Hydroxyl radical mapping of protein interfaces

超出提供的空间。确定蛋白质的哪些氨基酸残基形成小分子配体的结合部位，或者哪些氨基酸残基存在于蛋白质-蛋白质复合体的界面上，很难从脱辅基蛋白质的晶体结构中识别出来。最近，我们发展了结合重同位素标记的质谱学方法来定位蛋白质的活性部位。这些方法依赖于在存在和不存在配体的情况下测量溶剂可及性或蛋白质稳定性的变化，这是根据溶剂氚与酰胺NH的交换速率确定的，这一技术称为酰胺氢/氚交换或酰胺H/D-Ex。这项建议描述了开发一种补充酰胺I-I/D-Ex的方法，特别是羟基自由基介导的同位素交换成氨基酸侧链的脂肪族碳，这一技术称为烷基H/D-Ex。酰胺H/D-Ex表征了多肽主链的流动性和可及性，而烷基H/D-Ex通过揭示侧链的溶剂可及性提供了补充的结构信息。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。本研究的目的是证明烷基交换用于确定各种小分子/凝血酶和蛋白质/凝血酶复合体中的小分子-蛋白质界面，以及蛋白激酶A与小分子和多肽配体的相互作用。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)增加氚的掺入水平以改善技术的动态范围，2)使用烷基H/D-Ex来绘制蛋白质/蛋白质相互作用图，3)使用allcyl H/D-Ex来绘制配体-蛋白质相互作用图，4)将凯斯西部储备大学开发的烷基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I。建议的商业应用：基于结构的药物设计受到缺乏分析配体-蛋白质相互作用的低成本和快速方法的限制。烷基H/D-Ex技术代表了一种快速、通用、低成本的基于结构的药物设计方法。这项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改造，而该机构的工作重点将是加强烷基H/D-Ex的化学。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 I-I/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 pepfide/thrombin and protein/thrombin complexes; as well as, the intearetions 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 I for data analysis. PROPOSED COMMERCIAL APPLICATION: Structure based drug design is limited by the lack o4 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 hi_ 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. PERFORMANCE SITE ========================================Section End===========================================