Mapping the structure of an integral membrane protein under semi-denaturing conditions by laser-induced oxidative labeling and mass spectrometry.
Little is known about the structural properties of semi-denatured membrane proteins. The current study employs laser-induced oxidative labeling of methionine side chains in combination with electrospray mass spectrometry and optical spectroscopy for gaining insights into the conformation of bacteriorhodopsin (BR) under partially denaturing conditions. The native protein shows extensive oxidation at M32, M68, and M163, which are located in solvent-accessible loops. In contrast, M20 (helix A), M56/60 (helix B), M118 (helix D), M145 (helix E), and M209 (helix G) are strongly protected, consistent with the known protein structure. Exposure of the protein to acidic conditions leads to a labeling pattern very similar to that of the native state. The absence of large-scale conformational changes at low pH is in agreement with recent crystallography data. Solubilization of BR in SDS induces loss of the retinal chromophore concomitant with collapse of the binding pocket, thereby precluding solvent access to the protein interior. Tryptophan fluorescence data confirm the presence of a large protein core that remains protected from water. However, oxidative labeling indicates partial unfolding of helices A and D in SDS. Irreversible thermal denaturation of the protein at 100 degrees C induces a labeling pattern quite similar to that seen upon SDS exposure. Labeling experiments on refolded bacterioopsin reveal a native-like structure, but with partial unfolding of helix D. Our data suggest that noncovalent contacts with the retinal chromophore in native BR play an important role for the stability of this particular helix. Overall, the present work illustrates the viability of using laser-induced oxidative labeling as a novel tool for characterizing structural changes of membrane proteins in response to alterations of their solvent environment.
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
Yan
Y
Pan
Leonid
L
Brown
Lars
L
Konermann
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D008565:Membrane Proteins; D001436:Bacteriorhodopsins