Active-Site-Directed Photolabeling of the Melibiose Permease of Escherichia coli
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- 作者：Yves Ambroise ; Gé ; rard Leblanc ; and Bernard Rousseau
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：February 15, 2000
- 年：2000
- 卷：39
- 期：6
- 页码：1338 - 1345
- 全文大小：108K
- 年卷期：v.39,no.6(February 15, 2000)
- ISSN：1520-4995
文摘
Covalent photolabeling of the melibiose permease (MelB) of Escherichia coli has beenundertaken with the sugar analogue [3H]-p-azidophenyl 
-D-galactopyranoside ([3H]--PAPG) with thepurpose of identifying the domains forming the MelB sugar-binding site. We show that -PAPG is ahigh-affinity substrate of MelB (Kd = 1 × 10-6 M). Its binding to or transport by MelB is Na-dependentand is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl -D-galactopyranoside (-NPG). Membrane vesicles containing overexpressed histidine-tagged recombinantMelB were photolabeled in the presence of [3H]--PAPG by irradiation with UV light (
= 250 nm).Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in apolypeptide corresponding to MelB monomer. MelB labeling was completely prevented by an excess ofmelibiose or -NPG during the assay. Radioactivity analysis of CNBr cleavage or limited proteolysisproducts of the purified [3H]--PAPG-labeled transporter suggests that several domains of MelB are targetsfor labeling. One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of5.5 kDa. It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V,and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled aminoacid of this peptide. Labeling of loop 4-5 provides independent evidence that this specific domain playsa significant role in MelB transport. Comparison with the well-characterized equivalent domain of LacYsuggests that sugar transporters with similar structure and substrate specificity may have conserved domainsinvolved in sugar recognition.
-D-galactopyranoside ([3H]--PAPG) with thepurpose of identifying the domains forming the MelB sugar-binding site. We show that -PAPG is ahigh-affinity substrate of MelB (Kd = 1 × 10-6 M). Its binding to or transport by MelB is Na-dependentand is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl -D-galactopyranoside (-NPG). Membrane vesicles containing overexpressed histidine-tagged recombinantMelB were photolabeled in the presence of [3H]--PAPG by irradiation with UV light ( = 250 nm).Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in apolypeptide corresponding to MelB monomer. MelB labeling was completely prevented by an excess ofmelibiose or -NPG during the assay. Radioactivity analysis of CNBr cleavage or limited proteolysisproducts of the purified [3H]--PAPG-labeled transporter suggests that several domains of MelB are targetsfor labeling. One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of5.5 kDa. It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V,and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled aminoacid of this peptide. Labeling of loop 4-5 provides independent evidence that this specific domain playsa significant role in MelB transport. Comparison with the well-characterized equivalent domain of LacYsuggests that sugar transporters with similar structure and substrate specificity may have conserved domainsinvolved in sugar recognition.© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |