Purification and Biophysical Characterization of a Minimal Functional Domain and of an N-Terminal Zn<sup>2+</sup>-Binding Fragment from the Human Papillomavirus Type 16 E6 Protein
Bibliographic record
Abstract
The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.
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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".