Probing the Origins of Spectroscopic Responses to Analyte-Induced Conformational Changes in Fluorescently-Labeled Cod III Parvalbumin
Bibliographic record
Abstract
An emerging strategy for the development of reagentless biosensors is the coupling of fluorescence responses to analyte-induced conformational changes within fluorescently-labeled proteins. In this work, we have examined the absorbance and the steady-state and time-resolved fluorescence responses to Ca 2+ -induced conformational changes within cod III parvalbumin (C3P), which was labeled at cysteine-18 with the fluorescent probes fluorescein, acrylodan or nitrobenzoxadiazole (NBD). The basis of the analyte-induced responses was further characterized by examining reporter group accessibility and rotational reorientation dynamics in the presence and absence of analyte. We show that the fluorescence responses are often based on a combination of direct and indirect effects, and that changes in fluorescence quantum yield can be reinforced or opposed by simultaneous changes in absorbance, dramatically affecting the sensitivity of the observed signal to analyte concentration. In the case of NBD, the response is fully consistent with an increase in the nonradiative rate constant owing to increased exposure of the reporter group to solvent as a result of the analyte-induced conformational change. However, in the case of acrylodan and fluorescein, the response reflects a specific interaction between the probe and either the added Ca 2+ or the amino acid residues in the vicinity of Cys-18, which changes in response to protein conformation. Overall, the best performance is obtained from the NBD-labeled C3P, where a signal change of almost 50% was obtained upon complete binding of Ca 2+, providing a detection limit of 100 nM, a dynamic range of 2 orders of magnitude. The other two probes produced poor responses (<10% total change in intensity on binding Ca 2+ ), highlighting the difficulty associated with predicting the behavior of a labeled protein, and showing the complexity that exists at present in the design of sensitive biosensors based on labeled regulatory proteins.
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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".