Mass spectrometric analysis and mutagenesis predict involvement of multiple cysteines in redox regulation of the skeletal muscle ryanodine receptor ion channel complex
Bibliographic record
Abstract
Evgeniy V Petrotchenko1,2,4, Naohiro Yamaguchi1,3,4, Daniel A Pasek1, Christoph H Borchers1,2, Gerhard Meissner11Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA; 2University of Victoria, Genome BC Proteomics Centre, Victoria, British Columbia, Canada; 3Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA 4Contributed equally to the workAbstract: The tetrameric skeletal muscle ryanodine receptor ion channel complex (RyR1) contains a large number of free cysteines that are potential targets for redox-active molecules. Here, we report the mass spectrometric analysis of free thiols in RyR1 using the lipophilic, thiol-specific probe monobromobimane (MBB). In the presence of reduced glutathione, MBB labeled 14 cysteines per RyR1 subunit in tryptic peptides in five of five experiments. Forty-six additional MBB-labeled cysteines per RyR1 subunit were detected with lower frequency in tryptic peptides, bringing the total number of MBB-labeled cysteines to 60 per RyR1 subunit. A combination of fluorescence detection and mass spectrometry of RyR1, labeled in the presence of reduced and oxidized glutathione, identified two redox-sensitive cysteines (C1040 and C1303). Regulation of RyR activity by reduced and oxidized glutathione was investigated in skeletal muscle mutant RyR1s in which 18 cysteines were substituted with serine or alanine, using a [3H]ryanodine ligand binding assay. Three single-site RyR1 mutants (C1781S, C2436S, and C2606S) and two multisite mutants with five and seven substituted cysteines exhibited a reduced redox response compared with wild-type RyR1. The results suggest that multiple cysteines determine the redox state and activity of RyR1.Keywords: mass spectrometry, mutagenesis, ryanodine receptor, redox modification of cysteines
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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 | 0.000 |
| 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".