Open AccessA general method for determining helix packing in membrane proteinsin situ: Helices I and II are close to helix VII in the lactose permease ofEscherichia coli
Author(s) -
Jianhua Wu,
H. Ronald Kaback
Publication year - 1996
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.93.25.14498
Subject(s) - lactose permease , helix (gastropod) , transmembrane domain , residue (chemistry) , stereochemistry , chemistry , protein structure , permease , alpha helix , transmembrane protein , crystallography , biochemistry , escherichia coli , membrane , membrane protein , membrane transport protein , biology , ecology , receptor , snail , gene
It was previously shown that coexpression of the lactose permease ofEscherichia 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 proximityin 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.