The Application of Differential Scanning Fluorimetry in Exploring Bisubstrate Binding to Protein Arginine <i>N</i> ‐Methyltransferase 1
Bibliographic record
Abstract
Background Protein arginine N ‐methyltransferases (PRMTs) methylate polypeptide substrates using a methyl‐donor cofactor S ‐adenosyl‐L‐methionine (SAM). The binding mechanisms of both the cofactor and target substrates have been explored using transient and steady‐state kinetic experiments. However, many of these studies have found conflicting results due to the difficult and sometimes subjective interpretation of kinetic data. Therefore, a robust understanding of the PRMT bisubstrate mechanism can only be achieved through use of complementary structural and biophysical techniques. We hypothesize that thermal shift assays, which are widely used to monitor ligand‐receptor interactions in drug discovery processes, may be applied in a novel way to reveal order of bisubstrate binding to PRMTs. Methods We used differential scanning fluorimetry (DSF) to measure protein melting temperature ( T m ) changes in response to ligand binding. Protein unfolding was monitored using an environmentally sensitive dye SYPRO™ Orange, whose fluorescence increases when exposed to hydrophobic patches of unfolded protein. Cofactor analogues, peptide substrates, and an active site inhibitor were incubated with human PRMT1 to observe their individual and combined effects on enzyme stability; ligands which result in a higher T m interact with and stabilize the enzyme. Results We found that the cofactor analogues induce a positive shift in T m , indicating a stabilizing effect on PRMT1. Conversely, peptide substrates do not stabilize the enzyme and instead lead to a destabilizing, negative shift in T m . Together though, both cofactor and peptide substrate have an additive stabilizing effect on PRMT1. We also found that the active site inhibitor only interacts with PRMT1 in the presence of cofactor and causes a significant positive and stabilizing thermal shift. Conclusions We demonstrate for the first time that DSF can be used to explore the order of substrate binding to enzymes. Our results corroborate other structural, biophysical, and kinetic data which demonstrate that cofactor binding must precede target substrate binding for catalysis to occur. The enzyme:cofactor:substrate complex forms a stable structure that is conducive to methyl transfer. This technique is a valuable complement to kinetic experiments that will contribute to a sound understanding of enzyme kinetic mechanisms.
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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 | 0.001 | 0.001 |
| 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 | 0.000 |
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".