- Main
Expression, Purification and Crystallization Studies of Lactose Permease Mutants
- Bajaj, Renata Alexandra
- Advisor(s): Schlinger, Barney A.
Abstract
Lactose permease (LacY) catalyzes oligosaccharide/H+ symport across the membrane. Until recently, all LacY structures demonstrate inward-open conformations with closed periplasmic side. We sought to obtain LacY crystals that diffract at higher resolution and/or provide previously unsolved conformation. The strategy was to employ hybrid proteins available in the laboratory in which cytochromeb562 or flavodoxin-1 of E. coli was fused to LacY. Of the sixteen constructs, large-scale expression/purification of two LacY/cytochromeb562 constructs was successful but crystallization proved difficult. The crystal structure of LacY double-Trp (G46W/G262W) mutant with B-D-galactopyranosyl-1-thio-B-D-galactopyranoside (TDG) was recently reported. This structure has almost occluded, outward-open conformation with a rotation at the thioester linkage in TDG as compared to the previous structures. We obtained crystals of this double-mutant with octyl B-D-galactopyranoside that diffracted to ~20 Å. A mutagenesis strategy is proposed to obtain LacY in fully outward-open conformation. Further, a transport mechanism involving a configurational change(s) in TDG is postulated.
Main Content
Enter the password to open this PDF file:
-
-
-
-
-
-
-
-
-
-
-
-
-
-