Hydroxyl radical mapping of protein interfaces

蛋白质界面的羟基自由基图谱

• 批准号: 6832739
• 负责人: Vernon E. Anderson
• 金额: $ 34.42万
• 依托单位: EXSAR CORPORATION
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832739
• 关键词: analytical chemistry; benzimidazoles; cesium; computer simulation; enzyme inhibitors; hirudins; hydroxyl radical; intermolecular interaction; liquid chromatography mass spectrometry; mathematical model; method development; model design /development; molecular dynamics; molecular site; peptides; physical model; protein structure function; proteomics; prothrombin; protonation; radiotracer; statistics /biometry; structural biology; surface property; thermolysin; thrombin

DESCRIPTION (provided by applicant): Determining which amino acid residues of a protein form the binding site for a small molecule ligand, or that are present at the interface of a protein-protein complex, is difficult to identify from the crystal structure of the apo-protein. Recently, we have developed mass spectrometric methods coupled with heavy isotope labeling for mapping the location of active sites of proteins. These methods rely on measuring changes in solvent-accessibility or protein stability in the presence and absence of ligands, as determined from the rates of exchange of solvent deuterons with amide NH, a technique referred to as amide hydrogen/deuterium exchange or amide H/D-Ex. This proposal describes development of a complementary method to amide H/D-Ex, specifically hydroxyl radical-mediated isotope exchange into aliphatic carbons of amino acid side chains, a technique referred to as alkyl H/D-Ex. While amide H/D-Ex characterizes the mobility and accessibility of the polypeptide backbone, the alkyl H/D-Ex provides complementary structural information by revealing the solvent accessibility of the side chains. The proposed research builds on previous proof of-concept research to understand the underlying chemistry of the approach. The goal of this research is to demonstrate the utility of alkyl exchange to define the small molecule-protein interfaces within various small peptide/thrombin and protein/thrombin complexes; as well as, the interactions of protein kinase A with small molecule and peptide ligands. Finally, the approach will be adapted for high-throughput analysis. The specific aims of this proposal are: 1) increase the level of deuterium incorporation to improve the dynamic range of the technique, 2) use alkyl H/D-Ex to map protein/protein interactions, 3) use allcyl H/D-Ex to map ligand-protein interactions, 4) to transfer the alkyl H/D-Ex technology that was developed at Case Western Reserve University to ExSAR, and 5) develop computational methods for data analysis. PROPOSED COMMERCIAL APPLICATION: Structure based drug design is limited by the lack of low-cost and rapid methods for the analysis of ligand-protein interactions. Alkyl H/D-Ex technology represents a structure-based drug design approach that is rapid, versatile, and low cost. This technology can be adapted to high throughput allowing it to be integrated into small molecule drug discovery programs throughout the pharmaceutical industry. ExSAR Corporation will participate in the adaptations required for high throughput and validation of the technology, while the work at the institution will focus on enhancing the chemistry of the alkyl H/D-Ex.

描述（由申请人提供）：确定蛋白质的哪些氨基酸残基形成小分子配体的结合位点，或存在于蛋白质-蛋白质复合物的界面处，难以从脱辅基蛋白的晶体结构中鉴定。最近，我们发展了质谱方法结合重同位素标记的定位蛋白质的活性位点。这些方法依赖于测量在存在和不存在配体的情况下溶剂可及性或蛋白质稳定性的变化，如由溶剂氘与酰胺NH的交换速率确定的，该技术被称为酰胺氢/氘交换或酰胺H/D-Ex。该提案描述了酰胺H/D-Ex的补充方法的开发，特别是羟基自由基介导的氨基酸侧链的脂肪族碳的同位素交换，该技术被称为烷基H/D-Ex。虽然酰胺H/D-Ex表征多肽主链的移动性和可及性，但烷基H/D-Ex通过揭示侧链的溶剂可及性来提供互补的结构信息。拟议的研究建立在以前的概念验证研究的基础上，以了解该方法的基本化学原理。本研究的目的是证明烷基交换的效用，以确定各种小肽/凝血酶和蛋白质/凝血酶复合物内的小分子-蛋白质界面，以及蛋白激酶A与小分子和肽配体的相互作用。最后，该方法将适用于高通量分析。这项建议的具体目标是：1）增加氘掺入水平以改善该技术的动态范围，2）使用烷基H/D-Ex绘制蛋白质/蛋白质相互作用，3）使用烷基H/D-Ex绘制配体-蛋白质相互作用，4）将凯斯西储大学开发的烷基H/D-Ex技术转移到ExSAR，以及5）开发用于数据分析的计算方法。拟定商业应用：基于结构的药物设计受到缺乏低成本和快速分析配体-蛋白质相互作用的方法的限制。Alkyl H/D-Ex技术代表了一种基于结构的药物设计方法，具有快速、通用和低成本的特点。该技术可以适应高通量，使其能够整合到整个制药行业的小分子药物发现计划中。ExSAR公司将参与高通量和技术验证所需的调整，而该机构的工作将集中在提高烷基H/D-Ex的化学性质上。