A general method for determining helix packing in membrane proteins in situ: helices I and II are close to helix VII in the lactose permease of Escherichia coli.
It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease. After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots. A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII. Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54. Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI). The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI. The method should be applicable to a number of different polytopic membrane proteins.
Wu
J
J
Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles 90095-1662, USA.
Kaback
H R
HR
D001426:Bacterial Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D017510:Protein Folding; D027981:Symporters
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease