A photochemical approach to the lipid accessibility of engineered cysteinyl residues.
Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replaced with cysteine, and the mutants were expressed in Escherichia coli and isolated in inner membranes. These membranes were dissolved in detergent and reconstituted into asolectin liposomes incorporating INA. The extent of light-induced reaction of INA with each cysteine was assayed by subsequent reaction with the gel-shifting, SH-specific methoxy-polyethylene glycol-2-pyridine disulfide. The pattern of apparent second-order rate constants for the photoreactions of eight substituted cysteines in the N-terminal helix conformed to other measures of lipid exposure. The pattern of the rate constants for the photoreactions of 15 cysteines in the first transmembrane helix had peaks every third residue, which partly conformed to other measures of lipid exposure.
Li
Jing
J
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032.
Shi
Lei
L
Karlin
Arthur
A
D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008027:Light; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D008958:Models, Molecular; D016296:Mutagenesis; D010777:Photochemistry; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds
D001386:Azides; D001426:Bacterial Proteins; D004220:Disulfides; D008055:Lipids; D008081:Liposomes; D015221:Potassium Channels; D013438:Sulfhydryl Compounds; C107790:prokaryotic potassium channel; D008623:Mercaptoethanol; C015298:iodonaphthylazide; D003545:Cysteine; D011188:Potassium