Arai K, Maeda T, Kawakita M, Ohnishi S, Kaziro Y
J Biochem. 1976 Nov;80(5):1047-55. doi: 10.1093/oxfordjournals.jbchem.a131360.
The conformational transitions of polypeptide chain elongation factor Tu (EF-Tu) associated with the ligand change from GDP to GTP and also with the displacement of GDP by elongation factor Ts (EF-Ts) have been investigated using the spin-labeling technique. Of the two reactive sulfhydryl groups in EF-Tu, the one essential for interaction with aminoacyl-tRNA was selectively labeled with various kinds of iodoacetamide or maleimide spin-labeling reagents. The electron spin resonance (ESR) spectra of EF-Tu-GDP labeled with these reagents generally consisted of two components, one narrow and one broad, corresponding to labels relatively weakly and strongly immobilized, respectively. The degree of immobilization and the ratio of the narrow to the broad components were different for each kind of label used. The spectra of spin-labeled EF-Tu-GDP changed markedly when its GDP moiety was replaced by GTP through incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], the broad component increasing at the expense of the narrow component. The reversible nature of the conformational change was confirmed with EF-Tu labeled with a maleimide reagent. The GTP-induced spectral change was reversed upon conversion of labeled EF-Tu-GTP to EF-Tu-GDP by addition of excess GDP. A similar type of spectral change was also observed when spin-labeled EF-Tu-GDP was incubated with EF-Ts to form labeled EF-Tu-EF-Ts complex. The extent of the spectral change induced by EF-Ts was even greater than that induced by GTP. These results, together with those obtained by studies with hydrophobic and fluorescent probes (Arai, Arai, Kawakita, & Kaziro (1975) J. Biochem. 77, 1095-1106) indicate that a reversible conformational change is induced in EF-Tu near the sulfhydryl group that is essential for interaction with aminoacyl-tRNA.
已使用自旋标记技术研究了多肽链延伸因子Tu（EF-Tu）与配体从GDP转变为GTP以及延伸因子Ts（EF-Ts）将GDP置换相关的构象转变。在EF-Tu中的两个反应性巯基中，与氨酰基-tRNA相互作用所必需的那个巯基用各种碘乙酰胺或马来酰亚胺自旋标记试剂进行了选择性标记。用这些试剂标记的EF-Tu-GDP的电子自旋共振（ESR）光谱通常由两个成分组成，一个窄峰和一个宽峰，分别对应于固定化程度相对较弱和较强的标记。每种所用标记的固定化程度以及窄峰与宽峰成分的比例都不同。当通过与磷酸烯醇丙酮酸和丙酮酸激酶[EC 2.7.1.40]孵育将其GDP部分替换为GTP时，自旋标记的EF-Tu-GDP的光谱发生了显著变化，宽峰成分增加，窄峰成分减少。用马来酰亚胺试剂标记的EF-Tu证实了构象变化的可逆性。通过加入过量的GDP将标记的EF-Tu-GTP转化为EF-Tu-GDP后，GTP诱导的光谱变化得以逆转。当自旋标记的EF-Tu-GDP与EF-Ts孵育形成标记的EF-Tu-EF-Ts复合物时，也观察到了类似类型的光谱变化。EF-Ts诱导的光谱变化程度甚至大于GTP诱导的程度。这些结果，连同通过使用疏水和荧光探针获得的结果（Arai，Arai，Kawakita，& Kaziro（1975）J. Biochem. 77，1095 - 1106）表明，在与氨酰基-tRNA相互作用所必需的巯基附近，EF-Tu中诱导了可逆的构象变化。
