PMID:15476391

Citation	Smith, MN, Crane, RA, Keates, RA and Wood, JM (2004) Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli. Biochemistry 43:12979-89
Abstract	ProP is an osmosensor and osmoregulatory transporter in Escherichia coli. Osmotic activation of ProP is attenuated 5-fold in the absence of soluble protein ProQ, but proQ lesions do not influence proP transcription or ProP levels. The mechanism by which ProQ amplifies ProP activity is unknown. Putative proQ orthologues are found in Gram-negative bacteria (only), but none have known functions. ProQ was overexpressed to low and high levels with and without a C-terminal histidine tag (His(6)). Plasmid-encoded ProQ or ProQ-His(6) complemented in-frame chromosomal deletion DeltaproQ676, restoring ProP activity. After overexpression, both proteins were poorly soluble unless cells were lysed in media of high salinity. ProQ copurified with DNA binding proteins of similar size (HU and a histone-like protein) by ion exchange and exclusion chromatographies, whereas ProQ-His(6) could be purified to homogeneity by nickel chelate affinity chromatography. Sequence-based analysis and modeling suggest that ProQ includes distinct N- and C-terminal domains linked by an unstructured sequence. The N-terminal domain can be modeled on the crystal structure of alpha-helical RNA binding protein FinO, whereas the C-terminal domain can be modeled on an SH3-like domain (beta-structure). Both ProQ and ProQ-His(6) appeared to be monomeric, though the higher Stokes radius of ProQ-His(6) may reflect altered domain interactions. The measured secondary structure content of ProQ (circular dichroism (CD) spectroscopy) contrasted with sequence-based prediction but was as expected if the spectrum of the C-terminal domain is analogous to those reported for SH3 domains. The CD spectrum of ProQ was pH- but not NaCl-sensitive.
Links	PubMed Online version:10.1021/bi048561g
Keywords	Amino Acid Sequence; Carrier Proteins/biosynthesis; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Dimerization; Escherichia coli Proteins/biosynthesis; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Gene Deletion; Genetic Complementation Test; Histidine/genetics; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Osmolar Concentration; Plasmids; Protein Processing, Post-Translational; Protein Structure, Secondary; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Symporters/metabolism
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