Rational engineering of improved protein crystallization

改进蛋白质结晶的合理工程

• 批准号: 10249105
• 负责人: JOHN Francis HUNT
• 金额: $ 32万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249105
• 关键词: Address; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Applications Grants; Applied Research; Area; Basic Science; Biological Process; Biology; Buffers; Complex; Computer Analysis; Computing Methodologies; Coupled; Crystallization; Crystallography; Data; Data Analyses; Databases; Development; Engineering; Entropy; Epitopes; Failure; Goals; Impairment; Individual; Industrialization; Integral Membrane Protein; Laboratories; Macromolecular Complexes; Measurement; Mediating; Medical; Membrane; Membrane Proteins; Methods; Mutation; Preparation; Probability; Property; Protein Analysis; Protein Engineering; Proteins; Publishing; Research; Resistance; Roentgen Rays; Site; Software Engineering; Solubility; Speed; Statistical Data Interpretation; Structural Protein; Structure; Surface Properties; Synchrotrons; Thermodynamics; Twin Multiple Birth; Work; X ray diffraction analysis; aqueous; base; beamline; biophysical analysis; chemical property; comparative; computational chemistry; computational suite; computer studies; drug discovery; efficacy evaluation; experimental study; follow-up; improved; in vivo; insight; mutant; novel; physical property; preservation; programs; protein data bank; protein function; protein structure; structural genomics; success

This grant application addresses the major obstacle to using crystallographic methods to gain insight into biological function, which is the failure of most naturally occurring proteins to yield crystals suitable for x-ray structure determination. The goal of the current project is to develop methods for rational engineering of the sequence of a protein to produce high quality crystals. Structural genomics consortia systematically confirmed that crystallization is the major obstacle to determining the atomic structure of proteins using x-ray diffraction methods. Previously published work from the Hunt laboratory employed computational analysis of large-scale crystallization trials to demonstrate that protein surface properties, particularly the mean entropy of exposed sidechains, are a dominant determinant of crystallization propensity. This study identified a variety of sequence properties that correlate with crystallization success, including the content of several individual amino acids. However, every one of the amino acids that positively correlates with crystallization success negatively correlates with protein solubility, and vice-versa. This effect severely limits the efficacy of using single amino acid substitutions to engineer improved protein crystallization properties because crystallization probability is low unless the initial protein preparation is monodisperse and soluble. In this application, we propose to use a suite of computational methods to identify more complex sequence epitopes that promote successful protein crystallization without impairing solubility. Computational analyses will be used to select sites for introduction of such epitopes in a manner likely to preserve protein function and stability. These novel crystallization- engineering methods will be critically evaluated and optimized using studies in which the thermodynamic stability, solubility, and crystallization properties of purified mutant proteins are determined experimentally. Our preliminary data for these proposed studies support the efficacy of the approach while also showing that the crystallization propensity of a protein is not directly coupled to its thermodynamic solubility. Therefore, if the underlying stereochemical and thermodynamics mechanisms were sufficiently well understood, then it should be possible to engineer improved protein solubility in parallel with improved crystallization propensity. The twin objectives of the research proposed in this grant application are to elucidate these mechanisms while also generating rigorously validated methods for improving protein crystallization and solubility. Successful development of efficient methods for engineering improved protein crystallization would facilitate a wide variety of structural/functional biology projects, including projects focused on drug-discovery and structural characterization of macromolecular complexes.

该拨款申请解决了使用晶体学方法深入了解的主要障碍 生物功能，即大多数天然存在的蛋白质无法产生适合 X 射线的晶体 结构测定。当前项目的目标是开发合理工程的方法 蛋白质序列以产生高质量晶体。结构基因组学联盟系统证实 结晶是使用 X 射线衍射确定蛋白质原子结构的主要障碍 方法。亨特实验室之前发表的工作采用了大规模的计算分析 结晶试验证明蛋白质表面特性，特别是暴露的平均熵 侧链是结晶倾向的主要决定因素。本研究确定了多种序列 与结晶成功相关的特性，包括几种单独氨基酸的含量。 然而，每一种与结晶成功呈正相关的氨基酸都呈负相关 与蛋白质溶解度相关，反之亦然。这种效应严重限制了使用单一氨基的功效 酸替代以改善蛋白质结晶特性，因为结晶概率为 低，除非初始蛋白质制剂是单分散且可溶的。在此应用中，我们建议使用 一套计算方法来识别促进成功蛋白质的更复杂的序列表位 结晶而不损害溶解度。将使用计算分析来选择引入的地点 这些表位可能会保留蛋白质的功能和稳定性。这些新颖的结晶—— 将通过研究对工程方法进行严格评估和优化，其中热力学 纯化突变蛋白的稳定性、溶解度和结晶特性通过实验确定。我们的 这些拟议研究的初步数据支持该方法的有效性，同时也表明 蛋白质的结晶倾向并不与其热力学溶解度直接相关。因此，如果 潜在的立体化学和热力学机制已经被充分理解，那么它应该 可以在改善结晶倾向的同时提高蛋白质溶解度。双胞胎 本赠款申请中提出的研究目标是阐明这些机制，同时 生成经过严格验证的方法来改善蛋白质结晶和溶解度。成功的 开发改进蛋白质结晶的有效方法将促进多种 结构/功能生物学项目，包括专注于药物发现和结构生物学的项目 大分子复合物的表征。