Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

膜蛋白与高度结晶和可溶性蛋白质共结晶

基本信息

批准号：
8843009
负责人：
Gregory A. Weiss
金额：
$ 27.27万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8843009
关键词：
Affinity Affinity Chromatography Antibodies Area Binding Binding Proteins Binding Sites Biological Assay Caveolins Cells Chimeric Proteins Collaborations Crystal Formation Crystallization Detergents Development Disease Exhibits Foundations Freezing G Protein-Coupled Receptor Genes GTP-Binding Proteins Generations Goals Laboratories Letters Libraries Ligands Membrane Proteins Methods Molecular Conformation Muramidase Nature Phage Display Precipitation Production Property Protein Engineering Protein Overexpression Protein S Proteins Publishing Reagent S-crystallin Solubility Specificity Structural Biologist Structure System Techniques Therapeutic Thermodynamics Variant Work base biological systems design design and construction innovation molecular recognition novel strategies overexpression protein aggregation protein folding protein function protein purification protein structure receptor coupling research study structural biology success tool

项目摘要

DESCRIPTION (provided by applicant): Membrane proteins confound anything less than exceptionally heroic attempts aimed at solving their structures. The conventional approaches to membrane protein overexpression, purification, and crystallization typically fail due to problems with insolubility and folding. This project leverages large libraries of soluble and highly crystallizable proteins to identify binding partners for membrane proteins. Selectants from these libraries will provide affinity reagents for membrane protein co-expression, affinity purification and co-crystallization. Co-expression with the binding partner could help avoid membrane protein aggregation, and allow protein folding to take place. Affinity chromatography with the binding partner is aimed at assisting membrane protein purification, and co-crystallization aims to slow protein aggregation and precipitation during formation of crystals. The first specific aim focuses on design and construction of phage-displayed protein libraries for high affinity binding to membrane proteins. Strategic choice of proteins for library formation, such as the highly crystallizable protein lysozyme and the exceptionally soluble protein S-crystallin, for phage display will help insure the success of the project; additional libraries specifically tailored forG-protein coupled receptors (GPCRs) include variants of G- proteins and GPCR ligands. To obtain high affinity binding, thermal stability, solubility, and other properties, the second specific aim features a flow path of selections and screens. In the third specific aim, the affinity reagents from phage display are applied to the production of membrane proteins and their crystallization. By binding to and essentially freezing specific conformations of the membrane protein, the affinity reagents could offer powerful tools both for structural biology, but also other structure-function studies of membrane proteins. In summary, this proposal will define new approaches to protein engineering and molecular recognition, through development of new fusion proteins and their use in the recognition of membrane proteins.

描述（由申请人提供）：膜蛋白混淆了旨在解决其结构的异常英雄尝试。膜蛋白过表达，纯化和结晶的常规方法通常由于无法溶解性和折叠问题而失败。该项目利用大量可溶性和高度结晶的蛋白质库来鉴定膜蛋白的结合伴侣。这些文库中的选择者将为膜蛋白共表达，亲和力纯化和共结晶提供亲和力试剂。与结合伴侣共表达可以帮助避免膜蛋白聚集，并允许蛋白质折叠发生。与结合伴侣的亲和色谱旨在帮助膜蛋白纯化，共结晶旨在在形成晶体过程中慢慢蛋白质聚集和降水。第一个特定目标专注于设计和构建噬菌体蛋白质文库，以与膜蛋白的高亲和力结合。用于文库形成的蛋白质的战略选择，例如高度结晶的蛋白质溶菌酶和异常可溶的蛋白质S-晶状蛋白，用于噬菌体显示，将有助于确保项目的成功；其他专门定制的宽松蛋白偶联受体（GPCR）的文库包括G-蛋白和GPCR配体的变体。为了获得高亲和力结合，热稳定性，溶解度和其他特性，第二个特定目标具有选择和屏幕的流程路径。在第三个特定目的中，噬菌体显示的亲和力试剂应用于膜蛋白的产生及其结晶。通过与膜蛋白的结合并基本上冻结特定构象，亲和力试剂可以为结构生物学提供强大的工具，也可以为膜蛋白的其他结构 - 功能研究提供。总而言之，该建议将通过开发新的融合蛋白及其在识别膜蛋白中的使用来定义蛋白质工程和分子识别的新方法。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Continuous flow biocatalysis.

DOI：
10.1039/c7cs00906b
发表时间：
2018-07-30
期刊：
Chemical Society reviews
影响因子：
46.2
作者：
Britton J ;Majumdar S ;Weiss GA
通讯作者：
Weiss GA

Accelerating Enzymatic Catalysis Using Vortex Fluidics.

DOI：
10.1002/anie.201604014
发表时间：
2016-09-12
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
0
作者：
Britton J;Meneghini LM;Raston CL;Weiss GA
通讯作者：
Weiss GA

Vortex Fluidic Chemical Transformations.

DOI：
10.1002/chem.201700888
发表时间：
2017-09-27
期刊：
Chemistry (Weinheim an der Bergstrasse, Germany)
影响因子：
0
作者：
Britton J;Stubbs KA;Weiss GA;Raston CL
通讯作者：
Raston CL

Shear-stress-mediated refolding of proteins from aggregates and inclusion bodies.

DOI：
10.1002/cbic.201402427
发表时间：
2015-02-09
期刊：
CHEMBIOCHEM
影响因子：
3.2
作者：
Yuan, Tom Z.;Ormonde, Callum F. G.;Kudlacek, Stephan T.;Kunche, Sameeran;Smith, Joshua N.;Brown, William A.;Pugliese, Kaitlin M.;Olsen, Tivoli J.;Iftikhar, Mariam;Raston, Colin L.;Weiss, Gregory A.
通讯作者：
Weiss, Gregory A.

Chemically Modifying Viruses for Diverse Applications.