GROUNDING — h09 hydrogel literature audit
Every passage below was retrieved during this run and is persisted verbatim. Evidential weight is stated per item: STATES = source directly asserts the claim; VARIANT = source is adjacent and a bridging derivation is required (derivation given inline); ABSENCE = source was searched and the value is not there (this is itself evidence).
G1 — WH2 × F-actin side-binding Kd: fresh 2026-08 check
G1a. Local corpus, rag search
Exact query run:
rag search "WH2 domain binds the side of actin filament dissociation constant"
Index path of top hit:
~/Knowledge/BookLibrary/mineru-output/2005_chereau_wh2_actin_pnas/2005-chereau-wh2-actin-pnas/vlm/2005-chereau-wh2-actin-pnas.md
(duplicate copy also indexed under
Actin_bound_structures_of_Wiskott_Aldrich_syndrome_protein_Chereau_D_Kerff_F_Gra/.../vlm/...md)
Verbatim passage (Chereau et al. 2005, PNAS 102(46):16644–16649, doi 10.1073/pnas.0507021102, Results and Discussion, PDF p.6):
“The WH2–actin structures show how WH2 could play a role in both nucleation and elongation (Fig. 4A). The WH2 domains of spire, similar to those in WASP/WAVE, are of the short kind (≈17 aa), and are connected by short linkers, which has two important implications. First, the actin-binding interface of these WH2 domains does not interfere with intersubunit contacts in F-actin and can thereby coexist with the actin filament, as illustrated by a superimposition of a WH2–actin structure onto two consecutive subunits of Holmes’ filament model (32) (Fig. 4B). Second, consecutive WH2 domains must bind actin subunits along the same filament strand because the size of the linkers cannot support connections between actin subunits on opposite filaments strands.”
And (same paper, Results and Discussion, PDF p.5):
“We have established here that WH2 is significantly shorter, in particular in WASP/WAVE (≈17 aa), than Tβ (≈44 aa). The extra amino acids of Tβ form a C-terminal α-helix that adapts this domain for its actin-monomer-trapping function by capping both ends of actin (10), a function that would be at odds with the role of WH2 in filament nucleation (see below).”
Evidential weight: ABSENCE + VARIANT.
- ABSENCE for the audit target: the passage is the paper’s most F-actin-relevant statement and it reports no dissociation constant for WH2 against a preformed filament. The corpus’s most on-target retrieval yields structure, not affinity.
- VARIANT, and a correction to the vault’s prior reasoning: the 2026-04-25 audit justified the literature absence with the argument that the WH2 binding groove is “partially occupied by the next monomer in the long-pitch helix,” so that no coherent side-binding surface exists. Chereau states the opposite geometry — the short WH2 interface does not interfere with F-actin intersubunit contacts and can coexist with the filament. The absence of a measured Kd therefore does not rest on steric impossibility. It rests on the fact that nobody has run the titration. That is a weaker but more honest basis, and it is recorded here rather than smoothed over.
G1b. Web, 2026-08
Exact query run (WebSearch):
WH2 domain F-actin filament side binding dissociation constant Kd measured cosedimentation
Result set returned: Chereau 2005 PNAS; Dominguez 2016 “The WH2 Domain and Actin Nucleation – Necessary but Insufficient” (PMC4884163); Paunola/Quinlan/Lappalainen “The β-Thymosin/WH2 Domain” (Cell); Ducka 2010 Spire WH2 structures (PNAS 10.1073/pnas.1005347107); “Side-binding proteins modulate actin filament dynamics” (eLife 04599); two Rng2 actin-binding-domain preprints; VgrG-1 ACD (bioRxiv 226605).
Verbatim from the search synthesis:
“the search results don’t contain specific measurements of KD values for WH2 domain binding to the side of F-actin filaments using cosedimentation assays. The results focus more on G-actin binding or general WH2 interactions, rather than the specific side-binding to pre-formed F-actin filaments”
The two numeric Kd values surfaced are both G-actin, not F-actin: ~50 nM (VgrG-1 ACD WH2-like domain) and 1.5 µM (ciboulot D1, low ionic strength).
Evidential weight: ABSENCE. No 2026 publication has closed this gap. The eLife “side-binding proteins modulate actin filament dynamics” hit is about cofilin/tropomyosin-class side binders, not WH2 — an off-target neighbour, not a measurement of the audit target.
Combined verdict G1: CONFIRMED NOT MEASURED, re-confirmed 2026-08-21. Cumulative distinct search attempts across the vault’s audit history and this run: 7 (2026-04-24, logged in the prior audit) + 2 (this run, one local corpus, one web) = 9. Well past the 5-attempt stop rule.
G2 — closest measured analog (flagged as ANALOG, never as the measurement)
Source: sources/lit/2026-04-23-husson-wh2-multifunctionality.md (type: lit),
derived from Husson et al. 2010 on WH2 multifunctionality.
Verbatim from the vault note, lines 48–58:
Tβ4 × F-actin (side-binding):
- Kd = 5–10 mM (weak cooperative binding at [Tβ4] > 20 μM) … Implication for WH2 × F-actin:
- WH2 lacks the C-terminal α-helix that interferes with F-actin in Tβ4
- No direct measurement of WH2 × F-actin Kd reported in this paper
- The paper does not support the existence of a low-μM WH2 × F-actin interaction
Evidential weight: VARIANT / ANALOG. Tβ4 is a different domain family
(≈44 aa vs WH2 ≈17 aa) and Chereau (G1a) establishes that the extra Tβ4 residues
form the very α-helix that changes its filament behaviour. The 5–10 mM figure is
therefore a bracketing analog for a related domain, not a WH2 measurement. It
is legitimate as a conservative floor in a sensitivity model — which is exactly
how phase4j_avidity_kd_model.py lines 69–70 use it, labelled
"kd_mono_floor": "Husson 2010 — Tβ4 × F-actin = 5–10 mM (closest measured analog)".
It would be fabrication to report it as WH2’s Kd. It is not so reported anywhere
in the vault, and must not become so.
G3 — derived constant G_TO_F_ACTIN_SCALE = 10_000 (Phase 4m)
Location: hypotheses/h09-hydrogel/scripts/phase4m_iptm_kd_analysis.py:44, with
its own inline derivation at lines 42–43:
# Tβ4 G-actin/F-actin scaling factor: Tβ4 × G-actin = 760 nM, Tβ4 × F-actin = 5–10 mM# → scaling factor = (5e-3 to 10e-3) / 760e-9 = 6,580× to 13,160×. Use mid 10,000×.
Both inputs are primary-sourced and grep-verifiable:
- Tβ4 × G-actin 760 nM →
sources/lit/2026-04-23-chereau-wh2-actin-pnas.md(Fig. 1C ITC table; the same table carries WASP 250 ± 30 nM and WAVE2 52 ± 3 nM, lines 61–62). - Tβ4 × F-actin 5–10 mM →
sources/lit/2026-04-23-husson-wh2-multifunctionality.md:49.
Evidential weight: VARIANT with an explicit bridging derivation. The ratio is measured for Tβ4 and then transposed onto WH2. The transposition assumes that the G→F affinity penalty is a property of the actin surface rather than of the domain — which G1a’s Chereau passage actively undercuts, since the two domains are stated to engage the filament differently. This is the single largest unstated assumption in the h09 Phase 4m chain and is recorded here as an analog bridge, not as a sourced constant.
G4 — constants confirmed grep-verifiable against sources/lit/ this run
| constant | value | file (all type: lit) | grep line |
|---|---|---|---|
| WH2(WASP) × G-actin Kd | 250 ± 30 nM | sources/lit/2026-04-23-chereau-wh2-actin-pnas.md | 61 |
| WH2(WAVE2) × G-actin Kd | 52 ± 3 nM | same | 62 |
| Tβ4 × F-actin Kd (analog) | 5–10 mM | sources/lit/2026-04-23-husson-wh2-multifunctionality.md | 49 |
| C_eff, 3400 Da PEG, r = 39 Å | 13.4 mM | sources/lit/1998-kramer-karpen-polymer-ligand-dimers-nature.md | 61 |
| polyvalent formalism + 10–100× entropic shortfall | Mammen eqn 1.16 | sources/lit/1998-mammen-polyvalent-interactions-angew.md | 38, 68 |
| RWM permeability (TMPA) | 1.9 × 10⁻⁸ cm/s | sources/lit/2001-salt-ma-quantification-rwm-permeability.md | 44 |
| ST clearance t½ | 60 min | same | 45 |
| OHC bundle stiffness ± HTC | 5.12 / 2.05 pN/nm | sources/lit/dulon-2019-htc-bundle-mechanics.md | 39–40 |
Evidential weight: STATES for every row — the value appears verbatim in a
sources/lit/ file carrying canonical type: lit frontmatter. Grep commands and
their output are reproduced in runs/hypc/verify.txt.
G5 — absences carried forward from prior rounds (not re-litigated this run)
Both were established by the 2026-04-25 audit with specific enumerated evidence, and neither is the kind of claim that new literature would silently overturn in four months. They are cited, not re-searched, and that choice is stated openly:
- STRC_NORMAL_OHC_M = 1 µM — CONFIRMED NOT QUANTIFIED. Krey 2019 eLife
(PMC6855842) all supplementary materials checked (MaxQuant output, expression
heatmaps, 186-gene scRNA-seq list, antibody table); STRC absent from all. Chick
utricle dataset; STRC’s HTC role is mammalian-cochlea-specific. Recorded in
sources/lit/2026-04-23-krey-2019-stereocilia-proteomics-elife.md. - HTC links per bundle ≈ 19, per-link stiffness 0.16 pN/nm/link — EXTERNAL DERIVATION, not from the cited paper. Cartagena-Rivera 2019 (PMC6382404; previously misattributed in the vault as “Dulon 2019”) reports the aggregate 3.07 pN/nm HTC contribution but reports no link count and no per-link value. The 19 comes from an architectural assumption (N_links ≈ N_stereo − 1 for a 20-stereocilium OHC). Legitimate as an assumption; not a measurement.