Structural and functional analysis of ProQ: an osmoregulatory protein of Escherichia coli.
Transporter ProP of Escherichia coli senses extracellular osmolality and responds by mediating cytoplasmic accumulation of organic solutes such as proline. Lesions at the proQ locus reduce ProP activity in vivo. ProQ was previously purified and characterized. Homology modeling predicted that ProQ possesses an alpha-helical N-terminal domain (residues 1-130) and a beta-sheet C-terminal domain (residues 181-232) connected by an unstructured linker. In this work, we tested the structural model for ProQ, explored the solubility and folding of full length ProQ and its domains in diverse buffers, and tested the impacts of the putative ProQ domains on ProP activity in vivo. Limited tryptic proteolysis of ProQ revealed protease resistant fragments corresponding to the predicted N-terminal and C-terminal domains. Polypeptides corresponding to the predicted N- and C-terminal domains could be overexpressed and purified to near homogeneity using nickel affinity, size exclusion and reversed phase chromatographies. Circular dichroism spectroscopy of the purified proteins revealed that the N-terminal domain was predominantly alpha-helical, whereas the C-terminal domain was predominantly beta-sheet, as predicted. The domains were soluble and folded in neutral buffers containing 0.6 M NaCl. The N-terminal domain was soluble and folded in 0.1 M MES (2-[N-morpholino]-ethane sulfonic acid) at pH 5.6. Despite high solubilities, the proteins were not well folded in Na citrate (0.1 M, pH 2.3). The ProQ domains and the linker were expressed at physiological levels, singly and in combination, in bacteria lacking the chromosomal proQ locus. Among these proteins, the N-terminal domain could partially complement the proQ deletion. The full length protein and a variant lacking only the linker restored full activity of the ProP protein.
Smith
Michelle N
MN
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Kwok
Stanley C
SC
Hodges
Robert S
RS
Wood
Janet M
JM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D014357:Trypsin / Q000378:metabolism; D014882:Water-Electrolyte Balance
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C118132:ProQ protein, E coli; D016601:RNA-Binding Proteins; D014357:Trypsin