EVALUATION OF SECONDARY STRUCTURE OF OXLT, THE OXALATE TRANSPORTER OFOXALOBACTER-FORMIGENES, BY CIRCULAR-DICHROISM SPECTROSCOPY

OxlT, the oxalate/formate exchange transporter of Oxalobacter formigen es, was purified as a histidine-tagged variant, OxlTHis, using Ni2+-li nked affinity chromatography. OxlTHis was readily obtained in high pur ity (greater than or equal to 95%) and reasonable yield (greater than or equal to 60%), and showed kinetic and biochemical features characte ristic of its parent, OxlT, including an unusually high maximal veloci ty (60 mu mol/min per mg of protein at 4 degrees C). Circular dichrois m spectroscopy of purified OxlTHis identified the a-helix as its domin ant 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 (A be, K., Ruan, Z.-S., and Maloney, P. C. (1996) J. Biol. Chem. 271, 678 9-6793). In either octyl glucoside/lipid or dodecylmaltoside/lipid mic elles, solubilized OxlTHis showed a striking substrate-induced stabili zation of function, and at saturating levels of substrate (1000 x K-D) activity recoverable by reconstitution disappeared with a half-life o f 7 days at 23 degrees C. Measurement of changes of ellipticity at 222 nm as a function of time and substrate concentration showed that main tenance of function was attributable to a substrate-induced stabilizat ion of the alpha-helical ensemble with a K-D of 10 mu M for the 1:1 bi nding of oxalate to OxlTHis.