title: Transmembrane helix I and periplasmic loop 1 of Escherichia coli ProP are involved in osmosensing and osmoprotectant transport.
abstract: Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter. ProP is regulated by osmotic pressure via a membrane potential-dependent mechanism. A homology model predicts that ionizable and polar residues, highly conserved among ProP homologues, cluster deep within the N-terminal helix bundle of ProP. Chemical labeling of introduced cysteine (Cys) residues supported the homology model by confirming the predicted positions of transmembrane helix I (TMI) and periplasmic loop 1. Replacements of residues in the putative polar cluster impaired or altered ProP function, suggesting that they are important for osmosensing and may interact with the transport substrates. Asn34, Glu37, Phe41, Tyr44, and Ala48 line the most polar face of TMI; Tyr44 is on the periplasmic side of the putative polar cluster, and Ala59 is in periplasmic loop 1. The N-ethylmaleimide reactivities of Cys introduced at positions 41, 44, 48, and 59 increased with osmotic pressure, whereas the reactivities of those at cytoplasm-proximal positions 34 and 37 did not. Replacements of polar cluster residues that blocked transport also affected the NEM reactivity of Cys44 and its osmolality dependence. This report and previous work suggest that conformational changes associated with osmosensing may shift the equilibria between outward- and inward-facing transport pathway intermediates.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 488 Gordon Street, Guelph, Ontario, Canada.
authors.forename: Robert A B
authors.initials: RA
authors.lastname: Keates
authors.forename: Doreen E
authors.initials: DE
authors.lastname: Culham
authors.forename: Yaroslava I
authors.initials: YI
authors.lastname: Vernikovska
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Zuiani
authors.forename: Joan M
authors.initials: JM
authors.lastname: Boggs
authors.forename: Janet M
authors.initials: JM
authors.lastname: Wood
mesh_terms: D001692:Biological Transport / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009995:Osmosis / Q000502:physiology; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C079244:ProP protein, E coli; D027981:Symporters; D011392:Proline