Functional estimation of loop-helix boundaries in the lactose permease of Escherichia coli by single amino acid deletion analysis

被引:16
作者
Wolin, CD
Kaback, HR
机构
[1] Univ Calif Los Angeles, HHMI, Inst Mol Biol, Dept Physiol, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, HHMI, Inst Mol Biol, Dept Microbiol & Mol Genet, Los Angeles, CA 90095 USA
关键词
D O I
10.1021/bi0025767
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mutants with single amino acid deletions in the loops of lactose permease retain activity, while mutants with single deletions in transmembrane helices are inactive, and the loop-helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic deletion of single residues [Wolin, C. D., and Kaback, H. R. (1999) Biochemistry 38, 8590-8597]. The experimental approach is applied here to the remainder of the permease. Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions. In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted. Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues. Surprisingly, deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual deletion of only three residues in this region abolishes activity. Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts.
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页码:1996 / 2003
页数:8
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