Secondary structure components and properties of the melibiose permease from Escherichia coli: A Fourier transform infrared spectroscopy analysis

The structure of the melibiose permease from Escherichia coli has been inve stigated by Fourier transform infrared spectroscopy, using the purified tra nsporter either in the solubilized state or reconstituted in E. coli lipids . In both instances, the spectra suggest that the permease secondary struct ure is dominated by alpha-helical components (up to 50%) and contains beta- structure (20%) and additional components assigned to turns, 3(10) helix, a nd nonordered structures (30%). Two distinct and strong absorption bands ar e recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the tra nsporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helica l bands of comparable intensity. In contrast, a single intense band, center ed at similar to 1656 cm(-1), is recorded from the inactive permease in Tri ton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functi onal permease, distinct signals at 1660 and 1653 cm(-1) arise from two diff erent populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the s econdary structure is modified during the early step of sugar transport. Fi nally, the observation that similar to 80% of the backbone amide protons ca n be exchanged suggests high conformational flexibility and/or a large acce ssibility of the membrane domains to the aqueous solvent.