Hydroxyl radical mapping of protein interfaces

超出所提供的空间。确定蛋白质的哪个氨基酸残基形成了小分子配体的结合位点，或者哪些氨基酸残基存在于蛋白质-蛋白质复合物的界面，很难从载脂蛋白的晶体结构中识别出来。最近，我们开发了质谱方法结合重同位素标记来绘制蛋白质活性位点的位置。这些方法依赖于测量在配体存在和不存在的情况下溶剂可及性或蛋白质稳定性的变化，由溶剂氘与酰胺NH的交换速率确定，这种技术称为酰胺氢/氘交换或酰胺H/D-Ex。本提案描述了一种酰胺I-I/D-Ex的补充方法的发展，特别是羟基自由基介导的同位素交换到氨基酸侧链的脂肪碳，这种技术被称为烷基H/D-Ex。酰胺H/D-Ex表征多肽主链的迁移性和可接近性，而烷基H/D-Ex通过揭示侧链的溶剂可接近性提供互补的结构信息。拟议的研究建立在先前的概念验证研究的基础上，以了解该方法的潜在化学原理。本研究的目的是证明烷基交换在各种小肽/凝血酶和蛋白质/凝血酶复合物中定义小分子-蛋白质界面的效用；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。本提案的具体目标是：1)提高氘结合水平以提高技术的动态范围，2)使用烷基H/D-Ex来绘制蛋白质/蛋白质相互作用图，3)使用全烷基H/D-Ex来绘制配体-蛋白质相互作用图，4)将凯斯西储大学开发的烷基H/D-Ex技术转移到ExSAR， 5)开发用于数据分析的计算方法1。建议的商业应用：基于结构的药物设计受到缺乏低成本和快速的配体-蛋白质相互作用分析方法的限制。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，使其能够集成到整个制药行业的小分子药物发现项目中。ExSAR公司将参与该技术的高通量和验证所需的调整，而该机构的工作将侧重于增强烷基H/D-Ex的化学性质。网站性能 ======================================== 节结束 ===========================================

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===========================================