Kinetics of Early Events in Protein Folding
蛋白质折叠早期事件的动力学
基本信息
- 批准号:6740870
- 负责人:
- 金额:$ 27.38万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1998
- 资助国家:美国
- 起止时间:1998-05-01 至 2006-04-30
- 项目状态:已结题
- 来源:
- 关键词:bacterial geneticsbacterial proteinschemical kineticscomputer simulationconformationfluorescence resonance energy transferfluorescent dye /probemicroorganism culturemodel design /developmentnuclear magnetic resonance spectroscopynucleasephysical modelprotein biosynthesisprotein foldingprotein purificationprotein sequenceprotein structure functionsite directed mutagenesisstructural biologythermodynamics
项目摘要
DESCRIPTION (provided by applicant): Many small proteins show evidence for conformational changes prior to the rate-limiting step in the formation of the
densely packed native structure. Despite intense study, our understanding of
the physical properties and mechanistic role of these early folding
intermediates remains incomplete. In particular, little is known about the
chain topology and tertiary structural preferences in early intermediates,
which are critical for understanding how folding is initiated and directed
along productive channels. A major goal of this project is to elucidate the
structural properties of the transient states and kinetic barriers encountered
during early stages of folding of two representative model proteins, protein G
and staphylococcal nuclease. Initial stages of folding extending well into the
microsecond time scale will be explored by coupling advanced rapid mixing
methods with structurally informative conformational probes, such as intrinsic
and extrinsic fluorescence probes, and protection of individual amide hydrogens
from solvent exchange monitored by NMR. The involvement of specific residues
and interactions in stabilizing transient states and barriers in folding of
protein G will be explored by combining these kinetic methods with
site-directed mutagenesis. The results will identify key structural features
involved in each stage of folding of this prototypic single-domain protein.
Detailed insight into the formation of hydrophobic clusters, specific
fluorescence quenching interactions and long-range distance distributions
during folding of staphylococcal nuclease will be obtained by kinetic analysis
of variants with engineered tryptophan residues and fluorescence energy
transfer methods. Complementary information on the formation of hydrogen bonded
structure during early stages of folding will be obtained by combining
ultrarapid quenched-flow methods with NMR-detected hydrogen exchange. The
insight into fast folding events for these and other proteins with diverse
structural properties will provide a firm experimental basis for testing
theoretical and computational models of protein folding, structure prediction
and de novo protein design.
描述(由申请人提供):许多小蛋白质在形成限速步骤之前显示出构象变化的证据
密集的天然结构。尽管进行了大量的研究,我们的理解
这些早期折叠的物理特性和机械作用
中间体仍然不完整。尤其是,人们对以下方面知之甚少
早期中间体的链拓扑和三级结构偏好,
这对于理解折叠是如何启动和定向的至关重要
沿着生产渠道。该项目的一个主要目标是阐明
瞬态的结构特性和遇到的动力学障碍
在两个代表性模型蛋白 G 蛋白折叠的早期阶段
和葡萄球菌核酸酶。折叠的初始阶段很好地延伸到
将通过耦合先进的快速混合来探索微秒时间尺度
具有结构信息的构象探针的方法,例如内在的
和外在荧光探针,以及单个酰胺氢的保护
通过 NMR 监测溶剂交换。特定残基的参与
以及稳定瞬态的相互作用和折叠中的势垒
将通过将这些动力学方法与
定点诱变。结果将确定关键的结构特征
参与该原型单域蛋白折叠的每个阶段。
详细了解疏水簇的形成,具体
荧光猝灭相互作用和长程距离分布
通过动力学分析获得葡萄球菌核酸酶折叠过程中的
具有工程色氨酸残基和荧光能量的变体
转移方法。关于氢键形成的补充信息
折叠早期阶段的结构将通过组合获得
具有核磁共振检测氢交换的超快速骤冷流方法。这
深入了解这些蛋白质和其他蛋白质的快速折叠事件
结构特性将为测试提供坚实的实验基础
蛋白质折叠的理论和计算模型、结构预测
和从头蛋白质设计。
项目成果
期刊论文数量(0)
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会议论文数量(0)
专利数量(0)
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{{ truncateString('HEINRICH RODER', 18)}}的其他基金
Structural Plasticity and Functional Interactions of the Signaling Adapter NHERF
信号适配器 NHERF 的结构可塑性和功能相互作用
- 批准号:
9176248 - 财政年份:2016
- 资助金额:
$ 27.38万 - 项目类别:
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