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Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry,10.1016/j.jmb.2011.04.074,Journa

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry   (Citations: 2)
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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 I1 gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I2. Noncovalent retinal binding to I2 induces folding of helix D, thereby generating an intermediate IR⁎. 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 IR⁎⁎ 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.
Journal: Journal of Molecular Biology - J MOL BIOL , vol. 410, no. 1, pp. 146-158, 2011
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