Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli

ProlP 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)). Plasmidencoded 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-HiS6 could be purified to homogeneity by nickel chelate affinity chromatography. Sequence-based analysis and modeling suggest that ProQ includes distinct N- and C-terininal domains linked by an unstructured sequence. The N-terminal domain can be modeled on the crystal structure of a-helical RNA binding protein FinO, whereas the C-terminal domain can be modeled on an SH3-like domain (beta-structure). Both ProQ and ProQ-HiS6 appeared to be monomeric, though the higher Stokes radius of ProQ-HiS6 may reflect altered domain interactions. The measured secondary structure content of ProQ (circular dichroism (CD) spectroscopy) contrasted with sequencebased 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.