FLUORESCENCE OF NATIVE SINGLE-TRP MUTANTS IN THE LACTOSE PERMEASE FROM ESCHERICHIA-COLI - STRUCTURAL-PROPERTIES AND EVIDENCE FOR A SUBSTRATE-INDUCED CONFORMATIONAL CHANGE

Six single-Trp mutants were engineered by individually reintroducing e ach of the native Trp residues into a functional lactose permease muta nt devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87: 1638-1642), and fluorescent propertie s were studied with respect to solvent accessibility, as well as alter ations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the p resence of acrylamide only and no quenching by iodide. Of the six sing le-Trp mutants, only Trp 33 exhibits a significant change in fluoresce nce (ca. 30% enhancement) in the presence of the substrate analog beta ,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with p urified single-Cys W33 --> C permease labeled with 2-(4'-maleimidylani lino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behav ior with an apparent K-D of 71 mu M. The effect of substrate binding o n the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled prot ein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change i n acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J. Frillingos S. Kaback HR, 1995a, Biochemistry 34:8257-8 263) that ligand binding induces a conformational change at the C-term inus of helix I such that Pro 28 and Pro 31, which are on one face, be come more accessible to solvent, whereas Trp 33, which is on the oppos ite face, becomes less accessible to the aqueous phase. The findings r egarding accessibility to collisional quenchers are also consistent wi th the predicted topology of the six native Trp residues in the permea se.