Sequence and Environmental Determinants of the Protein Energy Landscape

蛋白质能量景观的序列和环境决定因素

• 批准号: 9152809
• 负责人: SUSAN MARQUSEE
• 金额: $ 35.83万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9152809
• 关键词: Affect; Amino Acid Sequence; Binding; Binding Proteins; Biological Models; Biology; Complex; Complex Mixtures; Controlled Environment; Coupled; Data; Dependence; Environment; Evolution; Explosion; Family; Goals; Health; Homologous Gene; Human; Human Genome; Ligands; Lytic; Measurement; Measures; Mechanics; Medicine; Molecular; Molecular Conformation; Monitor; Nature; Peptide Hydrolases; Peptide Sequence Determination; Play; Population; Problem Solving; Protein Conformation; Proteins; Ribonuclease H; Ribosomes; Role; Signal Transduction; Signaling Protein; Source; Specific qualifier value; Structure; System; Time; Variant; fitness; human genome sequencing; new technology; non-Native; polypeptide; protein folding; protein function; protein misfolding; reconstruction; research study; single molecule; single-molecule FRET; three dimensional structure; tool

 DESCRIPTION (provided by applicant): Decoding the information in the primary sequence of a protein is one of the most fundamental challenges in modern biology. A protein's sequence encodes more than just the native structure; it encodes the entire energy landscape - an ensemble of conformations whose energetics and dynamics are finely tuned. The goal of this proposal is a molecular, quantitative, and predictive understanding of the relationship between sequence and the energy landscape together with an understanding of how the environment modulates this landscape. A major hurdle in going from sequence to function is our lack of understanding of the non-native or high- energy regions of the landscape and how they are modulated by the environment. High-energy conformations are important for directing the stability and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. While, many sequences can encode the same structure, their function and dynamics can vary dramatically - due to changes in the landscape. Small variations in a sequence can have effects that range from undetectable to pathological. Soon we will have access to thousands of human genomes, and without our ability to interpret variation, the potential of these data to impact medicine and human health will never be fully appreciated. It is imperative, therefore, that we have an understanding and control over the relationship between sequence and the energy landscape. Modulations of the energy landscape are not easily detected due to the small populations and transient nature of the high-energy species. The experiments outlined here are aimed at understanding how changes in the sequence and the environment affect the energy landscape. Aim 1: Quantitative measures of protein folding and stability in complex environments a. Develop a quantitative bench-top measure of protein stability on the ribosome and other complex mixtures. b. Measure conformational dynamics of ribosome-bound polypeptide chains c. Monitor translational coupled folding using HaloTag as a model system Aim 2: Probe the energy landscape through evolution and sequence modulation a. Use Ancestral Sequence Reconstruction (ASR) to explore changes in the landscape of RNase H over time. We will evaluate the energy landscapes of these resurrected proteins to determine how optimizations of the energy landscape, and thus function/fitness, occur over evolutionary time. b. Use Ancestral Sequence Reconstruction to evaluate the rate-limiting step by using the alpha-lytic protease family of both kinetically stable and thermodynamically stable proteases Aim 3: Probe the energy landscape through single molecule mechanical unfolding a. Single molecule mechanical studies to probe the energy barriers in protein folding

 描述（由适用提供）：在蛋白质的主要顺序中解码信息是现代生物学中最根本的挑战之一。蛋白质的序列不仅仅是天然结构的编码。它编码整个能量景观 - 构型的合奏，其能量和动力学经过精心调节。该提案的目的是对序列与能量景观之间关系的分子，定量和预测性理解，以及对环境如何调节该景观的理解。从序列到功能的主要障碍是我们对景观的非本地或高能区域以及环境如何调节它们的方式缺乏了解。高能量会议对于指导蛋白质的稳定性和折叠很重要，并且该集合的调节在错误折叠，蛋白质信号传导，催化活性和变构中起作用。尽管许多序列可以编码相同的结构，但由于景观的变化，它们的功能和动力学可能会大大变化。序列中的小变化可能具有从无法检测到病理的范围。很快，我们将获得数千种人类基因组，如果没有解释变异的能力，这些数据可能会影响医学和人类健康的潜力。因此，当务之急是我们对序列与能量格局之间关系的理解和控制。由于高能物种的少量和瞬时性质，不能轻易检测到能量景观的调节。此处概述的实验旨在了解序列和环境的变化如何影响能量格局。目标1：在复杂环境中蛋白质折叠和稳定性的定量测量。开发核糖体和其他复合物混合物上蛋白质稳定性的定量基准测量。 b。测量核糖体结合多肽链的构象动力学c。使用Halotag作为模型系统目标2：通过进化和序列调制a探测能量格局的监视器耦合a。使用祖先序列重建（ASR）探索随着时间的推移RNase H景观的变化。我们将评估这些回收蛋白质的能量景观，以确定能量景观的优化以及功能/适应性如何在进化时间内发生。 b。使用祖传序列重建来评估速率限制步骤，通过使用两个动力学稳定的α-散裂蛋白酶家族 和热力学稳定蛋白的目标3：通过单分子机械展开探测能量景观。单分子机械研究以探测蛋白质折叠中的能屏障