FOURIER-TRANSFORM INFRARED-ANALYSIS OF PURIFIED LACTOSE PERMEASE - A MONODISPERSE LACTOSE PERMEASE PREPARATION IS STABLY FOLDED, ALPHA-HELICAL, AND HIGHLY ACCESSIBLE TO DEUTERIUM-EXCHANGE

The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose s ymporter. In this study, we have characterized a novel monodisperse, p urified preparation of lactose permease, as well as functionally recon stituted lactose permease, using spectroscopic techniques. The purific ation of monodisperse lactose permease has been aided by the developme nt of a lacY gene product containing an amino-terminal six histidine a ffinity 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-n itriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatograph y. The hydroxyapatite step was shown to be essential in reducing aggre gation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG E) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully rec onstituted and has been shown to be active for lactose transport. Four ier transform infrared (FT-IR) spectroscopy has been performed on mono disperse 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 st ably folded at 20 degrees C; thermal denaturation is first detected at 70 degrees C. Because the purified protein is also readily susceptibl e to H-2 exchange, these results suggest that the protein is conformat ionally flexible and that H-2 exchange is facilitated as the result of conformational fluctuations from the folded state.