Replicate-resolved pressure–cooling responses of tobacco mosaic virus coat protein reveal robust volumetric changes and non-dominant conformational redistribution

High hydrostatic pressure can alter protein structure through changes in system volume, hydration, packing, and conformational sampling. We combined a sequential pressure and pressure–cooling molecular-dynamics pathway for tobacco mosaic virus coat protein (TMVcp) with replicate-resolved analysis of Native (1 bar, 300 K), high hydrostatic pressure (HHP; 2500 bar, 300 K), and HHP plus low temperature (HHP+LT; 2500 bar, 255 K). Three independently velocity-initialised 20 ns replicates were analysed per condition after discarding the first 200 ps, yielding 89,109 frames. Relative to Native, mean box volume decreased by 9.15% under HHP and 11.49% under HHP+LT, while density increased by 10.07% and 12.98%. Water occupancy within 0.35 nm increased by 10.73% and 18.45%, respectively; after normalization for bulk densification, the residual increase was near-null for HHP (+0.6%) but persisted for HHP+LT (+4.9%). In pooled Cartesian PCA, PC1–PC2 captured 38.5% of total variance but did not separate same-condition from across-condition replicate pairs (Jensen–Shannon divergence 0.771 versus 0.768; p = 0.476). Condition-associated signal instead concentrated in PC3–PC4, with mean divergence of 0.545 within conditions and 0.773 across conditions; selection-aware exact permutation retained significance (p = 0.0143). Pairwise C-alpha RMSD and backbone-dihedral PCA supported condition-associated differences, although first-half versus second-half divergences indicated incomplete convergence. Endpoint epitope comparisons showed residue-level enrichment for Native and HHP+LT, but not HHP. Overall, TMVcp exhibited robust volumetric responses, condition-dependent near-protein hydration, and reproducible but non-dominant conformational redistribution without implying equilibrium convergence or causal antibody remodelling. These findings distinguish pressure-driven densification from cooling-associated local hydration effects while preserving appropriate mechanistic caution. Multi-panel figure detailing molecular dynamics simulations, PCA analyses, and structural comparisons of TMV coat protein under varying conditions.The figure has three sections. Section 1 shows simulated systems for TMV coat protein: Native, High Hydrostatic Pressure (HHP), and HHP with Low Temperature (HHP+LT). Key parameters like pressure and temperature are represented. Section 2 features replicate-resolved dynamics with PCA scatter plots (PC1, PC2, PC3, PC4), highlighting mixed and distinct clusters across conditions. Histograms and dot plots detail box volume changes and water counts near the protein. Section 3 includes structural models emphasizing epitope prediction areas and provides statistical data on residue overlaps and reactive shares, presented in bar charts.