Combining protein complementation and resonance energy transfer assays to determine if three different proteins are part of the same G protein signaling complex in living cells
Bibliographic record
Abstract
The N‐ and C‐terminal fragments of yellow fluorescent protein (YN and YC) expressed separately or together are not fluorescent. However, if YN and YC are brought together by fusing them to proteins that associate, a fluorescent protein can be reconstituted in a process called bimolecular fluorescence complementation (BiFC). If this fluorescent protein is close to (<10 nm) a luciferase (Luc) tagged protein, bioluminescence resonance energy transfer (BRET) occurs when the energy produced by Luc is transferred to the fluorescent protein in a process we call BiFC/BRET. G protein‐mediated signal transduction involves the interaction of a receptor, a heterotrimeric G protein and an effector. BiFC/BRET experiments were used to determine if three separate G protein signaling components are part of the same complex in vivo . BiFC occurred when various G protein β and γ subunits tagged, respectively, with YN and YC were co‐expressed and the BiFC was plasma membrane localized. BiFC was detected between tagged Gβ 1–4 and Gγ 2 , Gγ 3 , Gγ 7 and Gγ 11 . YN‐Gβ 1 and YC‐Gγ 2 subunits together acted as an acceptor in BiFC/BRET experiments when co‐expressed with either adenylyl cyclase or the Kir3.1 K + channel subunit tagged with Luc. BiFC/BRET was observed in the absence of a receptor agonist and these interactions were sensitive to receptor stimulation. These results demonstrate the feasibility of using BiFC to identify novel interacting proteins for Gβγ subunits, and of combining BiFC and BRET to demonstrate the simultaneous presence of three signaling proteins in a complex.
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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 | 0.001 | 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".