Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…In contrast to the behavior observed for many watersoluble proteins, it was found that oxidative modifications occurred exclusively at methionine residues, resulting in methionine sulfoxide (MetO) formation [56,57]. While this chemical selectivity is somewhat surprising, it is in line with the fact that sulfur-containing residues are generally most prone to oxidation [15,58] (BR does not contain cysteine).…”

“…SDS induces hydrolytic retinal loss and release of the chromophore into the solvent. We suggested that this step is accompanied by collapse of the chromophore binding pocket and partial unfolding/extrusion of helices A and D. In contrast, helices B, C, E, F, and G were proposed to remain largely intact, although some unraveling at the helix termini could not be excluded [56].…”

“…Unfortunately, considerable uncertainties continue to exist regarding the structural properties of SDS-denatured BR. For example, the extent to which individual helices become unraveled in SDS remains a matter of debate [55,56].…”

“…Our group has previously explored the properties of native and SDS-denatured BR by ·OH labeling [56]. In contrast to the behavior observed for many watersoluble proteins, it was found that oxidative modifications occurred exclusively at methionine residues, resulting in methionine sulfoxide (MetO) formation [56,57].…”

“…SDS solubilization results in marked changes of the BR oxidation pattern, with M20 [A] and M118 [D] becoming significantly more solvent accessible [56]. Based on those ·OH labeling data and the results of UV-Vis and fluorescence experiments we proposed a structural model of SDS-denatured BR.…”