PMID:9174357

Citation	Frillingos, S, Wu, J, Venkatesan, P and Kaback, HR (1997) Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli. Biochemistry 36:6408-14
Abstract	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.
Links	PubMed Online version:10.1021/bi970233b
Keywords	Antibodies, Monoclonal/chemistry; Binding Sites, Antibody/genetics; Cysteine/genetics; Escherichia coli/chemistry; Escherichia coli/enzymology; Escherichia coli Proteins; Ligands; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Conformation; Symporters
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