Skip to main content
eScholarship
Open Access Publications from the University of California

UCLA

UCLA Electronic Theses and Dissertations bannerUCLA

Expression, Purification and Crystallization Studies of Lactose Permease Mutants

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
For improved accessibility of PDF content, download the file to your device.
Current View