Engineering of Proteins for Crystallography

晶体学蛋白质工程

基本信息

批准号：
8187572
负责人：
Zygmunt S Derewenda
金额：
$ 43.7万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8187572
关键词：
Address Affect Algorithms Amino Acid Sequence Amino Acids Behavior Bioinformatics Biology Cloning Collaborations Collection Commit Communicable Diseases Communities Complex Crystallization Crystallography Data Deposition Development Drug Delivery Systems Drug Design Engineering Entropy Evaluation Failure Feasibility Studies Feedback Gene Expression Generations Glutamine Goals Heteronuclear NMR Human Individual Investigation Label Laboratories Membrane Proteins Methodology Methods Molecular Mutagenesis Mutation NMR Spectroscopy Nature Online Systems Output Performance Phase Physical Chemistry Physiology Planet Mars Preparation Production Property Protein Engineering Protein Structure Initiative Proteins Protocols documentation Publishing Recombinant Proteins Relative (related person)Research Research Personnel Resources Roentgen Rays Sampling Screening procedure Site-Directed Mutagenesis Solubility Solutions Solvents Speed Staging Structural Biologist Structure Surface Surface Properties Synthetic Genes Testing Time Variant Work base computer studies cost data mining design design and construction globular protein improved lysylglutamic acid molecular mechanics next generation novel protein complex protein structure research study response statistics structural biology structural genomics success technology development tool

Address Affect Algorithms Amino Acid Sequence Amino Acids Behavior Bioinformatics Biology Cloning Collaborations Collection Commit Communicable Diseases Communities Complex Crystallization Crystallography Data Deposition Development Drug Delivery Systems Drug Design Engineering Entropy Evaluation Failure Feasibility Studies Feedback Gene Expression Generations Glutamine Goals Heteronuclear NMR Human Individual Investigation Label Laboratories Membrane Proteins Methodology Methods Molecular Mutagenesis Mutation NMR Spectroscopy Nature Online Systems Output Performance Phase Physical Chemistry Physiology Planet Mars Preparation Production Property Protein Engineering Protein Structure Initiative Proteins Protocols documentation Publishing Recombinant Proteins Relative (related person)Research Research Personnel Resources Roentgen Rays Sampling Screening procedure Site-Directed Mutagenesis Solubility Solutions Solvents Speed Staging Structural Biologist Structure Surface Surface Properties Synthetic Genes Testing Time Variant Work base computer studies cost data mining design design and construction globular protein improved lysylglutamic acid molecular mechanics next generation novel protein complex protein structure research study response statistics structural biology structural genomics success technology development tool

展开

项目摘要

In spite of its enormous successes, structural biology is severely limited by the low success rates of macromolecular crystallization. Many valuable, biomedically important targets elude crystallization attempts, and overall high costs of structure determination are due primarily to the labor and time-intensive screening of targets at the stages of protein production and crystallization. Our research will address this bottleneck, through development of protein engineering strategies that allow for rational design of target variants with enhanced solubility, stability, and crystallizability, and implementation of web-based, publicly available servers that facilitate application of these strategies. During the previous phase, we demonstrated that protein crystallization can be rationally induced by surface engineering based on the premise of surface entropy reduction (SER), i.e. mutagenesis of large, polar and solvent exposed amino acids, such as Lys, Glu and Gln, with small residues, e.g. Ala. Further, we designed and implemented the first generation XtalPred and SERp servers, which offer automated evaluation of protein's propensity to crystallize and design of variants with enhanced crystallizability based on the SER strategy. These tools have been used successfully by thousands of investigators world-wide, and helped solve nearly 170 crystal structures, including those of novel globular and membrane proteins, complexes and drugtargets in drug design pipelines. We now propose to pursue further experimental and computational studies of the relationships between physical chemistry of the protein surface and its solution properties. Specifically, we will investigate how surface entropy reduction affects protein solubility and stability. We will design and implement second generation XtalPred and SERp algorithms, with numerous new features, to achieve higher success rates for prediction of protein crystallizability and for design of variants with higher crystallizability and solubility. Finally, to validate the methods, we will test them using a selection of biologically relevant protein targets.

尽管成功取得了巨大的成功，但结构生物学受到低成功率的严重限制大分子结晶。许多有价值的，生物医学上重要的目标避免了结晶的尝试，总体而言，确定结构的高成本主要是由于劳动力和时间密集型筛查在蛋白质产生和结晶阶段的靶标。我们的研究将解决这个瓶颈，通过制定蛋白质工程策略，允许使用增强的溶解度，稳定性和结晶性，以及基于Web的公开服务器的实现这有助于应用这些策略。在上一阶段，我们证明了蛋白质结晶可以通过表面合理诱导基于表面熵减少（SER）的前提，即大型，极性和溶剂暴露的氨基酸，例如Lys，Glu和Gln，带有小残留物，例如ala。此外，我们设计了并实施了第一代XTALPRED和SERP服务器，这些服务器提供了自动评估蛋白质基于Ser的结晶和设计具有增强性的变体的倾向战略。这些工具已被全球成千上万的调查人员成功使用，并帮助解决了将近170个晶体结构，包括新型球状和膜蛋白，复合物以及药物设计管道中的药品量。我们现在建议追求进一步的实验和计算研究蛋白质表面的物理化学及其溶液特性之间的关系。具体而言，我们将研究表面熵的还原如何影响蛋白质溶解度和稳定性。我们将设计和实施具有许多新功能的第二代Xtalpred和SERP算法实现较高的成功率，以预测蛋白质的结晶性和较高的变体的设计结晶性和溶解度。最后，为了验证方法，我们将使用一系列生物学测试它们相关蛋白质靶标。