title: Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality.
abstract: ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Doreen E
authors.initials: DE
authors.lastname: Culham
authors.forename: Yaroslava
authors.initials: Y
authors.lastname: Vernikovska
authors.forename: Natalia
authors.initials: N
authors.lastname: Tschowri
authors.forename: Robert A B
authors.initials: RA
authors.lastname: Keates
authors.forename: Janet M
authors.initials: JM
authors.lastname: Wood
authors.forename: Joan M
authors.initials: JM
authors.lastname: Boggs
mesh_terms: D000818:Animals; D001692:Biological Transport; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008954:Models, Biological; D008968:Molecular Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D027981:Symporters / Q000737:chemistry / Q000502:physiology*; D014867:Water / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C079244:ProP protein, E coli; D011522:Protons; D027981:Symporters; D014867:Water