Temporal Resolution of Activity-Related Solvation Dynamics in the TIM Barrel Enzyme Murine Adenosine Deaminase
Bibliographic record
Abstract
Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen–deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein–solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent-exposed helix–loop–helix moiety that presents the hydrophobic face of its long α-helix to the backside of the bound substrate. Herein, we pursue the time and temperature dependence of solvation dynamics at the surface of mADA for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with a close-to-native kinetic behavior. Single site-specific tryptophan mutants were back-inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays that reflect time-dependent environmental reorganization around the photoexcited state of Trp* display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent-exposed region near the helix–loop–helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for E a ( k Stokes shift ); further, when Phe61 within the long helix positioned behind the bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in E a ( k Stokes shift ) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, E a ( k cat ). The reported experimental findings implicate a solvent-initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.
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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".