title: Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange.
abstract: The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from beta-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% alpha-helical and stably folded at 20 degreesC; thermal denaturation is first detected at 70 degreesC. Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, College of Biological Sciences, University of Minnesota, St. Paul 55108, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Patzlaff
authors.forename: J A
authors.initials: JA
authors.lastname: Moeller
authors.forename: B A
authors.initials: BA
authors.lastname: Barry
authors.forename: R J
authors.initials: RJ
authors.lastname: Brooker
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000302:isolation & purification; D003902:Detergents; D003903:Deuterium / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D027981:Symporters
chemical_list: D000596:Amino Acids; D003902:Detergents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D008823:Micelles; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D003903:Deuterium