Rational engineering of improved protein crystallization

这份拨款申请解决了使用晶体学方法深入了解 生物功能，这是大多数天然存在的蛋白质不能产生适合X射线的晶体 结构测定当前项目的目标是制定合理的工程方法， 蛋白质序列，以产生高质量的晶体。结构基因组学财团系统地证实 结晶是用X射线衍射确定蛋白质原子结构的主要障碍 方法.亨特实验室先前发表的工作采用了大规模的计算分析， 结晶试验表明，蛋白质的表面性质，特别是暴露的平均熵， 侧链是结晶倾向的主要决定因素。这项研究确定了各种序列 与结晶成功相关的性质，包括几种单独氨基酸的含量。 然而，每一种与结晶成功正相关的氨基酸都是负相关的。 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氨基 酸取代以工程化改进蛋白质结晶性质，因为结晶概率 低，除非初始蛋白质制剂是单分散和可溶的。在本申请中，我们建议使用 一套计算方法来鉴定促进成功蛋白质更复杂序列表位 结晶而不损害溶解度。将使用计算分析来选择引入 这些表位以可能保持蛋白质功能和稳定性的方式存在。这些新颖的结晶- 工程方法将严格评估和优化使用的研究，其中热力学 纯化的突变蛋白的稳定性、溶解性和结晶性质通过实验确定。我们 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， 蛋白质的结晶倾向不直接与其热力学溶解度相关。因此如果 基本的立体化学和热力学机制得到充分理解，那么它应该 可以设计改进的蛋白质溶解度，同时改进结晶倾向。双 这项研究的目的是阐明这些机制，同时也 产生用于改善蛋白质结晶和溶解性的严格验证的方法。成功 开发用于工程化改进的蛋白质结晶的有效方法将促进各种各样的 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。

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.