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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Khanal, Anil Pan, Yan Brown, Leonid S. Konermann, Lars |
| Description | Country affiliation: Canada Author Affiliation: Khanal A ( Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.) |
| 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. |
| File Format | HTM / HTML |
| ISSN | 10765174 |
| Issue Number | 12 |
| Volume Number | 47 |
| e-ISSN | 10969888 |
| Journal | Journal of Mass Spectrometry |
| Language | English |
| Publisher | Wiley |
| Publisher Date | 2012-12-01 |
| Publisher Place | Great Britain (UK) |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Spectroscopy Discipline Chemistry Techniques Bacteriorhodopsins Chemistry Deuterium Exchange Measurement Methods Spectrometry, Mass, Electrospray Ionization Archaeal Proteins Metabolism Chromatography, Gel Deuterium Analysis Halobacterium Salinarum Models, Molecular Molecular Weight Protein Folding Protein Structure, Secondary Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Spectroscopy Medicine |
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