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Volume 272, Number 47, Issue of November 21, 1997 pp. 29566-29571
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices

(Received for publication, July 25, 1997, and in revised form, September 13, 1997)

Paula Braun , Bengt Persson ^§ , H. Ronald Kaback ^¶ and Gunnar von Heijne

From the  Department of Biochemistry, University of Stockholm, S-106 91 Stockholm, Sweden; the ^§ Department of Engineering and Natural Sciences, Växjö University, S-351 95 Växjö, Sweden; and ^¶ Departments of Physiology and Microbiology & Molecular Genetics, Howard Hughes Medical Institute, University of California, Los Angeles, California 90095-1662

A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of ~2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.

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