Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1788, Issue 5, Pages 1108-1115Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2009.01.010
Keywords
Membrane protein; Osmosensing; Fourier-transform infrared spectroscopy; Major facilitator superfamily; ProP; Lac permease
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ProP, an osmoprotectant symporter from the major facilitator superfamily was expressed, purified and reconstituted into proteoliposomes that are amenable to structural characterization using infrared spectroscopy. Infrared spectra recorded in both (H2O)-H-1 and (H2O)-H-2 buffers reveal amide 1 band shapes that are characteristic of a predominantly alpha-helical protein, and that are similar to those recorded from the well-characterized homolog, lactose permease (LacY). Curve-fit analysis shows that ProP and LacY both exhibit a high alpha-helical content. Both proteins undergo extensive peptide hydrogen-deuterium exchange after exposure to (H2O)-H-2, but are surprisingly thermally stable with denaturation temperatures greater than 60 degrees C. 25-30% of the peptide hydrogens in both ProP and LacY are resistant to exchange after 72 h in (H2O)-H-2 at 4 degrees C. Surprisingly, these exchange resistant peptide hydrogens exchange completely for deuterium at temperatures below those that lead to denaturation. Our results show that ProP adopts a highly alpha-helical fold similar to that of LacY, and that both transmembrane folds exhibit unusually high temperature-sensitive solvent accessibility. The results provide direct evidence that ProP adopts a structure consistent with other major facilitator superfamily members. (C) 2009 Elsevier B.V. All rights reserved.
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