Affinity Labeling of Nucleotide Sites in Proteins

蛋白质中核苷酸位点的亲和标记

• 批准号: 9423108
• 负责人: Roberta Colman
• 金额: $ 30.05万
• 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: 1995
• 资助国家: 美国
• 起止时间: 1995-01-15 至 1998-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9423108&HistoricalAwards=false
• 关键词: Affinity; Labeling; Nucleotide; Sites; Proteins

9423108 Colman Nucleotides have a multi-functional role in cellular metabolism. For example, they are directly involved in kinase involved in kinase reactions, they participate in dehydrogenase reactions as part of the coenzyme molecule, they function as regulators of many allosteric enzymes, they are involved (through the effects of cyclic AMP) in the action of several hormones, and they contribute to the aggregation of platelets. The aim of this study is to develop the tools which will allow a systematic exploration of the amino acid residues in the nucleotide sites of proteins. The general approach of affinity labeling will be used based on reagents previously synthesized in this laboratory as well as on 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 will thus provide the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents on adenylosuccinate synthetase, as well as on adenylosuccinate lyase, will be examined. Further, the reactions of cAMP derivatives with platelet cAMP phosphodiesterases will be explored. The aggregation of blood platelets is induced by ADP, presumably by binding to receptor sites on the platelet surface membrane. Affinity labeling with the purine nucleotide analogues will be used to characterize the ADP receptor protein of platelet membranes. For each protein, the kinetics of the affinity labeling reaction will be examined, the extent of reagent incorporation will be determined and radioactive peptides will be isolated from proteolytic digests of labeled enzyme to identify the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzyme will be evaluated. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has already been used to react at specific sites of more than 50 proteins; it is likely that the newer compounds which we develop will similarly have broad applicability in the labeling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes. In addition, these nucleotide analogues will have applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. %%% Nucleotides have a multifunctional role in cellular metabolism. For example, nucleotides participate directly in numerous enzymatic reactions, they function as activatos or inhibitors of many regulatory enzymes, and the"second messenger" cyclic AMP carries out the action of several hormones. The aim of this study is to develop the tools which will allow a systematic exploration of the region of an enzyme which binds nucleotides. The general approach of "affinity labeling" will be used in which we attach to a natural nucleotide a reactive portion (called an alkylating agent) which is capable of forming a permanent link to a protein. The nucleotide moiety of the new reagent will direct the compound to a specific binding site on the protein, and the reactive portion will make the binding irreversible so that the binding site can be isolated and characterized. We will now study the effects of these nucleotide alkylating agents ontwo enzymes ctitical for purine nucleotide biiosynthesis and on an enzyme important in the degradation of the regulatory nucleotide cyclic AMP. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has aleady been used to react at specific sites of more than 50 proteins; it is likely that the newer compounds which we develop will similarly have b road applicability in the lab eling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes. In addition, these nucleotide analogues will hve applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. ***

9423108 Colman核苷酸在细胞代谢中具有多功能作用。 例如，它们直接参与激酶反应中的激酶，它们作为辅酶分子的一部分参与脱氢酶反应，它们作为许多变构酶的调节剂发挥作用，它们参与（通过环AMP的作用）几种激素的作用，它们有助于血小板的聚集。本研究的目的是开发工具，这将允许在蛋白质的核苷酸位点的氨基酸残基的系统探索。亲和标记的一般方法将基于本实验室先前合成的试剂以及在嘌呤或核糖环的不同位置掺入能够与蛋白质共价反应的烷化剂的新试剂来使用;核苷酸部分因此将为结合位点内的蛋白质的化学修饰提供特异性。将检查这些核苷酸烷化剂对腺苷酸琥珀酸合成酶以及对腺苷酸琥珀酸裂解酶的影响。此外，将探讨cAMP衍生物与血小板cAMP磷酸二酯酶的反应。血小板的聚集由ADP诱导，推测是通过结合到血小板表面膜上的受体位点。嘌呤核苷酸类似物的亲和标记将用于表征血小板膜的ADP受体蛋白。 对于每种蛋白质，将检查亲和标记反应的动力学，测定试剂掺入的程度，并从标记酶的蛋白水解酶中分离放射性肽，以鉴定修饰的残基。的催化和配体结合特性，以及修饰的酶的构象特征将进行评估。我们描述的第一类核苷酸类似物，氟磺酰基苯甲酰基核苷，已经被用于在超过50种蛋白质的特定位点上反应;我们开发的新化合物很可能同样在蛋白质中核苷酸位点的标记中具有广泛的适用性，并且这些研究将作为使用这些化合物来探测其他酶的原型。此外，这些核苷酸类似物将在生物技术工业中具有应用，其中它们可用于检测克隆的核苷酸结合蛋白的表达，并为合理设计用作靶向特定核苷酸需要酶的特异性抑制剂的药物提供知识基础。 %核苷酸在细胞代谢中具有多功能作用。 例如，核苷酸直接参与许多酶促反应，它们作为许多调节酶的激活剂或抑制剂发挥作用，并且“第二信使”环AMP执行几种激素的作用。 本研究的目的是开发的工具，这将允许一个系统的探索区域的酶结合核苷酸。 将使用“亲和标记”的一般方法，其中我们将能够与蛋白质形成永久连接的反应性部分（称为烷化剂）连接到天然核苷酸。 新试剂的核苷酸部分将化合物引导至蛋白质上的特异性结合位点，并且反应性部分将使结合不可逆，使得结合位点可以被分离和表征。 我们现在将研究这些核苷酸烷化剂对嘌呤核苷酸生物合成的两种关键酶和对调节核苷酸环AMP降解的重要酶的影响。 我们描述的第一类核苷酸类似物，氟磺酰基苯甲酰基核苷，已经被用于在50多种蛋白质的特定位点上反应;我们开发的新化合物很可能在蛋白质核苷酸位点的实验室中具有类似的B路适用性，这些研究将作为使用这些化合物探测其他酶的原型。 此外，这些核苷酸类似物将在生物技术工业中具有应用，其中它们可用于检测克隆的核苷酸结合蛋白的表达，并为合理设计用作针对特定核苷酸需要酶的特异性抑制剂的药物提供知识基础。 ***