Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation.
Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.
Culham
Doreen E
DE
Department of Microbiology and Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph N1G 2W1, Ontario, Canada.
Hillar
Alexander
A
Henderson
James
J
Ly
Anh
A
Vernikovska
Yaroslava I
YI
Racher
Kathleen I
KI
Boggs
Joan M
JM
Wood
Janet M
JM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D014644:Genetic Variation; D008698:Mesylates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D015202:Protein Engineering / Q000379:methods; D021381:Protein Transport / Q000235:genetics; D011522:Protons; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D002264:Carboxylic Acids; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008698:Mesylates; C436864:Oregon Green 488 carboxylic acid; C079244:ProP protein, E coli; D011522:Protons; D011994:Recombinant Proteins; D027981:Symporters; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine