Structural characterization of the osmosensor ProP.
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 (1)H(2)O and (2)H(2)O buffers reveal amide I 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 (2)H(2)O, 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 (2)H(2)O 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.
Department of Biochemistry, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5.
Wajid M H
WM
Sayeed
John E
JE
Baenziger
D055592:Biophysical Phenomena; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D009004:Monosaccharide Transport Proteins / Q000737:chemistry; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011510:Proteolipids; D017550:Spectroscopy, Fourier Transform Infrared; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; C079244:ProP protein, E coli; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D017666:Deuterium Oxide