Chemistry Publications
Site-Directed Mutagenesis Combined with Oxidative Methionine Labeling for Probing Structural Transitions of a Membrane Protein by Mass Spectrometry
Document Type
Article
Publication Date
11-2010
Journal
Journal of the American Society for Mass Spectrometry
Volume
21
Issue
11
First Page
1947
Last Page
1956
URL with Digital Object Identifier
http://dx.doi.org/10.1016/j.jasms.2010.08.004
Abstract
Exposure of the membrane protein bacteriorhodopsin (BR) to SDS induces partial breakdown of the native conformation. The exact structural properties of this SDS state remain a matter of debate, despite its widespread use in BR folding experiments. The current work employs hydroxyl radical (·OH) labeling in conjunction with mass spectrometry (MS)-based peptide mapping for probing the solvent accessibility of individual BR segments in the presence of SDS. Previous work revealed methionine sulfoxide formation to be the dominant oxidative pathway. Those data suggested extensive unfolding of helices A and D in SDS. Unfortunately, the lack of Met residues in helices C and F implies that no direct information on the behavior of the latter two elements could be obtained. Here, we address this problem by employing two variants with additional Met residues, L93M (helix C) and V179M (helix F). The oxidation behavior of the resulting 11 methionines can be grouped into three categories: (1) extensively labeled both in native BR and in SDS (loop residues M32, M68, and M163); (2) protected in the native state but not in SDS (M20, M118); (3) always protected (M56, M60, M93, M145, M179, M209). These data show that a solvent-inaccessible core is retained in SDS. This core consists of partially intact helices B, C, E, F, and G. The termini of these helices are highly dynamic and/or unraveled, particularly on the cytoplasmic side. Overall, this work demonstrates how the use of engineered ·OH labeling sites can provide insights into structural properties of membrane proteins.
