Watty A, Weise C, Dreger M, Franke P, Hucho F
Institut für Biochemie, Fachbereich Chemie, Freie Universität Berlin, Germany.
Eur J Biochem. 1998 Mar 1;252(2):222-8. doi: 10.1046/j.1432-1327.1998.2520222.x.
To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.
为了获取来自电鳐电组织的烟碱型乙酰胆碱受体的结构信息，我们用激动性双功能试剂[¹⁴C]亚氨代辛二酸二甲酯修饰并交联赖氨酸残基。该试剂标记暴露的赖氨酸残基，尤其是那些位于配体结合位点附近的残基，并交联距离不超过交联剂长度11埃的赖氨酸残基。使用这种方法，我们鉴定出一个位于βLys177和βLys191之间的交联，表明其间的13个氨基酸形成一个环，这两个残基位于表面。αLys76和αLys77这两个相邻的赖氨酸残基之间也发生了交联，表明这些相邻的赖氨酸残基不参与β折叠结构。受体中存在的97个赖氨酸残基中共有21个被[¹⁴C]亚氨代辛二酸二甲酯修饰。因此，这些残基位于可及的膜外表面。αLys76和αLys179这两个赖氨酸残基被大量标记。由于[¹⁴C]亚氨代辛二酸二甲酯具有激动特性[瓦蒂，A.，梅特费塞尔，C. & 胡乔，F.（1997年）《美国国家科学院院刊》94，8202 - 8209]，这可能是因为它们与配体结合位点距离很近。
