Single Amino Acid Mutations Alter the Excitation and Emission of mCherry Fluorescent Protein
Bibliographic record
Abstract
The second-generation fluorescent protein mCherry has an excitation and emission maximum of 587 nm and 610 nm. The discovery of DsRed in Discosoma sp. and the creation of its second-generation variants marked a significant advance in the field of biology due to its use in microscopy. New and improved proteins will increase the diversity of microscopy experiments. The emission of light in mCherry can be attributed to the excitation of electrons held in four amino acids that form a fluorophore: Tyr-67, Gly-68, Arg-95, and Glu-215. The mCherry fluorophore folds via several post-translational modifications which alter the polypeptide backbone to create an imidazolinone ring. In mCherry, an emission red-shift results from protonation of Glu-215 as it shares a hydrogen bond with the nitrogen of the imidazolinone ring. This interaction modifies the distribution of electron density in the fluorophore. The mRFP variant mStrawberry, with an excitation maximum of 574 nm and emission of 596 nm, differs from mCherry by 6 mutations, including M66T and Q213L. These mutations adjacent to the fluorophore contain significant changes in the size, charge and behavior of the amino acids. We expect mutations near the fluorophore to alter the electron distribution controlling the excitation and emission of the fluorophore. In this paper we use site directed mutagenesis to separately induce these mutations into the plasmid bound mCherry protein and express the protein in Rosetta cells. Using a fluorescent spectrophotometer we expect to observe the blue and red shift previously observed after M66T and Q213L mutations in mStrawberry respectively. * Indicates faculty mentor.
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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 | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".