G3BP1 intrinsically disordered region analysis and phosphorylation effects

PRIMARY OUTCOME

characterization of IDR contributions to LLPS regulation

EXPECTED OUTCOMES

Quantitative predictions: (1) CD spectra: FL G3BP1 shows mixed α/β (NTF2, RRM domains) + random coil (IDRs). ΔAcidic, ΔNTR show increased α-helical content (loss of disordered signal). (2) Phase separation: FL requires 8-12 μM protein + 15 ng/μL RNA for droplets. ΔAcidic and ΔNTR: threshold increases to 15-20 μM (reduced propensity). ΔC-term: no LLPS up to 50 μM (IDR3 essential). (3) Salt sensitivity: FL critical salt ~200 mM. ΔAcidic: critical salt increases to 250-300 mM (less charge-driven). (4) Phosphorylation: S149E or T192E reduces LLPS propensity 40-60% (higher concentration required). Double phosphomimetic (S149E/T192E) additive effect (70-80% reduction). S/T→A mutants hyperstable (droplets at 5 μM). (5) SAXS: FL Rg ~45-50 Å (extended). ΔC-term Rg ~30-35 Å (more compact, loss of IDR3). (6) Mass spec: PKA phosphorylates S149 >90%, CDK1 phosphorylates T192 >80%. Phosphorylated G3BP1 shows 2-3 fold higher critical concentration for LLPS.

SUCCESS CRITERIA

Primary: FL G3BP1 undergoes LLPS (OD600 >0.3 at 10 μM + 20 ng/μL RNA, p<0.001 vs. no RNA). ΔC-term abolishes LLPS (OD600 <0.1 under same conditions, p<0.001 vs. FL). Secondary: (1) ΔAcidic and ΔNTR increase critical concentration >1.5-fold (p<0.01). (2) Phosphomimetics reduce LLPS: S149E or T192E shifts critical concentration >40% higher (p<0.01). (3) CD confirms disorder: IDR peptides show <10% α-helix + β-sheet, >80% random coil. (4) SAXS: Kratky plot shows extended coil (no plateau) for FL, more compact for ΔC-term. (5) Phosphorylation site occupancy >80% by mass spec. (6) Phase diagrams reproducible across 3 protein batches (critical points within 20%). Mechanistic insight: IDR3 essential, IDR1/2 modulatory, phosphorylation inhibits LLPS by charge repulsion.

PROTOCOL

Protein constructs: Recombinant human G3BP1 full-length (FL, aa 1-466) and IDR deletion mutants: ΔAcidic (Δ1-139), ΔNTR (Δ1-231), ΔC-term (Δ366-466). IDR1 (Acidic, 1-139), IDR2 (NTF2-like to RRM spacer, 140-231), IDR3 (C-terminal, 366-466). Express as His-SUMO fusions in E. coli, purify by Ni-NTA + SUMO protease cleavage. Phosphorylation sites: S149, T192 in IDR2; S232 in IDR3. Generate phosphomimetics (S→E, T→E) and non-phosphorylatable mutants (S/T→A). Disorder prediction: Run PONDR-FIT, IUPred2A, AlphaFold2 to confirm IDR regions. Circular dichroism: Measure secondary structure (far-UV CD, 190-260 nm) for FL and mutants. IDRs should show random coil signature (minimum at ~200 nm). Phase separation assays: Mix 5-20 μM G3BP1 with polyU RNA (5-50 ng/μL) in phase separation buffer (150 mM NaCl, pH 7.5, 10% PEG-8000). Image droplets by DIC microscopy, quantify turbidity (OD600), count droplets per field. Vary salt (50-300 mM NaCl) and crowding (0-20% PEG) to map phase diagrams. Phosphorylation effects: Treat recombinant G3BP1 with PKA kinase (phosphorylates S149) or CDK1 (phosphorylates T192) in vitro. Confirm by mass spectrometry (LC-MS/MS, phospho-enrichment). Compare phase behavior of phosphorylated vs. unmodified G3BP1. SAXS: Small-angle X-ray scattering to measure radius of gyration (Rg) and shape. IDRs should have expanded Rg vs. folded domains. n=3 independent protein preps.

LINKED HYPOTHESES

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[G3BP1 intrinsically disordered region analysis and phosphorylation effects](http://scidex.ai/artifact/exp-746b1779-10da-48db-bb4b-02784ddb16cb)

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