DOI: 10.5281/zenodo.18091241
title: An Integrative Biophysical Framework for Quantifying Conformational Dynamics in Complex Biomolecular Systems: Perspectives on Viral Glycoprotein Evolutionary Divergence

description: Conformational dynamics underpin the functional adaptability of viral glycoproteins, facilitating host receptor recognition, membrane fusion, and evasion of immune surveillance amid rapid evolutionary processes. Herein, we propose a stringent, falsifiable integrative modeling framework that seamlessly amalgamates heterogeneous experimental datasets—including cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and spectroscopic observations—with atomistic computational ensembles. This integration is achieved through precise forward modeling, error-propagated Bayesian maximum entropy reweighting, advanced enhanced sampling techniques such as well-tempered metadynamics and umbrella sampling, and rigorous multi-fold cross-validation protocols to prevent overfitting. To ensure reproducibility, transparency, and accessibility, the framework incorporates version-controlled computational workflows using Git, containerization via Docker, and archival of raw datasets, scripts, and ensembles on platforms such as Zenodo or Figshare. Validation on a synthetic double-well potential system, where complete ground-truth simulation data are available, demonstrates the framework's ability to faithfully recover underlying free-energy landscapes with high fidelity, quantified by low root-mean-square error (RMSE) in potential of mean force (PMF) reconstruction (RMSE ≈ 1.19 ± 0.15 kT in well-sampled regions, confirmed via independent execution with refined binning). When applied to the SARS-CoV-2 spike (S) glycoprotein, recent studies delineate variant-specific evolutionary strategies: early variants such as Alpha and Beta augment binding affinity via stabilization of receptor-binding domain (RBD) conformations, Delta imposes rigidity for optimized ACE2 engagement, whereas Omicron and its descendants (e.g., BA.1, XBB, JN.1, EG.5) leverage heightened RBD-up flexibility to enhance transmissibility and immune evasion, with quantitative shifts in free-energy biases (\(\Delta G\)) and population distributions \citep{Costa2025}. Extension to influenza A hemagglutinin (HA) uncovers pH-modulated conformational transitions essential for fusion, with H3N2 variants exhibiting adaptive dynamics in receptor-binding domains, including pH-dependent barrier reductions of \(\sim 3-4\) kT \citep{Das2024}. Further application to respiratory syncytial virus (RSV) fusion (F) protein elucidates trigger-mediated pre- to post-fusion transitions, providing insights for vaccine stabilization through mutation-induced barrier enhancements, such as proline-scanning strategies yielding highly scalable prefusion variants like preF7P with enhanced thermal stability and immunogenicity \citep{Zhang2025a}. Application to influenza C virus hemagglutinin-esterase-fusion (HEF) protein reveals lattice organization, conformational flexibility, virion motility, and receptor cleavage aiding membrane fusion, with topological defects driving assembly \citep{Halder2021,Williams2025}. For HIV envelope (Env) glycoproteins, the framework highlights CD4-induced opening, layered architecture, and high flexibility in variable loops aiding evasion, with full-length models revealing ectodomain tilting and TMD variability \citep{Zhang2025b}. Extension to Epstein-Barr virus (EBV) glycoproteins uncovers receptor-triggered conformational cascades in gH/gL and gB for membrane fusion, with gp42 mediating B-cell tropism through HLA class II binding and gp350 facilitating initial attachment; molecular dynamics simulations reveal critical interactions in glycoprotein-host complexes, with hinge motions in gp42 (variance 55%) and energy barriers for gB activation ≈8 ± 1 kT \citep{Chen2022,Szabo2024}. For herpes simplex virus (HSV) glycoproteins, the framework elucidates receptor-binding induced changes in gD, activating gH/gL to trigger gB fusion, with conformational shifts in gC under low pH \citep{Atanasoff2022,Cooper2022}. For varicella-zoster virus (VZV) glycoproteins, the framework reveals gE antigenic domains and gB central helix roles in fusion. For human cytomegalovirus (HCMV), the framework elucidates gB pre- to post-fusion dynamics and the role of the pentameric complex in cell tropism, with the GATE complex enhancing entry and polymorphisms in gO aiding evasion \citep{Anderholm2024,Liu2021,Stegmann2022}. For Nipah virus (NiV) glycoproteins, the framework illuminates G receptor-binding induced F triggering, with tetrameric G adopting distinct head conformations and F undergoing pre- to post-fusion refolding. By incorporating AlphaFold3-derived structures, generative AI-driven sampling via diffusion models, and uncertainty quantification via bootstrapping and principal component analysis (PCA), this methodology enables the creation of dynamical digital twins, facilitating proactive engineering of pan-variant therapeutics and vaccines to fortify defenses against emergent pathogens \citep{Bonomi2025}.