Evaluation of secondary structure of OxlT, the oxalate transporter of Oxalobacter formigenes, by circular dichroism spectroscopy

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 x K(D)) 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 K(D) of 10 μM for the 1:1 binding of oxalate to OxlTHis.