title: Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies.
abstract: Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Anil
authors.initials: A
authors.lastname: Khanal
authors.forename: Yan
authors.initials: Y
authors.lastname: Pan
authors.forename: Leonid S
authors.initials: LS
authors.lastname: Brown
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D003903:Deuterium / Q000032:analysis / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods*; D019617:Halobacterium salinarum / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D019843:Archaeal Proteins; D001436:Bacteriorhodopsins; D003903:Deuterium