071
• 发表时间: 2010
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Sodt,AlexJ;Head-Gordon,Teresa
• 通讯作者: Head-Gordon,Teresa