MAPPING OF RHODOPSIN TRANSDUCIN INTERACTION SITES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。视紫红质是视网膜视杆细胞中存在的一种完整的膜蛋白。光诱导的视紫红质构象变化是导致光感知的信号转导通路中的起始事件。这个信号通路中的一个关键事件是蛋白转导蛋白与光激活的视紫红质的结合。鉴于这种蛋白质-蛋白质相互作用的关键作用，Khorana小组已经指导了研究，以了解这两种蛋白质之间接触位点的确切性质。由于转导蛋白和视紫红质之间的相互作用是短暂的，研究结合了光交联法以共价复合体的形式捕捉这种相互作用，从而允许进一步纯化和分析复合体。具体地说，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部位。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，每个分子只含有一个半胱氨酸残基。该半胱氨酸提供了与双官能团光活化交联剂NETDB的一端反应的巯基。NETDB的另一端含有一个双氮烯基团，在紫外光激活后可以与转导蛋白反应。在交联化和去除未结合的转导蛋白之后，通过用二硫苏糖醇还原从视紫红质中去除交联化的转导蛋白。得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酶消化转导蛋白后用反相色谱检测交联肽的灵敏手段。这种分析是针对每个独立的视紫红质半胱氨酸变异体进行的。目前正在寻求两种通过质谱学定位交联点的方法。一种方法是利用离线层析技术，利用德克萨斯红发色团初步分离出交联肽。分离的组分首先通过基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF)进行分析，以确定与交联剂的存在相一致的质量增加的多肽。然后，通过串联质谱仪(MSMS)进一步分析任何已鉴定的交联肽，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质谱(LC-MSMS)在自动化过程中定位交联型氨基酸。第一种方法的优点是有更多的时间对每个候选多肽进行分析。然而，额外的处理步骤可能会导致低丰度样品的丢失。LC-MSMS方法最大限度地减少了由于处理而造成的样品损失，并将样品浓缩到色谱峰中，从而潜在地提高了灵敏度。然而，在这种方法中，每个多肽的MSMS分析时间都是有限的。根据为每个样本产生的数据的质量来确定要使用的具体方法。我们对从与视紫红质半胱氨酸突变体的交联反应中分离出来的七种转导蛋白消化酶进行了MS/MS分析。用纳米ESI-MSMS分析了所有变异体的高效液相色谱组分。从SDS-PAGE凝胶条带中电洗脱也被研究为回收交联蛋白的一种方法。Khorana团队目前正在开发一种替代策略来纯化交联肽，在串联MS分析之前利用交联肽的选择性浓缩；这种方法应该会增加多肽的产量。

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.