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. 2026 Aug 22;13:101541. doi: 10.1016/j.crfs.2026.101541

Investigating the structural changes of myosin under high pressure processing by molecular dynamics simulations

Xiaoyun Wang a,1, Jie Tang a,1, Hosahalli S Ramaswamy b, Hanying Duan a, Chao Wang a,⁎
PMCID: PMC13544339  PMID: 42699739

Abstract

High-pressure processing (HPP) exerts paradoxical effects on myosin, simultaneously promoting molecular unfolding and supramolecular aggregation, yet the underlying mechanisms remain unresolved at atomic resolution. This study employed all-atom molecular dynamics simulations at gradient pressures (0.1-450 MPa) using complementary monomer and motor domain contact models. The monomer exhibited pressure-induced disruption of intramolecular hydrogen bonds, increased per-residue flexibility (RMSF) and radius of gyration (Rg), and expanded the free energy landscape (FEL) toward higher-energy states, demonstrating a “local loosening–global stability” mechanism that exposes buried binding sites without global denaturation. The contact model, in contrast, underwent progressive interfacial compaction, evidenced by restricted Rg expansion, SASA reduction, and a broadened free energy landscape spanning diverse metastable sub-basins, yet the nature of this compaction shifted markedly with pressure: at 150 MPa, a specific, hydrophobically stabilized interface with favorable binding affinity was formed, whereas at ≥300 MPa this ordered packing collapsed, giving way to non-specific, electrostatically dominated, volume-constrained aggregation despite persistent structural compaction. These findings suggest that myosin functionality is governed by a shift from site-exposing monomer unfolding at moderate pressures (≤150 MPa) to the progressive collapse of specific interfacial packing and the onset of non-specific, volume-constrained aggregation at extreme pressures (≥300 MPa).

Keywords: Myosin, Molecular dynamics simulation, High-pressure processing, Binding free energy, Protein conformational dynamics

Graphical abstract

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Highlights

  • •

    All-atom MD simulations reveal a dual-response mechanism of myosin under HPP.

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    Pressure disrupts hydrogen bonds, inducing local loosening in monomers.

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    HPP drives myosin dimer compaction via hydrophobic interfacial dehydration.

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    Monomer unfolding exposes binding sites; dimer compaction occludes under HPP.

1. Introduction

Myofibrillar proteins (MPs), which account for 50–60% of the total protein content in animal skeletal muscle, are predominantly composed of myosin (50–55%) and actin (20–25%), and serve as the principal structural determinants governing the physicochemical and sensory properties of processed meat products (Tong et al., 2018; Ren et al., 2023). As the dominant functional component of MPs, myosin directly governs gel-forming capacity, emulsification behavior, and water-holding performance through its characteristic molecular flexibility and surface hydrophobicity, making it a primary target for processing-induced functional modification (Liu et al., 2021; Xu and Xu, 2021). High-pressure processing (HPP), operating within the range of 100–1000 MPa, has emerged as a promising non-thermal technology capable of modulating protein conformation without compromising heat-sensitive nutrients or volatile flavor compounds (Ekonomou and Boziaris, 2021). Unlike conventional thermal treatments, which often induce uncontrolled denaturation, HPP acts by selectively disrupting non-covalent interactions, including hydrogen bonds and hydrophobic associations, thereby enabling tunable modification of protein structure and functionality (Queirós et al., 2018; Zhang, 2025; Wang et al., 2025; Lin et al., 2026). This capacity to achieve targeted structural remodeling without chemical additives makes HPP an ideal platform for engineering advanced myosin-based systems with enhanced functional performance.

This study is the computational mechanistic companion to a recently published experimental study on HPP-treated chicken myofibrillar proteins (Wang et al., 2025). That study documented a biphasic structural and functional response to HPP: moderate pressure (≤150 MPa) induced partial unfolding of secondary structure, progressive exposure of hydrophobic residues and reactive sulfhydryl groups, and enhanced emulsifying performance, whereas pressures ≥ 300 MPa triggered extensive intermolecular aggregation via hydrogen bonding and disulfide crosslinking, impairing solubility and emulsification. Although these experimental findings map the macroscopic consequences of HPP on myosin structure and functionality, they cannot identify which specific intramolecular interactions are disrupted at each pressure threshold, nor explain why the same physical stimulus simultaneously drives molecular unfolding and dimer (hereafter referred to as the motor domain contact model) compaction. Conventional spectroscopic methods yield population-averaged signals that reflect thermodynamic end-states rather than dynamic conformational trajectories, leaving the atomic-level mechanisms unresolved. The present study therefore employs all-atom molecular dynamics simulations at gradient pressures (0.1–450 MPa) to provide the mechanistic framework that the experimental observations of Wang et al. (2025) necessitate but cannot be supplied by itself.

Resolving this paradox demands mechanistic insight at a resolution beyond what conventional experimental techniques can provide. Spectroscopic methods such as CD, intrinsic fluorescence, and UV-visible absorption spectroscopy yield population-averaged, time-integrated signals that reflect thermodynamic end-states rather than the dynamic conformational trajectories by which they are reached (Schuler and Eaton, 2008; Lu, 2005; Kelly et al., 2005). These ensemble measurements are inherently blind to transient intermediate states, site-specific structural rearrangements, and the atomistic mechanisms by which individual non-covalent interactions are sequentially disrupted or formed under pressure (Schuler and Eaton, 2008; Nijhawan et al., 2021; Dubois et al., 2020). Furthermore, protein structures deposited in crystallographic databases represent native-state conformations determined under ambient conditions, which cannot serve as accurate structural templates for probing the non-native conformational landscapes populated under extreme pressure (Berman et al., 2000). These combined limitations leave the most critical atomic-level questions unresolved: which intramolecular hydrogen bonds are preferentially ruptured under increasing pressure, and which surface residues mediate the intermolecular contacts that initiate aggregation? These questions define the core mechanistic “black box” in our current understanding of HPP-induced protein remodeling, a gap that can only be bridged by a computational approach capable of atomic-resolution, time-resolved structural analysis.

Molecular dynamics (MD) simulation is a precise computational tool, offering the unique ability to resolve protein conformational dynamics at atomic resolution across biologically relevant timescales under extreme conditions, such as high pressure (Hollingsworth and Dror, 2018; Hata et al., 2020). By numerically integrating Newton's equations of motion for all atoms in a solvated protein system, using established force fields such as ff19SB and realistic solvent models, MD simulation can faithfully reproduce the physical effects of extreme hydrostatic pressure through NPT ensemble simulations with tunable barostats (Case et al., 2005; Tian et al., 2019). Unlike experimental methods constrained by accessible length and time scales, MD can track the precise trajectories of every backbone atom and side-chain, quantify the evolution of hydrogen bond networks, and map the free energy landscape governing conformational transitions (Hollingsworth and Dror, 2018; Sinha et al., 2022). Critically, this computational approach allows the generation of realistic, pressure-modified structural templates that reflect the actual non-native conformational states of proteins under HPP (Hata et al., 2020; Paci, 2002). These structures are experimentally inaccessible through conventional structural databases, which contain only native protein conformations determined under ambient conditions, and cannot account for pressure-induced structural modifications observed experimentally, but are essential for understanding binding mechanisms (Yamada et al., 2015; Huang et al., 2021; Robeson et al., 2026). Therefore, MD simulations become indispensable for creating structural templates that reflect the actual conformational states of proteins under HPP conditions, enabling subsequent molecular docking analysis to elucidate specific binding mechanisms.

Building on these experimental and computational foundations, the present study advances a unified “Dual-Response Mechanism” hypothesis: HPP exerts qualitatively distinct and opposing structural effects depending on the level of protein organization examined. At the intramolecular (monomer) level, pressure-induced disruption of internal hydrogen bonds drives a “local loosening-global stability” response, wherein specific structural domains gain conformational freedom and expose previously buried residues while the overall protein fold is preserved. At the intermolecular (motor domain contact model) level, pressure promotes a hydrophobic interaction-driven “interfacial compaction” response, wherein the contact surface between adjacent molecules becomes progressively buried, structured, and stabilized. To test this hypothesis and characterize the associated molecular mechanisms, a myosin monomer model was constructed to probe intramolecular mechanical responses in isolation, specifically, to characterize how applied pressure disrupts the internal hydrogen bond network, induces local structural loosening, and drives the protein toward higher-energy conformational ensembles while preserving global fold integrity. In parallel, a myosin motor domain contact model was constructed to investigate intermolecular interactions under compression, elucidating how pressure overcomes steric barriers to drive interfacial compaction, enhance hydrophobic burial, and stabilize contact-model complexes in progressively lower-energy, more compact states. Together, these two complementary models provide a systems-level framework for mechanistically dissecting the unified, pressure-dependent biphasic response observed in macroscopic experiments (Wang et al., 2025). Gradient pressure conditions spanning 100KPa to 450 MPa were systematically applied in MD simulations, and the resulting conformational dynamics were comprehensively analyzed through root mean square deviation (RMSD) to track overall structural stability, root mean square fluctuation (RMSF) to identify pressure-sensitive flexible regions, radius of gyration (Rg) to quantify global structural compactness, solvent-accessible surface area (SASA) to monitor interfacial burial in the contact-model system, intramolecular hydrogen bond counting to link pressure to secondary structure disruption mechanistically, and free energy landscape (FEL) analysis to map the free energy distribution of accessible conformational states (Dong et al., 2021; Yekeen et al., 2023). The quantitative insights derived from these analyses not only establish a rigorous atomic-level theoretical foundation for understanding HPP effects in food protein systems but also provide mechanistically grounded guidance for the rational design of non-thermal processing protocols aimed at precisely engineering myosin gel structures, aggregation behavior, and ligand-binding properties in next-generation meat product applications.

2. Methods

2.1. System construction and pre-processing

The myosin crystal structure (PDB ID: 6YSY) was retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/). This structure represents skeletal muscle myosin motor domain in complex with the small-molecule inhibitor MPH-220 and the Mg·ADP·VO4 transition-state mimic, determined by X-ray crystallography at 3.25 Å resolution. MPH-220, ADP, VO4, and the Mg2+ ion were removed from the crystallographic coordinates prior to system construction, yielding the apo motor domain used in all subsequent simulations. The MD simulation system was constructed using AMBER 24. The protein was parameterized using the ff19SB force field, and the solvent environment was modeled using the TIP3P water model for all-atom simulation.

To simulate physiological ionic conditions, Na+ and Cl− counterions were added to a final concentration of 0.15 M using a Monte Carlo-based algorithm to achieve charge neutrality. Following system construction, energy minimization was performed for 5000 steps using the conjugate gradient method to eliminate local steric hindrance between atoms. The system was then gradually heated from 0 K to 298.15 K through a 500 ps linear heating process under the NVT ensemble, followed by pre-equilibration at this temperature to stabilize the thermodynamic state. The SHAKE algorithm was applied throughout all simulations to constrain hydrogen-containing bond vibrations, while periodic boundary conditions were employed to maintain solvent continuity, with an integration time step fixed at 2 fs.

2.2. Simulation of myosin monomer structural changes under HPP

To investigate the regulatory mechanism of HPP on myosin conformation, gradient high-pressure conditions were established under the NPT ensemble. The control group was maintained at standard atmospheric pressure (1.0 bar), while the high-pressure experimental groups were set at 150 MPa (1500 bar), 300 MPa (3000 bar), and 450 MPa (4500 bar). After pre-equilibration, MD simulations were conducted for 100 ns, with temperature maintained at 298.15 K using a Langevin thermostat.

2.3. Simulation of myosin motor domain contact model structural changes under HPP

To simulate the intermolecular interactions that drive pressure-induced macroscopic aggregation, a pre-aggregation intermediate model comprising two adjacent myosin motor domains was constructed. Although physiological myosin dimerization is naturally mediated by its tail coiled-coil region, modeling the direct contact between motor domains provides a necessary structural template for understanding the early stages of non-specific supramolecular aggregation under extreme hydrostatic pressure. A restricted docking strategy was employed, designating the two myosin molecules as the receptor and ligand, respectively. Based on AlphaFold-Multimer prediction module, the initial interaction model was generated through multiple sequence alignment and three-dimensional conformational space searching, with a confidence score (pLDDT) threshold set at >80 to ensure prediction reliability. All-atom energy optimization of the complex was performed using Amber 24 software: first, 5000 steps of the steepest descent method were executed in vacuum to eliminate atomic overlaps, followed by 10,000 steps of refined energy minimization using the conjugate gradient algorithm in an implicit solvent model (GB-OBC2). The ff19SB force field was used throughout the optimization process to parameterize protein backbone torsion angles, side-chain dihedral angles, and electrostatic interactions. Pressure conditions applied to the motor domain contact model simulations were consistent with those described for the monomer system above.

2.4. Hydrogen bonding, hydrophobic contact, interfacial residue, contact map, and binding free energy analyses

To further characterize the pressure-dependent structural stability and inter-subunit association of the myosin motor domain contact model, six additional trajectory-based analyses were performed using CPPTRAJ (AmberTools, v22–24) on representative simulation frames sampled at 10 ns intervals from each 100 ns production trajectory.

The total number of intramolecular and inter-chain hydrogen bonds was computed for each frame using the CPPTRAJ hbond command, with donor–acceptor pairs identified based on standard geometric criteria (distance ≤ 3.5 Å, angle ≥ 120°). The frame-wise hydrogen bond count was summed across all identified pairs to obtain the total hydrogen bond number as a function of simulation time, and reported as the mean ± standard deviation over the sampled frames.

Interfacial hydrophobic contacts between the two myosin subunits (heavy chain plus essential light chain; chain A: residues 1–922, chain B: residues 923–1844) were identified using the nativecontacts command, restricted to heavy atoms of hydrophobic residues (Ala, Val, Leu, Ile, Pro, Phe, Met, Trp) on each chain, with a distance cutoff of 4.5 Å. Both native (first-frame reference) and non-native (newly formed) contacts were included (savenonnative option) to capture the dynamic formation of the hydrophobic interface over the course of the simulation.

Interfacial residues were defined as residue pairs from the two subunits with any heavy-atom distance below 4.5 Å, identified using the nativecontacts command applied between the full residue ranges of chain A and chain B. Residue pairs were ranked by contact occupancy (fraction of sampled frames in which the contact was present) to determine the most persistent interfacial residues.

Residue–residue contact maps were generated using the CPPTRAJ matrix command with the dist and byres options, producing a symmetric 1844 × 1844 distance matrix averaged over all sampled frames, visualized as a two-dimensional heat map with the chain A/chain B boundary indicated.

Binding free energies between the two subunits were estimated using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method implemented in MMPBSA.py (AmberTools), using the GB-Neck2 model (igb = 8) with an ionic strength of 0.15 M. Receptor and ligand topologies were generated from the complex topology using ante-MMPBSA.py. Per-residue energy decomposition (idecomp = 1) was performed to identify individual residue contributions to the total binding free energy. Reported binding free energies (ΔGbind = Gcomplex − Greceptor − Gligand) represent enthalpic contributions only (molecular mechanics energy plus GB/SA solvation free energy), without entropic correction, consistent with common practice for computationally efficient relative comparison across simulation conditions.

Hydrogen bond counts, hydrophobic contact numbers, and interfacial residue pairs are reported as mean ± standard deviation (SD) across sampled frames. Because MM-GBSA binding free energies estimated from correlated trajectory frames are sensitive to autocorrelation, ΔGbind values are instead reported as mean ± standard error of the mean (SEM).

3. Results and discussion

3.1. The effect of HPP on myosin monomers

3.1.1. RMSD analysis

The conformational stability of myosin monomers under varying pressure conditions (150, 300, and 450 MPa) was systematically evaluated through analysis of structural parameters. RMSD analysis of the protein backbone relative to its initial conformation demonstrated that although pressure induced measurable structural perturbations, the overall protein fold remained largely preserved (Fig. 1-(a)). All pressure groups exhibited a cumulative increase in RMSD values throughout the 100 ns simulation period, confirming a positive correlation between pressure intensity and structural disruption. Importantly, the structural responses exhibited nonlinear pressure dependence. While the 150 MPa and 450 MPa groups remained within a relatively constrained RMSD range, the 300 MPa group experienced a pronounced conformational leap in the later stages of the simulation, with RMSD exceeding 6.0 Å. This distinct dynamic behavior suggests that 300 MPa acts as a critical thermodynamic threshold for the myosin monomer. At this critical point, pressure-induced water penetration likely triggers the cooperative unfolding of specific susceptible structural domains. Conversely, at 450 MPa, the immense hydrostatic pressure exerts an overriding volume-minimization effect, which paradoxically restrains further spatial expansion and locks the denatured protein into a compressed state, preventing catastrophic global unfolding.

Fig. 1.

Fig. 1

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Conformational stability and structural dynamics of the myosin monomer under gradient HPP treatments. (a) Time-dependent root-mean-square deviation (RMSD) profiles of the protein backbone; (b) Residue-specific root-mean-square fluctuation (RMSF) values across the 922-residue monomeric chain; (c) Time-dependent radius of gyration (Rg) trajectories indicating global spatial extension; (d) Evolution of intramolecular hydrogen bond counts over the 100 ns production trajectories.

Note: Simulations were performed under the NPT ensemble at a constant temperature of 298.15 K under four hydrostatic pressure conditions: 100KPa (cyan), 150 MPa (blue), 300 MPa (orange), and 450 MPa (red). Intramolecular hydrogen bonds in (d) were identified using CPPTRAJ based on standard geometric criteria (donor–acceptor distance ≤ 3.5 Å and donor–hydrogen–acceptor angle ≥ 120°).

The convergence of RMSD values across all pressure groups reflects a characteristic “local loosening-global stability” behavior, in which proteins accommodate energetic stress by reorganizing local structure without collapsing their overall fold. This pattern is consistent with the compressibility principle described under Le Chatelier's law: moderate hydrostatic pressure selectively disrupts weaker non-covalent interactions while the covalent backbone and globally folded architecture are preserved (Baldelli et al., 2024). Liu et al. (2021) similarly reported that HPP treatment of pork myofibrillar proteins elevated surface hydrophobicity and free sulfhydryl content without inducing complete unfolding. The stable RMSD plateau observed here is consistent with these macroscopic findings and with the preservation of overall protein composition documented by SDS-PAGE in Wang et al. (2025), corroborating at atomic resolution that moderate HPP induces local structural perturbation without catastrophic global unfolding.

3.1.2. RMSF analysis

While global structural stability was preserved, local protein dynamics were significantly altered by HPP treatment. RMSF analysis of individual amino acid residues revealed distinct pressure-sensitive regions exhibiting enhanced flexibility (Fig. 1-(b)). Under ambient pressure conditions, most residues displayed low RMSF values (typically < 3.0 Å), indicative of stable conformations. Upon application of 150 MPa, some residues (residues 1-20) showed marked increases in fluctuation amplitude. For instance, residue 1 exhibited an RMSF increase from 2.71 Å to 3.15 Å, while some regions around residue 300 showed slight decreases (e.g., from 1.06 to 1.04 Å), suggesting localized compaction effects. This trend was amplified at higher pressures, with residue 1 reaching an RMSF of 4.30 Å at 450 MPa, indicating that elevated pressure significantly enhanced conformational freedom in specific structural domains, likely by disrupting local stabilizing interactions.

The site-specific pattern of RMSF changes, which shows simultaneous local loosening in the terminal and loop regions alongside minor rigidification near residue 300, reflects the heterogeneous mechanical response of myosin's complex domain architecture to hydrostatic compression. This observation aligns with the general understanding that flexible loop regions and terminal segments are more susceptible to pressure-induced perturbation than structured core domains, owing to their lower packing density and fewer stabilizing contacts. This is consistent with findings from high-pressure MD studies on β-lactoglobulin, in which Kieserling et al. (2021) demonstrated via MD simulation that high hydrostatic pressure (HHP) induces a “molten globule”-like conformational state associated with enhanced interfacial adsorption capacity, while Huang et al. (2021) reported residue-level RMSF changes in β-lactoglobulin under 600 MPa conditions, confirming the site-dependent nature of pressure-induced flexibility modulation. The pressure-dependent enhancement of RMSF observed in specific myosin domains is consistent with the progressive increase in surface hydrophobicity documented experimentally by Wang et al. (2025), providing atomic-level mechanistic context for the macroscopic observation that HPP selectively increases the dynamic accessibility of functional surface regions.

3.1.3. Radius of gyration (Rg)

This localized structural loosening manifested as subtle protein expansion, quantified through Rg measurements. The Rg trajectories for all pressure groups demonstrated gradual, time-dependent increases with highly overlapping curves, indicating HPP-induced structural expansion or partial unfolding (Fig. 1-(c)). Despite the overlapping trends, all curves showed a consistent upward trajectory over time, suggesting that pressure treatment induced exposure of active regions. Notably, the fact that overall spatial extension of myosin monomers is not strongly differentiated by pressure intensity contrasts sharply with the motor domain contact model system (see Section 3.2), where extreme pressure structurally restricts the swelling behavior and favors tighter interfacial compaction. The gradual Rg increase observed here is consistent with the pressure-induced infiltration of water molecules into protein cavities, which weakens intramolecular hydrogen bonds and reduces the local packing density of hydrophobic cores (Collins et al., 2005; Grigera and McCarthy, 2010). This water penetration mechanism has been proposed as a key driver of the α-helix to random coil transition, as documented experimentally by CD spectroscopy (Boonyaratanakornkit et al., 2002; Lullien-Pellerin and Balny, 2002), because weakened backbone hydrogen bonds reduce the stability of helical secondary structures and allow greater conformational freedom in backbone dihedral angles. Taken together, the pressure-dependent increases in RMSF and the concurrent Rg expansion suggest that previously buried hydrophobic regions and polar residues become progressively accessible to solvent under HPP. This coordinated structural opening is consistent with the experimentally documented increases in surface hydrophobicity and reactive sulfhydryl exposure at moderate pressure reported by Wang et al. (2025), providing the atomic-level structural basis for those macroscopic observations.

3.1.4. Hydrogen bond analysis

The underlying mechanism for this behavior involved systematic disruption of the intramolecular hydrogen bond network. A clear inverse relationship was observed between applied pressure and hydrogen bond counts within myosin monomers (Fig. 1-(d)). The hydrogen bond count decreased from approximately 7.0 at ambient pressure to 5.0 at 150 MPa, 4.0 at 300 MPa, and reached a minimum of 2.0 at 450 MPa, maintaining a strict pressure-dependent hierarchy (ambient > 150 MPa > 300 MPa > 450 MPa) throughout the 100,000 ps simulation. This direct evidence links pressure application to destabilization of secondary structures (α-helices and β-sheets) maintained by these hydrogen bonds, providing a molecular explanation for the experimentally observed transition from ordered structures (α-helix, β-sheet) to disordered structures (β-turn, random coil). High pressure triggers a significant conformational shift by disrupting the hydrogen bond network that maintains the protein's secondary structure. As pressure increases, the α-helical structure unfolds due to the severing of intrachain hydrogen bonds, and its content systematically decreases. Simultaneously, the β-sheet content also decreases significantly due to the severing of these hydrogen bonds.

These findings are consistent with the secondary structure data reported by Wang et al. (2025), in which FTIR analysis documented progressive reductions in α-helix content (from 20.55% at ambient to 18.90% at 450 MPa) and concurrent increases in random coil content (from 29.94% to 36.28%), confirming that the H-bond disruption mechanism characterized here at atomic resolution underlies the macroscopic secondary structure transitions observed experimentally. This pressure-induced unfolding exposes internal hydrophobic residues and reactive sulfhydryls. Indeed, Zhang et al. (2015) observed that surface hydrophobicity and free-SH content of pork myofibrillar protein increased progressively under HPP, consistent with tertiary unfolding. The pressure-driven transition of α-helices into random coils has also been documented as pressure increases (Wang et al., 2025). Our observed decrease in α-helix content and increase in random-coil structures under HPP treatment is thus consistent with this established unfolding mechanism.

The strict pressure–hydrogen bond count negative correlation observed here provides the first atomic-level quantitative account of the mechanism by which HPP disrupts myosin secondary structure. These findings align closely with previous experimental evidence: Liu et al. (2021) documented that HPP treatment of myofibrillar proteins progressively reduced α-helix content and elevated disordered structure content across multiple meat sources, attributing these changes to intramolecular hydrogen bond disruption that allows α-helices to unravel into random coils. The review by Hata et al. (2020) further notes that water infiltration under pressure increases the density of water molecules around proteins and modifies protein–water hydrogen bonding networks, effectively competing for the N–H···O=C contacts that maintain helical geometry. The simulation data presented here extend these macroscopic observations by demonstrating that even moderate pressure (150 MPa) produces a ∼29% reduction in hydrogen bond count, consistent with the partial secondary structure disruption and surface hydrophobicity increase documented experimentally in the companion study (Wang et al., 2025). At 450 MPa, the near-complete elimination of intramolecular hydrogen bonds rationalizes the dramatic loss of ordered secondary structure and the onset of irreversible aggregation behavior observed at extreme pressures.

3.1.5. Free energy Landscape (FEL) analysis

The energetic consequences of these structural modifications were visualized using FEL analysis, which mapped the conformational states accessible to the protein under different pressure conditions (Fig. 2). At ambient pressure and lower pressures (100 kPa, 150 MPa), the landscapes showed concentrated distributions with predominant low-density regions (blue). As pressure increased (300 MPa, 450 MPa), there was a significant expansion of high-energy regions (red-colored areas), indicating that pressure forces the protein to populate a broader ensemble of less stable, structurally diverse conformations. This phenomenon results from pressure-induced hydrogen bond disruption, causing originally stable structures to become progressively loosened and allowing a greater proportion of high-energy conformations to emerge.

Fig. 2.

Fig. 2

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Free energy landscapes (FEL) of the myosin monomer under gradient HPP treatments. 2D projection and corresponding 3D surface representation of the relative free energy (G, in kcal/mol) profiles at (a) 100KPa, (b) 150 MPa, (c) 300 MPa, and (d) 450 MPa.

Note: Free energy landscapes were constructed using backbone RMSD (relative to the minimized starting structure) and the Radius of Gyration (Rg) as the collective variables (CVs). The 2D joint probability distribution P(RMSD, Rg) was calculated via Kernel Density Estimation (KDE) with a Gaussian kernel applied to trajectory frames sampled every 10 ps. Relative free energy was calculated using the Boltzmann relation G = −kBTln[P/Pmax] at T = 298.15 K, where the lowest-energy conformation (dark blue basin) is defined as G = 0 kcal/mol.

These simulation results collectively illustrate a “local loosening-global stability” phenomenon, wherein myosin monomers accommodate energetic stress through sacrificing local structural order - primarily via hydrogen bond cleavage - leading to increased regional flexibility and slight overall expansion while preserving the global fold. This adaptive mechanism allows the protein to maintain its overall structure while selectively increasing the dynamic accessibility of specific regions, which is critical for exposing potential ligand binding sites.

The FEL analysis provides thermodynamic context for the structural parameters described above. The progressive broadening of accessible conformational space at higher pressures, evidenced by the expansion of high-energy (red) regions, indicates that HPP shifts the Boltzmann population of myosin from a single, deep free energy minimum toward a flatter, more dispersed landscape populated by a diverse ensemble of partially unfolded states. This is mechanistically equivalent to the concept of “conformational entropy increase” under pressure stress, wherein the destabilization of stabilizing interactions allows previously inaccessible high-energy conformers to accumulate. FEL analysis has been established as a powerful tool for characterizing pressure-induced conformational heterogeneity in proteins (Strodel, 2021), and the FEL patterns observed here are qualitatively consistent with a “molten globule”-like intermediate state, retaining global topology while losing local order, which has been proposed as the active form for enhanced functional interactions in food processing contexts. The direct connection between expanded FEL space and increased ligand-accessible surface area explains why moderate HPP (150 MPa) enhances protein–flavor interactions while remaining below the threshold for irreversible aggregation. The occupation of these higher-energy conformational states represents a thermodynamic cost imposed by pressure, effectively destabilizing the native fold to generate a conformationally activated ensemble. This energy investment is functionally consequential: the elevated free energy of pressure-modified myosin lowers the enthalpic barrier for ligand accommodation, providing a mechanistic rationale for why moderate HPP conditions enhance rather than diminish protein–flavor binding capacity.

3.2. The effect of HPP on myosin motor domain association

3.2.1. Contact model RMSD and hydrogen bond analysis

In contrast to its effects on monomers, HPP promoted the formation of more compact and stable structures in myosin motor domain contact model. RMSD trajectory analysis revealed an initial structural adjustment period (0-70 ns) with significant conformational responses and continuous fluctuations, indicating a significant opening of contact structures, accompanied by reduced rigidity and the potential exposure of previously buried functional groups. Notably, after the 70 ns time point, the experimental groups transitioned from an initial rapid structural adjustment to a dynamically fluctuating phase, with RMSD values oscillating between 3.5 and 6.0 Å. This persistent fluctuation indicates that instead of freezing into a rigid state, the complex assembled into flexible yet persistent supramolecular complexes capable of structural readjustment under sustained high-pressure conditions (Fig. 3-(a)).

Fig. 3.

Fig. 3

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Interfacial compaction and morphological remodeling of the myosin motor domain contact model under gradient HPP treatments. (a) Time-dependent root-mean-square deviation (RMSD) profiles of the conplex backbone; (b) Time-dependent radius of gyration (Rg) trajectories of the overall contact model; (c) Solvent-accessible surface area (SASA) profiles of the model complex over the 100 ns simulation timescale.

Note: Compressive simulations of the pre-aggregation intermediate (comprising two adjacent motor domains, residues 1–1844) were conducted at 100KPa (cyan), 150 MPa (blue), 300 MPa (orange), and 450 MPa (red). SASA values in (c) represent the total solvent-exposed surface area of the dimer complex, computed using a water probe radius of 1.4 Å.

The biphasic RMSD trajectory of the contact model, an initial opening and fluctuation phase followed by convergence to a stable equilibrium, is mechanistically informative. The early high-fluctuation period (0–70 ns) likely corresponds to the initial disruption of native interfacial contacts and exposure of hydrophobic surfaces, consistent with experimental observations of increased surface hydrophobicity at elevated pressures (Wang et al., 2025). The subsequent convergence to 3.5-4.0 Å RMSD across all pressure groups reflects the formation of a new, pressure-stabilized interfacial architecture in which hydrophobic burial and intermolecular hydrogen bonding cooperatively establish a lower-energy aggregate state. This two-stage dynamics pattern resembles the nucleation-condensation mechanism of protein aggregation, in which an early disordered contact phase is followed by structural consolidation into a compact, thermodynamically favorable aggregate (Werner et al., 2020). The convergence to a common RMSD plateau despite different applied pressures suggests that the final aggregate architecture is structurally similar across pressure groups, with pressure primarily modulating the rate and thermodynamic driving force for compaction rather than the nature of the final state. The stable RMSD plateau observed after 70 ns suggests that the myosin motor domain contact model entered a dynamically stable state under pressure. To probe the molecular determinants underlying this inter-subunit stability, we next examined the pressure-dependent evolution of the hydrogen-bonding network.

The total number of hydrogen bonds (both intramolecular and inter-chain) was monitored across different pressure conditions (Fig. 4-(a)). Under ambient pressure (100KPa), the contact-model system maintained a mean of 973.4 ± 28.1 hydrogen bonds. Upon compression, the total hydrogen bond count exhibited a progressive, pressure-dependent increase, reaching 976.9 ± 27.1 at 150 MPa, 998.4 ± 25.2 at 300 MPa, and reaching a maximum of 1011.3 ± 32.8 at 450 MPa.

Fig. 4.

Fig. 4

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Thermodynamic, energetic, and interfacial residue-level characterization of the myosin contact-model interface under HPP. (a) Total system hydrogen bonds (intramolecular plus inter-subunit); (b) Number of active interfacial residue pairs across the Chain A–Chain B interface; (c) Symmetric 1844×1844 residue–residue distance contact maps averaged over the equilibrated trajectories; (d) Total number of interfacial hydrophobic contacts; (e) Estimated binding free energies (ΔGbind, in kcal/mol) calculated via MM-GBSA; (f) Energetic components of the binding affinity including van der Waals (VDWAALS), electrostatic (EEL), 1-4 electrostatic (1-4 EEL), polar solvation (EGB), and non-polar solvation (ESURF) terms.

Note: Statistical analyses in (a), (b) and (d) were performed on n = 11 representative frames sampled at 10 ns intervals (including the equilibrated starting time) across the 100 ns production trajectories, and are reported as mean ± standard deviation (SD). Hydrophobic contacts in (d) and interfacial residues in (b) were identified using the nativecontacts command with a heavy-atom distance cutoff of 4.5 Å. In (c), distance maps represent the symmetric residue distance matrix, with the red dashed lines denoting the boundary between Chain A (residues 1–922) and Chain B (residues 923–1844). MM-GBSA binding free energies in (e) were estimated from n = 10 production-phase frames (10-100 ns) using the GB-Neck2 model (igb = 8) and are reported as mean ± standard error of the mean (SEM) to minimize trajectory autocorrelation.

Interestingly, this upward trend in the contact-model system contrasts sharply with the dramatic disruption of backbone hydrogen bonds observed within individual myosin monomers (which decreased from ∼7.0 to ∼2.0 per residue, as discussed in Section 3.1.4). This disparity suggests a distinct cooperative response: while high hydrostatic pressure penetrates and unfolds the secondary structure helices of individual monomers, it simultaneously drives the two motor domain into closer proximity, compressing the system and facilitating the formation of dense, non-specific inter-subunit and compacted intramolecular polar networks. This structural consolidation under extreme pressure conditions (300 and 450 MPa) compensates for local unfolding and contributes to the global stabilization of the compacted aggregate.

3.2.2. Complex compaction: Radius of gyration and interfacial contact analysis

Consistent with the progressive interfacial compaction detailed in Section 3.2.3, the radius of gyration (Rg) of the complex followed a non-monotonic trajectory, increasing transiently at 150 MPa before undergoing pronounced compaction at ≥ 300 MPa (Fig. 3-(b)). To elucidate the residue-level basis of these global structural changes, we quantified the number of interfacial residue pairs (Fig. 4-(b)) and mapped the residue–residue distance matrices (Fig. 4-(c)). At 100KPa, the dimer interface was characterized by a highly localized contact region involving only 56 interfacial residue pairs, consistent with a relatively loose and flexible initial dimer configuration.

Under a moderate pressure of 150 MPa, the number of interfacial residue pairs surged dramatically to 242, coinciding with the transient expansion of Rg noted above. This suggests that moderate compression first drives an outward rearrangement of the monomeric domains, swelling the overall dimer envelope while simultaneously forcing a larger proportion of the protein surface into direct inter-chain contact. At extreme pressures of 300 and 450 MPa, Rg contracted sharply and SASA continued to decline, while the number of interfacial residue pairs, though modestly reduced from its 150 MPa maximum, remained exceptionally high at 231 and 196, respectively, indicating that the expanded interface established under moderate compression is largely retained even as the global contact model subsequently undergoes compaction.

The contact maps (Fig. 4-(c)) visually substantiate this pressure-induced interfacial expansion, which reaches its greatest extent at 150 MPa and is largely preserved, albeit with modest attenuation, at 300 and 450 MPa. The off-diagonal quadrants, representing inter-subunit (chain A–chain B) contacts, showed a substantial increase in density and a widening of contact regions at elevated pressures compared to the ambient state. This spatial proximity of the two subunits across broad domain regions indicates that compressive hydrostatic stress effectively eliminates interfacial void volumes and drives the subunits into a highly compacted, interfacially paired state, which macroscopically manifests as the protein aggregation observed experimentally at pressures ≥ 300 MPa.

3.2.3. Complex SASA analysis

Complementing the radius of gyration and interfacial contact trends described above, systematic decreases in solvent-accessible surface area (SASA) were observed as pressure increased from 100 kPa to 450 MPa (Fig. 3-(c)). The SASA reduction confirmed that larger portions of protein surfaces became buried at intermolecular interfaces, shielded from solvent interaction. This process was driven by enhancement of hydrophobic interactions, forcing expulsion of water molecules from the interface and resulting in more buried surface areas with reduced solvent accessibility, stabilizing the contact model in a lower-energy, more strongly bound state.

The pressure-dependent SASA decrease provides direct quantitative evidence that HPP promotes interfacial burial as a mechanism of contact-model stabilization. From a thermodynamic perspective, the burial of hydrophobic residues at the protein–protein interface reduces the unfavorable water-exposure enthalpy of nonpolar side chains, while the associated expulsion of interfacial water molecules generates a favorable entropy contribution. This hydrophobic collapse mechanism is analogous to the hydrophobic effect driving protein folding, applied here at the intermolecular rather than intramolecular level. Chen et al. (2023) demonstrated experimentally that modulating hydrophobic interactions in myofibrillar protein systems directly controls the extent and stability of protein aggregation, with stronger hydrophobic contacts producing larger, more stable aggregates. This is corroborated by Wang et al. (2025), in which surface hydrophobicity increased monotonically with pressure (BPB binding: 12.2 mg at ambient to 32.9 mg at 450 MPa) while emulsifying stability simultaneously declined, consistent with progressive interfacial burial reducing the proportion of hydrophobic surface available for oil-water interfacial adsorption. Furthermore, the pressure-driven elimination of interfacial void volume reduces the partial molar volume of the contact model relative to the separated monomers, providing thermodynamic stabilization in accordance with the volume-minimization principle governing high-pressure chemistry. Together with the Rg and RMSD data, the SASA trajectory establishes a self-consistent molecular picture of HPP-driven dimer compaction.

3.2.4. Thermodynamic and energetic characterization of the myosin interfacial association

To dissect the physical forces driving the pressure-dependent compaction of the contact model and to evaluate the biological validity of our contact-model interface, we calculated the interfacial hydrophobic contacts (Fig. 4-(d)) and estimated the binding free energies (ΔGbind) using the MM-GBSA method (Fig. 4-(e) & (f)).

Under ambient conditions (100KPa), the contact model exhibited a strong enthalpic binding free energy (ΔGbind = −51.25 ± 6.66 kcal/mol). This favorable free energy was driven predominantly by van der Waals packing (VDWAALS = −155.76 kcal/mol); the combined electrostatic and solvation terms (EEL + EGB + ESURF = +104.5 kcal/mol) were net destabilizing at this pressure, offsetting a substantial portion of the van der Waals contribution (Fig. 4-(f)). The hydrophobic contacts at the interface were relatively low (4.0 ± 2.4) at this stage, indicating that the ambient-pressure interface is held together primarily through shape-complementary van der Waals packing rather than specific electrostatic pairing.

Upon the application of moderate pressure (150 MPa), a striking transition occurred: the number of interfacial hydrophobic contacts peaked significantly at 11.9 ± 6.9, while the binding free energy remained favorable at −34.07 ± 3.44 kcal/mol. Notably, this increase in specific hydrophobic contacts was accompanied by an overall reduction in total van der Waals packing energy (VDWAALS = −119.78 kcal/mol, versus −155.76 kcal/mol at ambient pressure), suggesting that the newly formed hydrophobic contacts compensate for a broader loosening of the ambient-pressure interface rather than adding further stabilization on top of it. This peak in hydrophobic contacts nonetheless provides direct atomic-level evidence for pressure-induced “hydrophobic collapse” at the interface, where moderate compressive stress drives the exposure and subsequent mutual burial of hydrophobic residues (Ala, Val, Leu, Ile, Pro, Phe, Met, Trp). This hydrophobic interface sealing reduces solvent accessibility, as evidenced by the SASA reduction, and establishes a stable, hydrophobically driven pre-aggregation intermediate.

In stark contrast, when the pressure was increased to extreme levels (≥300 MPa), the specific interfacial hydrophobic contacts dropped precipitously to nearly zero (0.2 ± 0.6 at 300 MPa and 0.0 ± 0.0 at 450 MPa). Concurrently, the MM-GBSA binding free energy became progressively weaker, at −11.23 ± 2.17 kcal/mol at 300 MPa and −4.44 ± 2.05 kcal/mol at 450 MPa (Fig. 4-(e)). Energy component analysis (Fig. 4-(f)) revealed that this weakening arose from two mechanistically distinct regimes: the initial decline from 150 to 300 MPa was driven primarily by a further loss of van der Waals packing (VDWAALS decreasing from −119.78 to −96.38 kcal/mol), coinciding with the near-complete collapse of specific hydrophobic contacts, while electrostatic repulsion (EEL) actually eased over this interval (from +396.27 to +256.04 kcal/mol). Beyond 300 MPa, however, electrostatic repulsion rose sharply, reaching +476.21 kcal/mol at 450 MPa; although this was substantially offset by a correspondingly more favorable polar solvation term (EGB = −401.13 kcal/mol), the residual imbalance further eroded the already-weakened binding free energy.

These thermodynamic findings provide a highly nuanced and rigorous mechanism for the biphasic behavior of myosin under HPP. Moderate pressure (150 MPa) optimizes the structural templates by facilitating specific, functional hydrophobic interactions. However, extreme pressures (≥300 MPa) thermodynamically destabilize the specific, ordered contact-model interface predicted by AlphaFold (as indicated by the near-zero binding energy and loss of hydrophobic contacts). Instead, the system is forced into a non-specific, disordered, and volume-constrained amorphous state, driven by Le Chatelier's volume-minimization principle rather than specific thermodynamic pairing. This explains why extreme HPP treatment leads to irreversible, highly heterogeneous, and poorly soluble protein aggregates in processed meat products.

3.2.5. Contact-model free energy Landscape analysis

Thermodynamic landscape analysis through FEL mapping revealed that HPP triggered a significant expansion of the accessible conformational space, indicative of increased conformational entropy (Fig. 5). At ambient pressure, the complexes were predominantly confined to a sharp, highly localized energy basin, representing a relatively rigid initial predicted interfacial complex. As pressure increased to 150 MPa and beyond, the low-energy regions noticeably broadened and diversified into multiple sub-basins. This phenomenon demonstrates that extreme hydrostatic stress forces the adjacent molecules away from their initial highly ordered contact interface to populate a diverse ensemble of metastable conformations. Molecularly, this structural diversification reflects the disruption of specific native inter-chain hydrogen bonds and the compensatory formation of non-specific hydrophobic contacts, which drive the proteins into heterogeneous, amorphous aggregate states.

Fig. 5.

Fig. 5

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Free energy landscapes (FEL) of the myosin motor domain contact model under gradient HPP treatments. 2D projection and corresponding 3D surface representation of the relative free energy (G, in kcal/mol) profiles of the contact model at (a) 100KPa, (b) 150 MPa, (c) 300 MPa, and (d) 450 MPa. Note: Landscapes were constructed using dimer backbone RMSD and the overall Radius of Gyration (Rg) as the collective variables (CVs). The 2D probability density and Boltzmann-derived relative free energy (G) were mapped using identical methodology and sampling parameters as described in Fig. 2.

This pressure-induced broadening of the dimer FEL represents a critical thermodynamic mechanism that perfectly aligns with macroscopic experimental observations. The highly diversified energy landscape at ≥300 MPa explains why high-pressure treated myosin forms dense, heterogeneous aggregates that resist re-dispersion (Wang et al., 2025), as the system is kinetically trapped in multiple compact but structurally distinct aggregation states rather than a single ordered crystal. Taken together, the FEL analyses of both monomers and contact-model provide a comprehensive thermodynamic explanation for the biphasic functional behavior of myosin: moderate pressure facilitates monomer conformational flexibility and enhanced ligand accessibility, whereas extreme high pressure drives the contact model to explore and subsequently become irreversibly trapped in diversified, amorphous aggregate states that sterically occlude functional binding sites.

The MD simulation results were validated against experimental findings and our previously published work on pressure-induced MP structural changes, confirming the reliability of the computational models for subsequent molecular docking analysis (Wang et al., 2025). These findings revealed a critical dichotomy in HPP effects: inducing unfolding in monomers while promoting aggregation and compaction in contact model. This dual mechanism provides a predictive framework suggesting that functional consequences of HPP, including ligand binding, are governed by the balance between these opposing effects. At lower pressures, monomer-unfolding effects may dominate, exposing binding sites, whereas higher pressures favor contact-model compaction effects, potentially sequestering and shielding these sites.

3.3. Future perspective

The present study establishes a quantitative, trajectory-based computational framework for understanding the pressure-dependent structural remodeling of myosin. By incorporating direct contact map analysis, interfacial residue pairing, and MM-GBSA thermodynamic estimations, we have partially resolved the driving forces of myosin aggregation. Nonetheless, several important directions remain for future investigation.

First, while our 100 ns simulations resolved the initial, atomically detailed structural reorganization, the long-term kinetics of non-specific aggregation and the transition from pre-aggregation intermediates to large-scale fibrillar networks occur on microsecond-to-millisecond timescales. Future studies employing multi-microsecond all-atom simulations or coarse-grained modeling (e.g., using the MARTINI force field) are required to capture the complete aggregation pathway. Second, the residue-level structural changes predicted by these simulations, particularly the pressure-sensitive surface loops and the thermodynamic destabilization of the dimer interface, should be validated experimentally using high-pressure hydrogen-deuterium exchange mass spectrometry (HDX-MS), which can directly probe backbone accessibility under pressure. Finally, the generalizability of this dual-response mechanism should be systematically tested across other key myofibrillar protein components, such as actin and actomyosin complexes, to build a holistic, food-matrix-level molecular model.

4. Conclusion

In summary, this study employed all-atom molecular dynamics simulations and thermodynamic free energy calculations to mechanistically dissect the dual structural response of myosin under high-pressure processing. For the myosin monomer, a strict inverse relationship between pressure and hydrogen bonds was established, confirming a “local loosening–global stability” response that exposes functional surface domains.

Strikingly, this local loosening was accompanied by an opposing, global response at the inter-subunit level: total hydrogen bonding increased progressively with pressure (from 973.4 ± 28.1 at 100KPa to 1011.3 ± 32.8 at 450 MPa), while the number of interfacial residue pairs expanded from 56 at ambient pressure to a maximum of 242 at 150 MPa and remained above 190 even at 450 MPa, with residue–residue contact maps directly visualizing this pressure-driven interfacial consolidation. This contrast, intramolecular unfolding within each monomer coupled with inter-subunit compaction across the contact model, indicates that hydrostatic pressure redistributes, rather than uniformly disrupts, the myosin structural network.

Thermodynamically, this compaction was not a single uniform process. Moderate pressure (150 MPa) favored a distinct, specific hydrophobic sealing of the interface, marked by a peak in hydrophobic contacts (11.9 ± 6.9); the subsequent weakening of binding free energy from 150 to 300 MPa (−34.07 to −11.23 kcal/mol) was driven primarily by the loss of van der Waals packing, whereas the further decline to −4.44 ± 2.05 kcal/mol at 450 MPa reflected a sharp rise in electrostatic repulsion only partially offset by polar solvation. Together, these findings indicate that the ordered, AlphaFold-predicted dimer interface is progressively dismantled at extreme pressures, giving way to a non-specific, volume-constrained amorphous aggregate.

This computationally derived thermodynamic framework bridges molecular-level conformational transitions with the macroscopic aggregation behavior observed experimentally in HPP-treated myosin, and offers rational, mechanism-based guidance for tuning pressure protocols to preserve, or deliberately modulate, myofibrillar protein functionality in non-thermal food processing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by Guangzhou Key-Area Research and Development Project (2023B03J1275) and Key-Area Research and Development of Heyuan City (No. 2022010).

Contributor Information

Xiaoyun Wang, Email: wangxiaoyun0211@foxmail.com.

Jie Tang, Email: jietang@jnu.edu.cn.

Hosahalli S. Ramaswamy, Email: hosahalli.ramaswamy@mcgill.ca.

Hanying Duan, Email: tduhy@jnu.edu.cn.

Chao Wang, Email: chao_wang@jnu.edu.cn.

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