title: 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.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles 90095-1662, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: 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
chemical_list: 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