Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent
Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent
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DOI：
10.1073/pnas.051632898
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发表时间：
2001-04-24
影响因子：
11.1
通讯作者：
Khorana, FC
中科院分区：
文献类型：
--
作者：
Cai, K;Itoh, Y;Khorana, FC
光激活视紫红质与转导素Cn的相互作用是视觉信号转导的第一个事件。我们使用共价交联的方法来映射两种蛋白质之间相互作用的接触位点。在这里，我们使用光活化试剂，N-[（2-吡啶基二硫代）-乙基]，4-叠氮基水杨酰胺，该试剂通过与吡啶基硫代基团的二硫键交换反应连接到细胞质单半胱氨酸视紫红质突变体的SH基团上，然后衍生的视紫红质通过λ> 495 nm的照射与T复合。随后在λ 310 nm处照射复合物在两种蛋白质之间产生共价交联。T和一些单半胱氨酸视紫红质突变体之间的交联被证明，然而，仅详细研究了T和视紫红质突变体S240 C（胞质环V-VI）之间的交联位点，交联主要发生在T-α。为了鉴定T-α中的交联位点，使用的策略包括：（i）用N-乙基马来酰亚胺衍生交联蛋白中的所有游离半胱氨酸;（ii）还原连接两种蛋白的二硫键，并分离携带具有游离SH基团的交联部分的所有T-α物质;（iii）后者与含有生物素基的试剂马来酰亚胺-丁酰基-生物胞素的N-马来酰亚胺部分形成加合物;（iv）所得T-α衍生物的胰蛋白酶降解和通过抗生物素蛋白分离携带马来酰亚胺基-丁酰基-生物胞素的T-α肽。琼脂糖色谱法;和（v）通过基质辅助激光解吸/电离飞行时间质谱法鉴定分离的肽。我们发现交联主要发生在T-α中含有氨基酸序列310-313和342-345的两个C-末端肽。
Interaction of light-activated rhodopsin with transducin Cn is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide, The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at lambda > 495 nm. Subsequent irradiation of the complex at lambda 310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants, However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI), Crosslinking occurred predominantly with T-alpha. For identification of the sites of crosslinks in T-alpha the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T-alpha species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T-alpha derivatives and isolation of T-alpha peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T-alpha containing the amino acid sequences 310-313 and 342-345.