Overexpression, Purification, and Characterization of ProQ, a Posttranslational Regulator for Osmoregulatory Transporter ProP of <i>Escherichia coli</i>
Bibliographic record
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".