Frillingos S, Wu J, Venkatesan P, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1570, USA.
Biochemistry. 1997 May 27;36(21):6408-14. doi: 10.1021/bi970233b.
By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity. The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.
通过使用原位定点巯基修饰的半胱氨酸扫描诱变技术[弗里林戈斯，S.，& 卡巴克，H. R.（1996年）《生物化学》35卷，3950 - 3956页]，研究了大肠杆菌乳糖通透酶中配体或单克隆抗体（mAb）4B1结合所诱导的构象变化。在螺旋I、V、VII、VIII、X或XI中的31个单半胱氨酸替代突变体中，4B1结合改变了外向型膜囊泡中缬氨酸238突变为半胱氨酸（螺旋VII）、缬氨酸331突变为半胱氨酸（螺旋X）或单半胱氨酸355（螺旋XI）通透酶与N - 乙基马来酰亚胺（NEM）的反应性。此外，定点荧光光谱表明，mAb 4B1结合使通透酶中位置331（螺旋X）处于更疏水的环境。相比之下，配体结合引发的变化更为广泛，这表现为丙氨酸244突变为半胱氨酸、苏氨酸248突变为半胱氨酸（螺旋VII）、苏氨酸265突变为半胱氨酸（螺旋VIII）、缬氨酸315突变为半胱氨酸（螺旋X）、谷氨酰胺359突变为半胱氨酸或甲硫氨酸362突变为半胱氨酸（螺旋XI）通透酶的NEM反应性增强，而这些突变体的反应性均不受4B1结合的影响。此外，未观察到4B1对可能与底物直接接触的位置半胱氨酸148（螺旋V）、缬氨酸264突变为半胱氨酸、甘氨酸268突变为半胱氨酸或天冬酰胺272突变为半胱氨酸（螺旋VIII）的反应性有影响。关于通透酶的N端一半，4B1结合使突变体脯氨酸28突变为半胱氨酸或脯氨酸31突变为半胱氨酸（螺旋I）的反应性略有增加，而配体结合使反应性增加得更多。这些发现表明，4B1结合在通透酶中诱导的结构变化比配体结合诱导的结构变化要少得多。
