title: Evaluation of secondary structure of OxlT, the oxalate transporter of Oxalobacter formigenes, by circular dichroism spectroscopy.
abstract: OxlT, the oxalate/formate exchange transporter of Oxalobacter formigenes, was purified as a histidine-tagged variant, OxlTHis, using Ni2+-linked affinity chromatography. OxlTHis was readily obtained in high purity (>/=95%) and reasonable yield (>/=60%), and showed kinetic and biochemical features characteristic of its parent, OxlT, including an unusually high maximal velocity (60 micromol/min per mg of protein at 4 degrees C). Circular dichroism spectroscopy of purified OxlTHis identified the alpha-helix as its dominant secondary structural unit, encompassing 60-70% of OxlTHis residues and consistent with a model suggesting 60% of OxlT (OxlTHis) residues are involved in the construction of 12 transmembrane alpha-helices (Abe, K., Ruan, Z.-S., and Maloney, P. C. (1996) J. Biol. Chem. 271, 6789-6793). In either octyl glucoside/lipid or dodecylmaltoside/lipid micelles, solubilized OxlTHis showed a striking substrate-induced stabilization of function, and at saturating levels of substrate (1000 x KD) activity recoverable by reconstitution disappeared with a half-life of 7 days at 23 degrees C. Measurement of changes of ellipticity at 222 nm as a function of time and substrate concentration showed that maintenance of function was attributable to a substrate-induced stabilization of the alpha-helical ensemble with a KD of 10 microM for the 1:1 binding of oxalate to OxlTHis.
authors.affiliation: Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
authors.forename: D
authors.initials: D
authors.lastname: Fu
authors.forename: P C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D001426:Bacterial Proteins; D002352:Carrier Proteins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C099549:OxlT protein, bacteria