Abstract
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 approximately 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.
Highlights
Like globular proteins, integral membrane proteins appear to be highly resilient to point mutations, both in their membrane-spanning and extra-membraneous domains [1]
We demonstrated that Ala insertion scanning could be used to identify segments in the glycophorin A transmembrane helix that contribute to the dimerization of this protein in vitro [3]
The insertion into helix X only slightly reduced the lactose permease level in the membrane, and the Ala insertion in helix I resulted in similar levels of lactose permease as seen for the wild-type protein
Summary
Integral membrane proteins appear to be highly resilient to point mutations, both in their membrane-spanning and extra-membraneous domains [1]. A detailed Ala insertion scan of transmembrane helix III, a segment previously shown to be able to accommodate cysteine residues in every position without significant effects on permease expression or activity [13], shows that a central core of ϳ9 residues is crucial for stability and/or activity. Ala insertions outside this central core have surprisingly mild effects, and the distal segments in helix III appear to play only a minor role in supporting protein stability and lactose uptake activity. A Trp scan of the core region identifies residues Ile and Gly as important for the stability of the permease
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