MAPPING OF RHODOPSIN TRANSDUCIN INTERACTION SITES

视紫红质转导蛋白相互作用位点的绘图

• 批准号: 7369215
• 负责人: H. GOBIND KHORANA
• 金额: $ 0.09万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369215
• 关键词: MAPPING; RHODOPSIN; TRANSDUCIN; INTERACTION; SITES

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rhodopsin is an integral membrane protein that is present in rod cells of the retina. A light-induced conformational change in rhodopsin is the initiating event in the signal transduction pathway leading to light perception. One critical event in this signaling pathway is the binding of the protein transducin to light-activated rhodopsin. Given the critical role this protein-protein interaction, the Khorana group has directed studies towards understanding the precise nature of contact sites between these two proteins. Since the interaction between transducin and rhodopsin is short-lived, the studies incorporate photocrosslinking to capture this interaction as a covalent complex, thus allowing for further purification and analysis of the complex. Specifically, the presence of the crosslinker on transducin facilitates location of the site of interaction with relatively high precision. The binding reaction is carried out in vitro using purified recombinant rhodopsin and transducin. A series of rhodopsin molecules have been generated which contain only one cysteine residue each. This cysteine provides a sulfhydryl group that reacts with one end of the bifunctional photoactivatable crosslinker NETDB. The opposite end of NETDB contains a diazirene group that can react with transducin following activation by UV light. Subsequent to crosslinking and removal of unbound transducin, crosslinked transducin is removed from rhodopsin by reduction with dithiothreitol. Reaction of Texas Red maleimide with the free sulfhydryl group of the bound crosslinker provides a sensitive means of detecting crosslinked peptides by reversed-phase chromatography after trypsin digestion of transducin. This analysis is performed for each independent rhodopsin cysteine variant. Two approaches to localize the crosslinked sites by mass spectrometry are being pursued. One approach utilizes off-line chromatography to initially isolate crosslinked peptides using the Texas Red chromophore. Isolated fractions are first analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) to identify peptides that have a mass increase consistent with the presence of the crosslinker. Any identified crosslinked peptides are then further analyzed by tandem mass spectrometry (MSMS) in order to determine the specific amino acid residue that is covalently bonded to the crosslinker. The second approach utilizes on-line chromatography tandem MS (LC-MSMS) to localize the crosslinked amino acid in an automated process. The first approach has the advantage of allowing more time for analysis of each candidate peptide. However it is possible that the extra handling steps could lead to loss of low abundance samples. The LC-MSMS approach minimizes sample loss due to handling and concentrates samples into chromatographic peaks, thereby potentially increasing sensitivity. However, the MSMS analysis time for each peptide is limited in this approach. The particular method to be used is determined based on the quality of data produced for each sample. We have performed MS/MS analysis on seven transducin digests isolated from crosslinking reactions with rhodopsin cysteine variants. HPLC fractions of all the variants were analyzed by nanoESI MSMS. Electroelution from SDS-PAGE gel bands has also been investigated as a method to recover the crosslinked protein. The Khorana group is currently developing an alternative strategy to purify cross-linked peptides, making use of selective enrichment of cross-linked peptides prior to tandem MS analysis; this approach should increase the yield of peptides.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。视紫红质是一种存在于视网膜杆状细胞中的完整膜蛋白。光诱导的视紫红质构象变化是导致光感知的信号转导途径的起始事件。该信号传导途径中的一个关键事件是蛋白质转导蛋白与光激活视紫红质的结合。鉴于这种蛋白质-蛋白质相互作用的关键作用，Khorana 小组将研究方向转向了解这两种蛋白质之间接触位点的精确性质。由于转导蛋白和视紫红质之间的相互作用是短暂的，因此研究结合了光交联来捕获这种相互作用作为共价复合物，从而允许进一步纯化和分析该复合物。具体而言，转导蛋白上交联剂的存在有助于以相对较高的精度定位相互作用位点。使用纯化的重组视紫红质和转导蛋白在体外进行结合反应。已经产生了一系列视紫红质分子，每个分子仅含有一个半胱氨酸残基。该半胱氨酸提供了一个巯基，可与双功能光活化交联剂 NETDB 的一端发生反应。 NETDB 的另一端包含二氮杂环基团，在紫外线激活后可以与转导蛋白发生反应。在交联并去除未结合的转导蛋白之后，通过用二硫苏糖醇还原将交联的转导蛋白从视紫红质中去除。德克萨斯红马来酰亚胺与结合交联剂的游离巯基的反应提供了一种在胰蛋白酶消化转导蛋白后通过反相色谱法检测交联肽的灵敏方法。对每个独立的视紫红质半胱氨酸变体进行该分析。目前正在寻求两种通过质谱法定位交联位点的方法。一种方法利用离线色谱法，利用德克萨斯红发色团初步分离交联肽。首先通过基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF) 分析分离的组分，以鉴定质量增加与交联剂存在一致的肽。然后通过串联质谱 (MSMS) 进一步分析任何鉴定的交联肽，以确定与交联剂共价键合的特定氨基酸残基。第二种方法利用在线色谱串联 MS (LC-MSMS) 在自动化过程中定位交联氨基酸。第一种方法的优点是可以有更多时间分析每个候选肽。然而，额外的处理步骤可能会导致低丰度样品的损失。 LC-MSMS 方法最大限度地减少了因处理而导致的样品损失，并将样品浓缩到色谱峰中，从而有可能提高灵敏度。然而，这种方法中每种肽的 MSMS 分析时间有限。要使用的特定方法是根据每个样本生成的数据的质量来确定的。我们对从视紫红质半胱氨酸变体的交联反应中分离出的七个转导蛋白消化物进行了 MS/MS 分析。所有变体的 HPLC 级分均通过 nanoESI MSMS 进行分析。还研究了 SDS-PAGE 凝胶条带的电洗脱作为回收交联蛋白的方法。 Khorana 小组目前正在开发一种纯化交联肽的替代策略，在串联 MS 分析之前利用选择性富集交联肽；这种方法应该会增加肽的产量。