title: Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry.
abstract: We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins.
authors.affiliation: Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7.
authors.forename: Yan
authors.initials: Y
authors.lastname: Pan
authors.forename: Leonid
authors.initials: L
authors.lastname: Brown
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D008565:Membrane Proteins; D001436:Bacteriorhodopsins; D010100:Oxygen