Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

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

“…The expected masses of intact wt BR, L93M BR, and V179M BR based on the amino acid sequences (including retinal) were calculated as 27,050 Da [59], 27,068 Da, and 27,082 Da, respectively. Low-temperature SEC/ESI-MS analysis [60] of all three protein variants confirmed these theoretical mass values to within Ϯ 1 Da. The amino acid substitutions of the two mutant proteins were further verified by tryptic peptide mapping and ESI-MS/MS (data not shown).…”

“…SDS denaturation dramatically enhances the degree of HDX, resulting in 75% deuteration within the same time interval (Figure 2b). A detailed analysis of these isotope labeling data is beyond the scope of the current work and has been reported elsewhere [60]. Unfortunately, applying the traditional proteolytic digestion/HDX-MS approach to BR is difficult [68], and hence we were unsuccessful in analyzing the BR deuteration behavior in a spatially resolved manner.…”

“…The results presented here are consistent with a scenario where this residual helicity is caused by partially intact helices B, C, E, F, and G. However, HDX measurements suggest that these helices are significantly more dynamic than in native BR. Specifically, it was found that the number of highly protected backbone hydrogens in SDS drops to one third of the native state value [60] (see also Figure 2b). The oxidative labeling data presented here imply that the central regions of the helices where most methionines are located remain fairly stable, such that HDX must be predominantly mediated by helix fraying at the termini.…”

“…Aliquots were removed at various time points, followed by acid quenching and flash freezing [60]. Zero time point controls (m 0 ) were performed by exposing the samples to a mixture of labeling and quenching buffer.…”

“…From previous work it is known that SDS disrupts the native purple membrane and solubilizes individual protein molecules in monomeric form [69,70]. Furthermore, SDS exposure results in hydrolysis of the Schiff base and release of the chromophore into the solvent [60].…”