Changes in the Kinetics and Emission Spectrum on Mutation of the Chromophore-Binding Platform in <i>Vibrio harveyi</i> Luciferase
Bibliographic record
Abstract
The recently proposed model for the bacteria luciferase-flavin mononucleotide complex identifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazine ring of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between the isopropyl side chain of alphaVal173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophore represents an important target to test the validity of the proposed model. Here, structure-function analysis of luciferase variants carrying single point mutations at position alpha173 have verified the functional layout of the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease in the stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account for changes in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position alpha173 to increase its polarity confirmed that alphaVal173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of the isoalloxazine chromophore (alphaA75G/C106V) into the alphaV173A, alphaV173C, alphaV173T, and alphaV173S mutants led to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted the emission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emission wavelength spectrum provides the basis toward engineering luciferase variants that emit different light colors with the proposed flavin-luciferase model complex as a design reference.
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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 | 0.001 |
| 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".