Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling.
Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule [Jung, K., Jung, H., Wu, J., Privé, G. G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys). In this report, these interactions are used to study dynamic aspects of the permease. Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure. Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase. The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide. With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Jung
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K
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Jung
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H
Kaback
H R
HR
D003545:Cysteine / Q000737:chemistry; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011510:Proteolipids; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D027981:Symporters
D003902:Detergents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D003545:Cysteine