Conformational dynamics of a membrane transport protein probed by H/D exchange and covalent labeling: the glycerol facilitator.
Glycerol facilitator (GF) is a tetrameric membrane protein responsible for the selective permeation of glycerol and water. Each of the four GF subunits forms a transmembrane channel. Every subunit consists of six helices that completely span the lipid bilayer, as well as two half-helices (TM7 and TM3). X-ray crystallography has revealed that the selectivity of GF is due to its unique amphipathic channel interior. To explore the structural dynamics of GF, we employ hydrogen/deuterium exchange (HDX) and oxidative labeling with mass spectrometry (MS). HDX-MS reveals that transmembrane helices are generally more protected than extramembrane segments, consistent with data previously obtained for other membrane proteins. Interestingly, TM7 does not follow this trend. Instead, this half-helix undergoes rapid deuteration, indicative of a highly dynamic local structure. The oxidative labeling behavior of most GF residues is consistent with the static crystal structure. However, the side chains of C134 and M237 undergo labeling although they should be inaccessible according to the X-ray structure. In agreement with our HDX-MS data, this observation attests to the fact that TM7 is only marginally stable. We propose that the highly mobile nature of TM7 aids in the efficient diffusion of guest molecules through the channel ("molecular lubrication"). In the absence of such dynamics, host-guest molecular recognition would favor semipermanent binding of molecules inside the channel, thereby impeding transport. The current work highlights the complementary nature of HDX, covalent labeling, and X-ray crystallography for the characterization of membrane proteins.
Department of Chemistry, The University of Western Ontario, London, ON, Canada N6A 5B7.
Yan
Y
Pan
Hadeesha
H
Piyadasa
Joe D
JD
O'Neil
Lars
L
Konermann
D000595:Amino Acid Sequence; D020346:Aquaporins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation
D020346:Aquaporins; D029968:Escherichia coli Proteins; C078043:GlpF protein, E coli