Cai K, Itoh Y, Khorana H G
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4877-82. doi: 10.1073/pnas.051632898.
Interaction of light-activated rhodopsin with transducin (T) 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 lambda310 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.
光激活视紫红质与转导蛋白（T）的相互作用是视觉信号转导的首个事件。我们采用共价交联方法来绘制这两种蛋白质相互作用中的接触位点。在此，我们使用一种光可激活试剂，N-[(2-吡啶二硫基)-乙基]，4-叠氮基水杨酰胺。该试剂通过与吡啶硫基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，然后经波长>495nm的光照使衍生化的视紫红质与T形成复合物。随后在波长310nm处照射该复合物，可在这两种蛋白质之间产生共价交联。已证实T与多个单半胱氨酸视紫红质突变体之间发生了交联。然而，仅对T与视紫红质突变体S240C（细胞质环V-VI）之间的交联位点进行了详细研究。交联主要发生在T(α)上。为了鉴定T(α)中的交联位点，所采用的策略包括：(i) 用N-乙基马来酰亚胺使交联蛋白质中的所有游离半胱氨酸衍生化；(ii) 还原连接这两种蛋白质的二硫键，并分离出所有带有游离SH基团的携带交联部分的T(α)物种；(iii) 使后者与含有生物素基团的试剂马来酰亚胺基丁酰生物素的N-马来酰亚胺部分形成加合物；(iv) 用胰蛋白酶降解所得的T(α)衍生物，并通过抗生物素蛋白-琼脂糖色谱法分离携带马来酰亚胺基丁酰生物素的T(α)肽段；(v) 通过基质辅助激光解吸/电离飞行时间质谱法鉴定分离出的肽段。我们发现交联主要发生在T(α)中两个C末端肽段上，其氨基酸序列为310-313和342-345。
