Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
Bibliographic record
Abstract
EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd) and maximum specific one-site binding (Bmax) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".