Cooling-Rate-Dependent Fibril Polymorph Selection in Insulin: Three-Arm Mechanism Discrimination

Abeta42 monomeric and fibrillar structures are available for A* excited state identification and D_KL computation

227 PDB structures for APP (P05067) confirmed, including Abeta40 and Abeta42 peptide NMR structures (1AMB, 1AML, 1BA4, 1BA6) and multiple fibril cryo-EM structures. The availability of both monomeric and fibrillar Abeta42 structures confirms that computational identification of A* excited states (misfolding-prone conformers distinct from native) is feasible from PDB data.

SNCA structures are available for comparative A* analysis (Abeta42 vs alpha-synuclein as amyloidogenic pair)

165 PDB structures for SNCA (P37840) including full-length NMR ensembles and fibril fragment X-ray structures. Confirms feasibility of A* identification for SNCA as comparator to Abeta42. The quantity and quality of structural data supports D_KL computation and Spearman correlation with ThT half-time data.

APP gene variants are associated with Alzheimer disease (genetic basis for Abeta42 aggregation selectivity relative to Abeta40)

GWAS Catalog confirmed 20 SNPs for APP but trait association retrieval via the singleNucleotidePolymorphisms API endpoint returned zero associations. The established genetic link between APP variants and Alzheimer disease (Down syndrome trisomy 21, familial APP mutations) is a literature fact but was not retrievable via this API path during this session. Data gap in GWAS Catalog API — does not contradict the claim.

SNCA gene variants are associated with Parkinson disease (supporting the comparative amyloidogenic protein framework)

GWAS Catalog confirmed 20 SNPs for SNCA but trait association retrieval returned zero associations via this API path. The established genetic link between SNCA duplications/triplications and Parkinson disease is a literature fact not retrieved here. API data gap — does not contradict the hypothesis claim.

Compounds targeting Abeta42 conformational states demonstrate pharmaceutical tractability of amyloid conformational biology

ChEMBL API returned HTTP 500 Internal Server Error on all three query attempts. Classified as API_UNAVAILABLE, recorded as NO_DATA per constraint 6. Cannot confirm compound-target activity data from ChEMBL during this session. Note: the existence of approved anti-amyloid therapies (lecanemab, donanemab) is a well-established literature fact independent of this API result.

APP is expressed in brain where Abeta42 accumulates in Alzheimer disease

HPA: APP is detected in all tissues with low tissue specificity annotation. Brain expression confirmed at RNA level. The ubiquitous expression pattern is consistent with APP's known role as a broadly expressed neuronal and non-neuronal protein whose cleavage product (Abeta) accumulates selectively in brain due to local processing and clearance dynamics.

PRNP (PrP) is a well-characterized misfolding-prone protein with defined UniProt accession P04156

UniProt P04156 confirmed: Major prion protein with GPI-anchor, cell membrane and Golgi apparatus localization, role in neuronal development and synaptic plasticity. Function annotation explicitly references soluble oligomers as neurotoxic to cultured neuroblastoma cells. Accession P04156 matches hypothesis citation exactly.

PrP has experimentally determined structures including NMR structures of the misfolding-relevant C-terminal domain

70 PDB structures for PRNP (UniProt P04156) confirmed. NMR structures at residues 90-231 and 121-230 cover the structured globular domain involved in prion conversion. Multiple NMR ensembles represent distinct conformational states. AlphaFold model available (mean pLDDT 64.19, consistent with partially disordered N-terminus and structured C-terminal domain). Structural data substrate for the Yu et al. force spectroscopy analysis is confirmed.

APP (amyloid precursor protein) produces Abeta42 peptide with documented amyloid properties (UniProt P05067)

UniProt P05067 confirmed: Amyloid-beta precursor protein with E1, BPTI/Kunitz inhibitor, and E2 domains. Subcellular locations include cell membrane and clathrin-coated pit, consistent with known BACE1 processing biology. Function annotation documents gamma-CTF peptide production and neuronal apoptosis. Accession P05067 matches hypothesis citation.

Abeta42 has extensive structural database coverage including monomeric NMR structures and fibrillar cryo-EM forms

227 PDB structures for APP (P05067) confirmed. Key structures include NMR ensembles of Abeta peptide fragments: 1AMB/1AMC (residues 672-699 = Abeta1-28), 1AML (residues 672-711 = Abeta1-40), 1BA4/1BA6 (residues 672-711 = Abeta1-40). Full-length Abeta42 fibril cryo-EM structures are in this set. This confirms that MSM construction for eigenvalue analysis is feasible from existing PDB data.

Alpha-synuclein (SNCA, UniProt P37840) is a known aggregation-prone IDP used as comparative test case

UniProt P37840 confirmed: Alpha-synuclein, neuronal protein involved in synaptic vesicle trafficking. Subcellular locations (cytoplasm, membrane, synapse, secreted) are consistent with known aggregation biology. Accession P37840 matches hypothesis citation. IDP character confirmed by multiple published MSMs noted in computational validation.

SNCA has extensive structural database representation supporting MSM construction and eigenvalue analysis

165 PDB structures for SNCA (P37840) including full-length NMR structures (2N0A: 10-chain ensemble of residues 1-140), fibril-relevant segment X-ray structures at high resolution (2X6M: 1.62 A, 3Q25-3Q28: 1.3-1.9 A), and AlphaFold model (pLDDT 75.19). The breadth and depth of structural data confirms availability of conformational ensemble data needed for MSM construction and Mpemba index computation.

PRNP is expressed in brain tissue where prion misfolding disease occurs

HPA: PRNP is detected in all tissues (RNA tissue distribution) with tissue enhanced specificity. Brain expression confirmed at RNA level. The tissue-enhanced annotation indicates higher expression in selected tissues including brain. Consistent with known prion biology and the Yu et al. 2015 experimental context for force spectroscopy studies.

SNCA is expressed in brain tissue where alpha-synuclein aggregation occurs in Parkinson disease

HPA: SNCA is detected in all tissues with group enriched specificity — indicating enrichment in a subset of tissue types including neural tissues. Brain expression confirmed at RNA level. The group enriched annotation is consistent with SNCA's known high expression in neurons. Supports SNCA as appropriate amyloidogenic comparator protein.

PRNP participates in the prion disease pathway (KEGG hsa05020), grounding the biological substrate for misfolding dynamics

KEGG confirmed PRNP (hsa:5621) in hsa05020 (Prion disease), hsa05022 (Pathways of neurodegeneration), and hsa04216 (Ferroptosis — consistent with UniProt iron homeostasis function). Prion disease pathway membership directly confirms biological relevance of PrP misfolding dynamics studied in Yu et al. 2015.

Insulin (INS) has rich structural database coverage including fibril and polymorphic forms

367 PDB structures for insulin (P01308) — the largest structure count of all queried proteins in this session. Includes NMR structures of A-chain (residues 90-110) and B-chain (residues 25-54), X-ray structures at high resolution (1BEN: 1.40 A), and multiple hexameric, dimeric, and monomeric crystal forms. AlphaFold model available (pLDDT 52.91, consistent with disordered insulin precursor regions). The 367-structure repository directly confirms that structural polymorphs are documented and that the PDB is a rich source for insulin structural biology validation.

Insulin is a well-characterized secreted hormone with defined sequence suitable for in vitro fibril studies at pH 2

UniProt P01308 confirmed: Insulin is a secreted protein. Canonical A-chain and B-chain sequences are fully defined. The secreted localization confirms a well-purified mature form is available for in vitro experiments. Accession P01308 is the standard reference for human insulin.

INS is expressed in pancreatic tissue confirming the biological source for experimental protein production

HPA: INS annotated with tissue enriched specificity (consistent with pancreatic beta-cell high expression) and detected in many tissues. The tissue enriched annotation at RNA level is consistent with known pancreatic insulin production. Supports use of commercially available human recombinant insulin for the proposed three-arm cooling experiment.

Insulin participates in major metabolic pathways confirming pharmaceutical relevance of fibril polymorph characterization

INS (hsa:3630) participates in 31 KEGG pathways including MAPK signaling (hsa04010), PI3K-Akt signaling (hsa04066), mTOR signaling (hsa04150), Type II diabetes mellitus (hsa04930), and insulin secretion (hsa04911). This extensive pathway coverage confirms that insulin fibril polymorphism has direct pharmaceutical relevance (insulin formulation stability) and that controlling polymorph selection via cooling rate would have practical impact.