Structural characterization of an integral membrane protein in its natural lipid environment by oxidative methionine labeling and mass spectrometry.
Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here.
Pan
Yan
Y
Department of Chemistry, The University of Western Ontario, London, Ontario N6A5B7, Canada.
Stocks
Bradley B
BB
Brown
Leonid
L
Konermann
Lars
L
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000032:analysis / Q000737:chemistry*; D019617:Halobacterium salinarum / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007834:Lasers; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008715:Methionine / Q000032:analysis / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010782:Photolysis; D053719:Tandem Mass Spectrometry / Q000379:methods
D008051:Lipid Bilayers; D008565:Membrane Proteins; D001436:Bacteriorhodopsins; D008715:Methionine; D006861:Hydrogen Peroxide