Fourier transform infrared analysis of purified lactose permease: A monodisperse lactose permease preparation is stably folded, α-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 symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from β-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% α-helical and stably folded at 20 °C; thermal denaturation is first detected at 70 °C. Because the purified protein is also readily susceptible to ²H exchange, these results suggest that the protein is conformationally flexible and that ²H exchange is facilitated as the result of conformational fluctuations from the folded state.