Evaluation of Secondary Structure of OxlT, the Oxalate Transporter of Oxalobacter formigenes, by Circular Dichroism Spectroscopy

• Published in: The Journal of biological chemistry 1997-01, Vol.272 (4), p.2129-2135
• Main Authors: Fu, DaXiong, Maloney, Peter C.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins; Carrier Proteins; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Kinetics; Membrane Proteins - chemistry; Membrane Transport Proteins; Oxalobacter formigenes; Protein Structure, Secondary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.4.2129

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 μmol/min per mg of protein at 4°C). Circular dichroism spectroscopy of purified OxlTHis identified the α-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 α-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 × KD) activity recoverable by reconstitution disappeared with a half-life of 7 days at 23°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 α-helical ensemble with a KD of 10 μM for the 1:1 binding of oxalate to OxlTHis.