Affinity Labeling of Nucleotide Sites in Proteins

蛋白质中核苷酸位点的亲和标记

• 批准号: 9728202
• 负责人: Roberta Colman
• 金额: $ 27万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728202&HistoricalAwards=false
• 关键词: Affinity; Labeling; Nucleotide; Sites; Proteins

Colman 9728202 1. Technical Nucleotides have a multi-functional role in cellular metabolism: involved in kinase reactions, participate in dehydrogenase reactions as part of the coenzyme molecule, function as regulators of many allosteric enzymes, engaged (through the effects of cyclic AMP) in the action of several hormones, and contribute to the aggregation of platelets. This study is aimed to develop tools for a systematic exploration of the amino acid residues in the nucleotide sites of proteins. The general approach of affinity is used, with reagents previously synthesized in this laboratory and new reagents which incorporate, at various positions of the purine or ribose ring alkylating agents capable of reacting covalently with the protein; the nucleotide moiety thus provides the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents are examined on several enzymes; adenylosuccinate synthetase and adenylosuccinate lyase, which catalyze successive reactions in the conversion of inosine monophosphate to AMP; and cyclic AMP phosphodiesterases, which catalyzes the hydrolysis of cAMP to form 5' - AMP. For each protein, the kinetics of the affinity labeling reaction is examined, the extent of reagent incorporation determined and the radioactive peptides isolated from proteolytic digests of labeled enzyme and identified with the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzymes are also evaluated. For most of these enzymes, the results of these enzymes in solution are compared with their determined crystal structures. The nucleotide analogs, fluorosulfonylbenzoyl nucleosides, have been tested on more than 50 proteins; with results promising for its general applicability to other enzymes. They are useful for the detection of nucleotide binding proteins expressed by cloning. The significance of this may rest with providing the k nowledge base for rational design of specific inhibitors targeted to particular nucleotide-requiring enzymes. 2. Non-technical Nucleotides have a multifunctional role in cellular metabolism, such as participating in enzymatic reactions, activating regulatory enzymes, and serving as "second messenger" like cyclic AMP. This study is aimed to develop the tools which allow a systematic exploration of the enzyme which binds nucleotides. A general approach of "affinity labeling" is used to attach a nucleotide moiety of the new reagent compound to a specific binding site on the protein, irreversibly, so that the binding site can be isolated and characterized. The effects of nucleotide alkylating agents on two purine nucleotide biosynthesis enzymes and on an enzyme important in the degradation of nucleotide cyclic AMP are studied. Nucleotide analogues such as fluorsulfonylbenzoyl nucleosides which react to proteins at specific sites are specifically probed. For biotechnology, these molecules can be used to detect expression of cloned proteins, and to provide the knowledge base for rational design of inhibitors for nucleotide-requiring enzymes.

科尔曼9728202 1.技术核苷酸在细胞代谢中具有多种功能：参与激酶反应，作为辅酶分子的一部分参与脱氢酶反应，作为许多变构酶的调节器，参与(通过环腺苷的作用)几种激素的作用，并有助于血小板的聚集。这项研究的目的是开发工具，系统地探索蛋白质核苷酸位点上的氨基酸残基。使用亲和力的一般方法，使用先前在本实验室合成的试剂和在不同位置加入能够与蛋白质共价反应的嘌呤或核糖环烷化剂的新试剂；因此，核苷酸部分为结合位置内的蛋白质的化学修饰提供了特异性。这些核苷酸烷基化试剂对几种酶的影响进行了检测：腺苷琥珀酸合成酶和腺苷琥珀酸裂解酶，它们催化肌苷一磷酸转化为AMP的连续反应；以及环AMP磷酸二酯酶，它催化cAMP的水解形成5‘-AMP。对于每个蛋白质，研究了亲和标记反应的动力学，测定了试剂的掺入程度，并从标记酶的蛋白水解液中分离出放射性多肽，并用修饰残基进行了鉴定。对修饰后的酶的催化和配体结合性能以及构象特征进行了评价。对于大多数这些酶，这些酶在溶液中的结果与它们确定的晶体结构进行了比较。这些核苷酸类似物，氟磺酰苯甲酰核苷，已经在50多种蛋白质上进行了测试；结果表明，它对其他酶的普遍适用性很有希望。它们可用于通过克隆表达的核苷酸结合蛋白的检测。其意义可能在于为针对特定核苷酸需求酶的特定抑制剂的合理设计提供知识基础。2.非技术性核苷酸在细胞代谢中具有多种功能，如参与酶反应、激活调节酶、充当环磷酸腺苷等“第二信使”。这项研究的目的是开发工具，允许系统地探索与核苷酸结合的酶。亲和标记的一般方法是将新试剂化合物的核苷酸部分不可逆地连接到蛋白质上的特定结合部位，以便分离和表征结合部位。研究了核苷酸烷化剂对两种嘌呤核苷酸生物合成酶和一种在核苷酸环状AMP降解中起重要作用的酶的影响。具体探讨了在特定位置与蛋白质反应的核苷酸类似物，如氟磺酰苯甲酰核苷。在生物技术方面，这些分子可用于检测克隆蛋白的表达，并为合理设计需要核苷酸的酶的抑制剂提供知识库。