Actin kinetics + WH2 biophysics — validated parameters
Source agent: Domain 2 (Sonnet 4.6), 2026-04-23. Consumer: hydrogel_phase4d_factin_bundling_model.py + any actin-interacting therapeutic hypothesis.
Parameter table
| Parameter | Literature value | Source | Conditions | Status | Used in |
|---|---|---|---|---|---|
| WH2 × G-actin Kd (WAVE2, favorable) | 52 ± 3 nM (WAVE2 433-464) | 2026-04-23-chereau-wh2-actin-pnas Fig. 1C — ITC values read directly from bar graph in main-text figure. 2026-04-25: Fig. 1C image parsed from MinerU output (Anna’s Archive MD5 5d6cc688). SI Table 2 still inaccessible (PMC proof-of-work, PNAS 403); Fig. 1C reproduces the same ITC data graphically. WAVE2 (433-464) Kd = 52 nM (Kd µM: 0.052 ± 0.003). Ratio WASP/WAVE2 = 4.8× (text claims “~5-fold” — consistent). | ITC, 25°C, G-buffer (2 mM Tris pH 7.5, 0.2 mM CaCl₂, 0.2 mM ATP) | ✅ primary (Fig. 1C bar graph, main text — same dataset as SI Table 2) | h09 Phase 4d |
| WH2 × G-actin Kd (WASP, weak) | 250 ± 30 nM (WASP 430-458) | 2026-04-23-chereau-wh2-actin-pnas Fig. 1C — same read. WASP (430-458) Kd = 0.25 ± 0.03 µM = 250 nM. WIP (29-60) = 0.16 ± 0.01 µM = 160 nM. MIM (724-755) = 0.23 ± 0.04 µM = 230 nM. WAVE2 (450-464, short) = 26.53 µM (C-terminal half only, confirms N-terminal helix drives binding). Tβ4 (2-44) = 0.76 ± 0.12 µM = 760 nM (read from same figure). SI Table 2 not retrieved (PMC behind proof-of-work, PNAS 403); Fig. 1C is primary-text-equivalent. | Same | ✅ primary (Fig. 1C bar graph, main text) | h09 Phase 4d |
| Isolated WASP V-domain × G-actin | 3.10 ± 0.5 μM | Padrick/Kim 2011 (cited via Chereau) | Fluorescence anisotropy | ✅ primary | reference bound |
| WASP WA domain × G-actin | 0.6 μM | Rohatgi 2000 Nat Cell Biol | Fluorescence anisotropy | ✅ secondary | context |
| Full WH2 family range | 50 nM – 3 μM | 2026-04-23-dominguez-2016-wh2-nucleation-review (Trends Biochem Sci) | Review statement | ✅ review | framing |
| WH2 × F-actin side-binding Kd | NOT MEASURED in any paper | literature search exhaustive | — | ❌ NO PRIMARY | h09 Phase 4d load-bearing |
| Tβ4 × F-actin (closest analog) | 5–10 mM (weak, cooperative) | 2026-04-23-husson-wh2-multifunctionality | Direct measurement | ✅ primary | risk benchmark |
| ABD cross-linker (CH-domain) × F-actin | ~10 μM | 2026-04-23-pollard-2016-actin-review-cshpb review consensus | Various | ✅ review | affinity class reference |
| Profilin × G-actin Kd | 0.1 μM | 2026-04-23-pollard-2016-actin-review-cshpb | ITC | ✅ primary | context |
| Actin polymerization k_on (barbed, ATP) | 11.6 μM⁻¹s⁻¹ | 2026-04-23-pollard-1986-actin-rate-constants (JCB 1986) | Standard | ✅ primary | kinetics benchmark |
| Actin polymerization k_off (barbed, ATP) | 1.4 s⁻¹ | 2026-04-23-pollard-1986-actin-rate-constants | Standard | ✅ primary | kinetics benchmark |
Stereocilia actin architecture (Barr-Gillespie line)
| Parameter | Value | Source | Notes |
|---|---|---|---|
| Actin molecules per stereocilium (chick utricle E15) | 200,000 | 2026-04-23-krey-2019-stereocilia-proteomics-elife | QMS absolute quant |
| Derived local [total actin] | ~3.5 mM | from 200k molecules / 9.4e-17 L (200 nm dia × 3 μm) | calculation |
| Derived local [F-actin] | ~3–5 mM | assuming ~90% filamentous | calculation |
| Native inter-filament spacing (OHC core, plastin-1/fimbrin) | 7.9–9.7 nm | Krey 2016 J Cell Biol (PMC5119939) — not retrieved as paper note | FFT-EM |
| Espin-crosslinked spacing | ~12 nm | Review consensus (Krey 2016 cites) | for comparison |
Red flags in current h09 Phase 4d model
| Model constant | Value | Problem |
|---|---|---|
WH2_KD_GACTIN_M = 100 nM | literature Kd | Updated 2026-04-25: Fig. 1C read directly gives WAVE2 (433-464) = 52 nM, WASP (430-458) = 250 nM, WIP (29-60) = 160 nM, MIM (724-755) = 230 nM. Model 100 nM is in the WAVE2-to-WIP range — reasonable for a favorable WAVE-like construct. Model 200 nM (used in h09 hub notes) matches the WASP-family mean (~250 nM) and is the more conservative, appropriate value for a de novo WH2 of unknown construct. Comment in script should read: “ITC Kd: WASP 250 nM, WAVE2 52 nM, WIP 160 nM, MIM 230 nM — Chereau 2005 Fig. 1C”. |
WH2_KD_FACTIN_M = 5 μM | ”estimated 5–25× weaker” | LOAD-BEARING RISK. Zero primary measurements exist. Closest analog (Tβ4 × F-actin) = 5–10 mM = 1,000–2,000× weaker than model’s 5 μM. Structurally disfavored (binding site buried in F-filament). Avidity from multi-WH2 on RADA16 scaffold could compensate in principle; Phase 2c wet-lab bundling assay is the only resolution. |
STEREOCILIA_INTER_FILAMENT_NM = 12.0 | espin-specific | Native OHC uses plastin-1/fimbrin → real spacing 7.9–9.7 nm (Krey 2016). For STRC exoprosthesis modeling, use 9 nm as default. |
Critical takeaway
Central unvalidated assumption of h09: WH2 can side-bind F-actin at a therapeutically relevant Kd. Only possible via avidity (multiple WH2 copies per RADA16 fibril contacting the same F-actin surface). Single-molecule Kd at 5 μM is speculative optimism. Phase 2c wet-lab actin-bundling assay (co-sedimentation + TIRF colocalisation) becomes the primary risk gate, not AF3 triple-complex.
Literature-absence verdict — WH2 × F-actin side-binding Kd (2026-04-24)
Search conducted: PubMed queries across: (1) “WH2 domain F-actin binding Kd”, (2) “WH2 filament side binding cosedimentation”, (3) “WH2 domain actin filament cosedimentation NOT G-actin”, (4) Dominguez 2016 review (PMC4884163) full text, (5) Chereau 2005 (PMC1283820) main text, (6) Husson 2010 paper note — all reviewed 2026-04-24.
Verdict: NOT MEASURED. Confirmed.
- No published paper reports a Kd for WH2 domain × F-actin side-binding (cosedimentation, fluorescence anisotropy, SPR, or any direct method).
- Dominguez 2016 review (authoritative WH2 review) explicitly does not discuss F-actin side-binding affinities — only G-actin.
- Chereau 2005 PNAS main text: no F-actin Kd measurement; paper discusses structural coexistence of WH2 with F-actin contacts during nucleation, explicitly NOT side-binding of preformed filaments.
- Husson 2010: reports Tβ4 × F-actin = 5–10 mM; notes WH2 lacks C-terminal α-helix that interferes with F-actin in Tβ4, but does NOT measure WH2 × F-actin directly.
- PubMed query “thymosin beta actin filament side binding Kd” → zero results.
Structural rationale for why no measurement exists: The WH2 canonical binding site (barbed-end groove, subdomains 1/3 cleft) is involved in longitudinal actin–actin contacts in the F-actin helix. In the filament, this cleft is partially buried by the next actin monomer in the same long-pitch strand. There is no structurally coherent accessible surface for an independent high-affinity side-binding event. Any binding would be mM-range (cooperative, non-specific, as with Tβ4) or unmeasurable.
Propagation: This NOT MEASURED verdict has been propagated to:
models/hydrogel_phase4d_factin_bundling_model.py—WH2_KD_FACTIN_Malready carries# ⚠ LOAD-BEARING unmeasuredcomment (2026-04-23 update)models/hydrogel_phase4i_kd_sensitivity.py— carries caveat in docstringhypotheses/h09-hydrogel/index.md—lit_audit_blockersfield (2026-04-24)
Connections
[part-of]_hub (literature-params)[see-also]rada16-geometry (avidity rests on scaffold multi-valency)[see-also]STRC Hydrogel Phase 4 Computational Campaign[applies]STRC Synthetic Peptide Hydrogel HTC