Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

SMFRET-GUIDED INTEGRATIVE BIOPHYSICS REVEALS ALLOSTERIC MODULES AS ENERGETIC CONTROL POINTS OF PROTEIN FREE-ENERGY LANDSCAPES Chara Sarafoglou 1,2 ; Yusran A Muthahari 1 ; Charalambos Pozidis 1 ; Andreas Kofidis 3 ; Mary Providaki 1 ; Alexis Molfetas 1 ; Mikis Mylonakis 4 ; Kostas Mavrakis 4 ; Reza Aditama 5 ; Rukman Hertadi 5 ; Giannis Zacharakis 4 ; Yannis Pantazis 3 ; Giorgos Gouridis 1 ; 1 Institute of Molecular Biology and Biotechnology , Structural Biology and Biophysics, Heraklion, Greece 2 University of Crete, Biology, Heraklion, Greece 3 Institute of Applied and Computational Mathematics, Heraklion, Greece 4 Institute of Electronic Structure and Lasers, Biophotonics and molecular imaging, Heraklion, Greece 5 Bandung Institute of Technology, Biochemistry, Bandung, Indonesia Free-Energy Landscapes (FELs) provide a unifying physical framework for understanding protein folding, allostery, ligand recognition, and functional trade-offs that underlie evolution. Yet a challenge remains: which structural and energetic units sculpt these landscapes, and how does their perturbation redirect protein behaviour across equilibrium and nonequilibrium biochemical processes? In my PhD, I addressed this challenge using substrate-binding proteins (SBPs) providing tractable model landscapes and single-molecule Förster Resonance Energy Transfer (smFRET) as the central experimental readout of conformational-state populations, transitions, and fluxes. Across folding, ligand binding, transport-coupled function, and evolutionary adaptation, a common principle emerged: coevolving allosteric modules act as energetic control points that define the wells, barriers, and connectivity of the FEL.By combining smFRET with time-varying mixture modeling in the NEXT-FRET framework (PNAS, doi:10.1073/pnas.2529979123), we tracked nonequilibrium population flow during folding of freely diffusing SBPs and resolved transient on-pathway intermediates. Rather than representing exclusively folding species, these intermediates correspond to wells within the same modular FEL that governs native-state dynamics and allostery. Signal peptides and chaperones reroute folding by selectively stabilizing these distinct intermediates and reshaping kinetic barriers. The same modules then fine-tune the native landscape to control function and evolvability. Integration with hydrogen–deuterium exchange mass spectrometry, molecular dynamics simulations, calorimetry, and transport-coupled assays revealed that perturbing them reweights conformational ensembles, switches binding mechanisms among induced fit, lock-and-key, and conformational selection, and tunes thermodynamically imposed functional trade-offs (bioRxiv, doi:10.1101/2025.10.08.680613). Extending this logic across related SBPs showed that affinity and specificity cannot be explained by binding-cleft chemistry alone. Instead, they emerge from coupling between local ligand contacts and the conformational free energy encoded by the allosteric network, providing a route to engineer protein function by reweighting pre-existing states rather than redesigning binding sites.Together, these studies unify folding, dynamics, function, and adaptation within a shared FEL framework.

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