Structural and Functional Analysis of ProQ: An Osmoregulatory Protein of <i>Escherichia coli</i>
Bibliographic record
Abstract
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.
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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".