The Journal of Physical Chemistry Letters

Intrinsic tryptophan fluorescence is widely used as a sensitive reporter of protein conformational dynamics, yet the molecular origin of its temperature-dependent modulation remains unclear. Here we investigate the conformational dynamics of Trp134 in bovine serum albumin (BSA) using molecular dynamics (MD) simulations, free-energy calculations based on umbrella sampling and WHAM, quantum mechanical (QM) calculations, and QM/MM approaches. MD simulations show that the global structure of BSA remains stable while temperature induces a gradual population shift from the Ia+ to the Ia- rotamer. The corresponding free-energy landscapes reveal that this shift arises from subtle changes in basin stability and transition barriers along the rotameric coordinate. In contrast, standalone QM calculations on isolated tryptophan predict different energetic trends, highlighting the sensitivity of rotamer stability to electronic-structure treatments and environmental effects. QM/MM calculations partially reconcile these differences by incorporating the protein environment. Together, these results suggest that temperature reshapes the rotamer free-energy landscape of Trp134, leading to population shifts that modulate intrinsic tryptophan fluorescence in proteins.

We present a new formulation of the low-field effect (LFE) in spin-correlated radical pairs based on a zero-field singlet-triplet basis for the isotropic spin Hamiltonian. The aim is to provide a description that is both formally rigorous and mechanistically transparent, especially in the regime of weak magnetic fields such as the geomagnetic field. For the standard model radical pair containing a single spin [Formula] nucleus, we show that the conventional singlet-triplet basis obscures the distinct dynamical roles of the hyperfine and Zeeman interactions. In the zero-field S-T basis, by contrast, the mechanism separates cleanly: isotropic hyperfine coupling mixes singlet-doublet and triplet-doublet states, whereas the weak-field Zeeman interaction mixes triplet-quartet and triplet-doublet states without directly introducing an additional singlet-triplet coupling. The LFE is therefore revealed as a sequential process in which a weak field unlocks access from a triplet-only manifold to a singlet-accessible triplet manifold, from which hyperfine-driven singlet-triplet interconversion can occur. We then generalize this picture to radical pairs with arbitrary isotropic hyperfine structures by identifying maximal, interior, and, when present, minimal triplet-only manifolds in the zero-field spectrum. Finally, we introduce a practical blockwise dark-state recruitment measure for the triplet-only zero-field state space made singlet-accessible by a weak field, and show how this quantity depends on hyperfine symmetry, including the effects of equivalent nuclei. The resulting framework provides both a simple physical picture of the LFE and a general route to estimating its structural upper bound for arbitrary radical pairs.

Biomolecular condensates play essential roles in cellular organization and are implicated in diverse pathological processes. Their formation is driven by liquid-liquid phase separation (LLPS), a process that requires coordinated multistep desolvation of biomolecular chains and multivalent inter-chain interactions. Although coarse-grained (CG) models with implicit solvent are widely used to probe LLPS thermodynamics and kinetics, they typically neglect explicit desolvation energetics, limiting their accuracy and mechanistic interpretability. Here, guided by all-atom simulations and experimental measurements, we develop a CG model that incorporates residue-level desolvation terms directly into the energy function and apply it to investigate LLPS of intrinsically disordered proteins. Incorporating explicit desolvation reshapes the phase diagram, yielding improved predictions of dense-phase packing density. Strikingly, we uncover a linear relationship between the temperature gap (simulation temperature relative to the critical point) and the extent of conformational compaction accompanying the dilute-to-dense phase transition, a result further supported by theoretical analysis. We also find that desolvation barriers accelerate early-stage coarsening dynamics while slowing chain mobility within mature condensates, whereas solvent-separated contact interactions exert the opposite effects. Together, this framework enables efficient and explicit treatment of desolvation in CG simulations and reveals how desolvation energetics shape both the thermodynamic landscape and kinetic property of biomolecular LLPS.

Protein kinases regulate signaling by recognizing short sequence motifs, and how these motifs bind influences both specificity and therapeutic strategies that target kinase pathways. Peptide-based inhibitors that engage substrate-recognition regions are attracting interest, but designing them requires an understanding of how a flexible peptide approaches and settles into the bound pose. Traditional studies have focused on the bound pose and affinities, whereas the steps that link the initial encounter with the bound pose have been explored less thoroughly because the relevant intermediates are too short-lived to capture experimentally and evolve on timescales that standard molecular dynamics cannot readily access. Here, we focused on Abl kinase and Abltide, the experimentally identified optimal substrate peptide for Abl kinase, and examined the sequence of events linking initial encounter to the bound pose using two-dimensional replica exchange (gREST/REUS), which selectively enhances flexibility in the peptide and its binding interface while also sampling progression along a distance coordinate. The resulting simulations yielded a detailed binding landscape, revealing five distinct encounter regions outside the substrate-binding site and six intermediate states that may connect the initial approach to the bound pose. Some encounter regions and intermediate states participate in the dominant binding pathways. During this process, EF/G/{beta}11 hydrophobic patch, together with G helix negative patch, plays a central role in guiding Abltide toward the substrate-binding site. These findings provide mechanistic insight into substrate recognition by protein kinases and offer a foundation for the rational design of peptide-based inhibitors.

The catalytic mechanism of the hairpin ribozyme has remained controversial for more than two decades, with different experimental approaches often supporting distinct mechanistic interpretations. In this work, we investigate the conformational landscape of the active site along several proposed reaction pathways using all-atom molecular dynamics simulations in explicit solvent combined with enhanced sampling techniques. Specifically, we employ Hamiltonian replica exchange simulations to extensively explore active-site conformations without relying on predefined collective variables, enabling a broad characterization of the structural ensembles associated with multiple protonation states along three candidate reaction mechanisms. Our simulations suggest that a dianionic general acid/general base pathway involving direct participation of A38 and G8 is unlikely to proceed through well-defined intermediates with catalytically competent geometries. In particular, states associated with G8 deprotonation and subsequent O2 deprotonation exhibit strongly distorted active-site arrangements that appear poorly suited for reaction progression. Although highly synchronous protontransfer steps cannot be excluded, the required deprotonation of G8 remains difficult to reconcile with neutral pH conditions. In contrast, monoanionic pathways in which the non-bridging oxygens of the scissile phosphate act as transient proton relays produce intermediates that sample geometries favorable for the nucleophilic addition and leaving-group elimination steps of the reaction. These mechanisms do not require direct catalytic involvement of G8 while remaining compatible with potential acid catalysis by protonated A38+. Our results provide a unified conformational perspective on competing mechanistic scenarios. The ensembles generated here offer a foundation for future QM/MM and ML/MM calculations aimed at quantitatively resolving the free-energy landscapes governing hairpin ribozyme catalysis. Finally, the present strategy could easily be applied to other biomolecular systems with high conformational plasticity, including other ribozymes.

The repair of photo-induced DNA lesions through nucleotide excision repair machinery is still the source of important questions. It has been observed that the repair rate of the different cyclobutane pyrimidine dimers, i.e. the photoproducts induced by dimerization of two {pi}-stacked pyrimidines (T<>T, T<>C, C<>T, C<>C), depends on the nucleobases involved in the lesion. TT derivatives (T<>T) are removed more slowly than those containing cytosine, especially in 5. Using all-atom molecular dynamics simulations and free-energy calculations, we demonstrate that the variation of the repair rate observed in human skin and in cultured cutaneous cell is associated to the recognition of the four lesions by the DDB2 protein moiety, and more specifically by the differential structural deformation induced on the complementary strand. Indeed, while C<>C and C<>T induce a larger deviation on the groove parameters, T<>T and T<>C, instead, affect DNA structure to a lesser extent. less affected. These effects then hamper differentially the downstream recruitment of the repair complexes. The observed DNA deformation correlates with the experimental repair rate and provides a structural rationale for the different repair rates of CPD by nucleotide excision repair machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/724087v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@cf6b6dorg.highwire.dtl.DTLVardef@195e35forg.highwire.dtl.DTLVardef@1829296org.highwire.dtl.DTLVardef@165baba_HPS_FORMAT_FIGEXP M_FIG C_FIG

Eukaryotic cap-dependent translation initiation is regulated by binding of the predominantly folded eukaryotic initiation factor 4E (eIF4E) to the intrinsically disordered eIF4E binding proteins (4E-BPs). Here, we report full-length atomistic conformational ensembles generated by IDPConformerGenerator and optimized by X-EISDv2 workflow for both apo 4E-BP2, the neuronal 4E-BP, and 4E-BP2 in complex with eIF4E, using data from single-molecule fluorescence and nuclear magnetic resonance (NMR), together with select coordinates from a 4E-BP1:eIF4E crystal structure. Structural sampling within dynamic complexes is often under-appreciated, with NMR and crystal structure data for 4E-BP:eIF4E suggesting different degrees of structural heterogeneity. Our ensemble models validated by solution spectroscopy data enable comparison of free 4E-BP2 and its complex with eIF4E. This shows a delocalization of contacts around canonical regions, which supports previous findings of unidirectional conditional occupancy of the binding sites. Two new contact regions emerged: one between the disordered N-termini of eIF4E and 4E-BP2, which may play an allosteric role in tuning the binding affinity, and the other between the C-terminus of 4E-BP2 and an extended region of eIF4E, which is consistent with the extended, dynamic binding interface that we reported previously. These results support a model of translation regulation in which the dynamic 4E-BP2:eIF4E complex facilitates accessibility of regulatory sites of 4E-BP2 when bound.

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.

The molten globule (MG) state is an intermediate in the unfolding pathway of proteins, typically triggered by denaturing agents such as urea, extreme pH, high pressure, or heat. The microscopic details of such states are far from understood. Here we study the MG states in protein Hen Egg-White Lysozyme (PDB ID: 1AKI) using microscopic constant pH molecular dynamics (CpHMD) simulations and experiments across a wide pH range. We observe that the titratable residues act as key drivers of conformational fluctuations, promoting the emergence of MG states at extreme pH. These states display partial unfolding, and small global structural changes (< 7% deviation). Hydration around the fluctuating acidic residues shows reduced water density and weakened hydrogen bonding at low pH. At high pH, hydration around acidic residues increases relative to pH = 7, whereas hydration around basic residues decreases. The translational and rotational dynamics of hydration water also exhibit pronounced pH dependence: the translational diffusion coefficient (Dtrans) increases linearly with decrease in pH in acidic medium and increases linearly with increasing pH in the basic regime. The rotational diffusion (Drot) shows similar dependencies on pH except a break at pH {approx} 4 corresponding to acidic residue pKa values. Our results may be useful to identify ligand binding of lysozyme in extreme pH conditions.

G-quadruplex (GQ) structures within the HIV-1 long terminal repeat (LTR) regulate viral transcription and represent promising antiviral targets; however, detailed mechanistic understanding of their ligand recognition at the molecular level remains limited and has largely been investigated under dilute conditions despite the crowded and compartmentalized nature of intracellular environment. Here, we investigate the interaction of the cationic porphyrin TMPyP4 with the HIV-1 LTR-III GQ under dilute conditions and inside protein-rich phase-separated condensates that mimic intracellular biocondensates. Steady-state and time-resolved fluorescence measurements reveal a dual binding behavior that is not discernible from absorption spectroscopy. A high-affinity guanine-rich binding mode leads to efficient fluorescence quenching through electron transfer from ground-state guanine to excited TMPyP4, whereas a weaker non-guanine binding mode gives rise to enhanced and long-lived emission. Nucleotide-specific control experiments validate the origin of these distinct binding environments. Molecular docking and molecular dynamics simulations further support preferential binding of TMPyP4 at the terminal G-quartet together with a secondary binding mode near the quadruplex-duplex junction. Importantly, both TMPyP4 and LTR-III GQ preferentially partition into the condensates, where the hybrid GQ structure, dual binding behavior, and associated excited-state signatures remain preserved despite the crowded and viscous environment. Although a slight reduction in binding affinity is observed inside the condensates, the overall binding mechanism remains largely preserved due to compensatory effects arising from the condensate microenvironment. Overall, this work demonstrates that ligand recognition of viral GQ remains preserved within protein condensates and establishes fluorescence spectroscopy as a sensitive approach for resolving hidden binding heterogeneity in GQ-ligand interactions.

Ultraviolet (UV) radiation induces DNA damage associated with cancer and aging, yet the sequence dependence of UV absorption remains to be investigated. Here, we present a systematic study of the UV absorption spectra of DNA based on all-electron Hartree-Fock calculations. We analyze all possible sequences up to four base pairs, as well as longer randomized sequences and genomic nullomers - motifs which are missing in a given genome. We observe a pronounced sequence dependence: cytosine- and guanine-rich motifs exhibit significantly enhanced absorption, whereas adenine-thymine-rich sequences absorb up to four times less in the mid-UV range. Notably, the human genome is biased toward adenine-thymine-rich sequences, giving it an increased susceptibility to UV-induced damage. In addition, we introduce a computational framework enabling spectral calculations of large DNA and RNA fragments, opening the door to large-scale optical analyses.

HIV-1 buds from infected cells as immature virion particles with a scattered envelope glycoprotein (Env) distribution on their envelope. It then undergoes maturation, during which the viral protease cleaves the Gag polyprotein at multiple sites, leading to structural reorganization of the viral particle and lateral redistribution of Env proteins, ultimately rendering the virion infectious. However, the underlying mechanism of maturation-induced Env reorganization remains elusive. In this study, we combine microsecond-long all-atom (AA), bottom-up coarse-grained (CG) molecular dynamics simulations, and diffusion model-based backmapping to investigate the structural organization and key interactions of Env in viral membranes. AA simulations of fully glycosylated Env embedded in HIV-1 mimetic asymmetric bilayers were first performed to characterize its conformational dynamics and Env-lipid interactions. We then developed a bottom-up CG model of glycosylated Env from that AA data and simulated the mature HIV-1 virion envelope containing multiple Env proteins. The CG simulations predict that Env proteins form clusters through interactions mediated by the cytoplasmic tail domain (CTD) and adopt diverse tilted conformations within these clusters. These CG simulations were then backmapped to AA resolution and further AA simulations were carried out to identify, in detail, the specific interacting residues in the Env clusters. Additionally, analysis of epitope accessibility shows that broadly neutralizing antibodies (bnAbs) targeting the V1/V2 and V3 loops may efficiently interact with Env clusters on the mature virion surface. Together, these results provide a molecular mechanism for Env oligomerization during viral maturation and offer new insights into the accessibility of bnAb epitopes on Env clusters.

The conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@dcf96aorg.highwire.dtl.DTLVardef@17acdc7org.highwire.dtl.DTLVardef@15bdc2borg.highwire.dtl.DTLVardef@1d39f3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Elucidating the structure and dynamics of insulin and its analogs has been of broad interest, while presenting challenges due to the unique structural dynamics of insulin (composed itself of two multiply cross-linked peptides A and B) and its ability to assemble in a variety of oligomeric structures under physiological conditions. Here, we present two distinct X-ray crystallographic structures of the long-acting human insulin analog detemir (INSD) resolved in hexameric and dodecameric (or dihexameric) states at 2.85 [A] and 2.70 [A] resolution, respectively, using diffraction data collected under ambient temperature conditions. Characterization of the collective dynamics of these oligomers using the Gaussian Network Model (GNM) reveals several key features: (i) Oligomerization imparts high cooperativity in structural dynamics evidenced by dissection of the cross-correlations at various hierarchical levels; (ii) detemir monomers conformational flexibility is highly suppressed within oligomeric constructs, the effect being particularly strong in the dihexamer due to the asymmetric packing of the hexamers and the presence of myristoyl groups at B peptides termini whose interactions imparts further heterogeneities; and (iii) a number of key residues retain, however, their intrinsic dynamics, to be deployed upon release from the oligomers. We distinguish in particular residues serving as hinge sites that mediates the conformational dynamics of the asymmetric units (dimers) and monomers (I2A-V3A and Y19 A -C20A, and L11B-L15B and Y26B of the respective peptides A and B), or as anchors supporting structural stability (disulfide-bridge forming cysteines, plus selected residues such as L16A, G8B and R22B-F24B. Overall, this study provides a structural-dynamic framework for gaining new insights into the dynamics of long-acting analog INSD and helps identify actionable sites for modulating insulin (analogs) dynamics toward designing more effective therapeutics.

Recent advances in cryo-electron microscopy and cryo-electron tomography have dramatically increased the number of class A G protein-coupled receptor (GPCR) structures, especially in previously inaccessible G protein-bound, active-like conformations. The increased structural diversity provides a unique opportunity to explore the conformational landscape underlying GPCR activation. To this end, we developed a machine learning (ML) framework that utilizes experimental structural data to elucidate the activation dynamics of class A GPCRs. We find that receptors can populate both inactive and active-like conformations even in the absence of ligand or G protein, providing a structural basis for agonist-free basal activity. Agonist binding shifts this conformational ensemble towards the active state but does not fully stabilize it. Instead, a stable active state is only established upon G protein binding, which locks the receptor in its active conformation. These results support a hybrid activation mechanism in which ligand binding follows conformational selection, while transducer engagement is governed by induced fit. Beyond clarifying class A GPCR activation, the openly available and modifiable ML framework provides a practical tool for analyzing newly determined structures, investigating the mechanisms of action of other GPCR classes and protein families, and guiding structure-based drug discovery in important pharmacological superfamilies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/714415v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19d1327org.highwire.dtl.DTLVardef@1549782org.highwire.dtl.DTLVardef@a6dfaaorg.highwire.dtl.DTLVardef@1a650ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

-Synuclein (Syn) is an intrinsically disordered protein whose interactions with lipid membranes are central to both its physiological function and its role in synucleopathies. While membrane charge, phase, and curvature are each known to influence Syn binding, these properties are typically examined independently, leaving their combined effects on both equilibrium and dynamic membrane association unresolved. Here, we systematically investigate how membrane phase and charge jointly regulate Syn binding, curvature sensitivity, and exchange dynamics using fluorescence microscopy, circular dichroism spectroscopy, and fluorescence recovery after photobleaching (FRAP), complemented by coarse-grained molecular dynamics simulations. Under zwitterionic conditions, Syn preferentially binds highly curved gel-phase membranes, driven by curvature-dependent enrichment of packing defects arising from faceted vesicle morphologies. Incorporation of anionic lipids selectively enhances binding in liquid-phase membranes while attenuating curvature-dependent partitioning in gel-phase membranes. Dynamic measurements reveal that membrane phase and charge also govern the stability of membrane-associated Syn, with gel-phase membranes and anionic lipids promoting kinetically stabilized states. Simulations show that curvature-induced defect formation is strongly amplified in gel-phase membranes but largely insensitive to charge. These findings establish that Syn-membrane interactions are governed by a cooperative interplay between membrane phase, curvature, and charge and highlight the importance of resolving both thermodynamic and kinetic contributions to protein-membrane binding.

Orthoflavivirus, such as West Nile Virus (WNV), dengue virus (DENV) and ZIKA virus (ZIKV), are globally distributed pathogens that pose substantial threats to human health. Currently, there are still no effective antiviral drugs for WNV or ZIKV. Despite the availability of two licensed DENV vaccines, their use remains limited due to potential risks, highlighting an urgent need for antiviral drug development. The highly conserved orthoflavivirus protease NS2B/NS3 is required for viral replication, making it a promising anti-flavivirus target. A major challenge, however, is the highly charged active site of this enzyme, which requires charged chemical matters with low bioavailability. An alternative and more attractive strategy is to target potential allosteric sites or folding intermediate states of the protease. In this work, we employ the topology-based coarse-grained G[o] modeling to explore the coupled binding and folding pathways of WNV NS2B/NS3 protease and study the effects of the widely used experimental construct with a G4SG4 linker between NS2B and NS3 on stability and folding. Our results provide a holistic conformational landscape of the protease binding and folding, including several key intermediate states. We find that the presence of the G4SG4 linker alters the folding pathways and destabilizes the NS2B C-terminus. The latter is consistent with experimental observations that the G4SG4 linked protease has lower activity and adopts an open state without the substrate in crystal structures. Together, these findings provide for the first time a complete picture of the binding and folding of the NS2B/NS3 protease and identify important folding intermediate states that could be targeted for allosteric antiviral drug development. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/722635v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@163c356org.highwire.dtl.DTLVardef@ad7b35org.highwire.dtl.DTLVardef@173ed8aorg.highwire.dtl.DTLVardef@1f026bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, cytochrome c peroxidase (CcP) generates a W191*+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base at position 232 (as Glu or His) recovers activity. E232 fluorination shifts the ET pH dependence to lower values, verifying that a hydrogen bond elevates the Y191* formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP*+, respectively, facilitate Y191* formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions reveal that W191*+ lies [~]15 mV in potential below ZnP*+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, ZnCcP Y,G191:E,H232 variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP*+/Trp*+ and the importance of a hydrogen bond to Y191* for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.

Avian phototransduction and magnetoreception have been proposed to involve shared retinal proteins, including interactions between long-wavelength opsin (LWO), the cone-specific heterotrimeric G protein (Gt), and cryptochrome 4a (Cry4a), yet structural information on avian phototransduction complexes is lacking. Here we present and critically assess two atomistic models of the European robin LWO-Gt complex generated by distinct modelling strategies. A full-complex prediction using AlphaFold3 yields a tightly packed, structurally stable interface but exhibits pronounced activation-like conformational features of the Gt-subunit that persist in simulations of the isolated protein, revealing a strong bias toward the active state. In contrast, a template-guided assembly based on single-chain predictions and an experimental rhodopsin-Gt reference structure forms a weaker interface and shows no intrinsic activation bias, while still displaying subtle activation-related dynamics. These results demonstrate that machine-learned complex prediction can encode functional states independently of the local interaction environment, thereby limiting its interpretability for signalling mechanisms that hinge on activation equilibria. Our findings highlight the need for explicit assessment of conformational-state bias when modelling regulatory protein assemblies and provide a structural framework for future studies of Cry4a-dependent modulation of retinal G-protein signalling in avian magnetoreception.

Disease-related missense mutations reshape protein conformational energy landscapes, thereby altering biological function. However, mechanistically linking sequence variation to changes in conformational dynamics remains challenging for both experimental and computational approaches. Here, we introduce an internal-coordinate-based, essential-dynamics-refined elastic network model (ICed-ENM) that improves the physical fidelity of normal modes while capturing subtle mutation-induced side-chain effects and preserving computational efficiency. By constraining bond-length and bond-angle fluctuations and refining mode subspaces against experimentally observed collective motions, ICed-ENM provides a stable, structure-encoded description of intrinsic protein dynamics. Building on this framework, we developed a systematic mutation-scanning analysis that quantifies mutation impact as changes in vibrational entropy, providing a dynamic measure of mutation-induced redistribution within conformational energy landscapes. Validation against all-atom molecular dynamics simulations demonstrates that residues predicted as mutation hot spots induce substantial reshaping of free-energy landscapes, consistent with altered intrinsic conformational dynamics. Extending this analysis across a curated protein structure dataset reveals global patterns of mutation sensitivity across diverse structural and physicochemical contexts. Notably, these trends align with large-scale public mutation datasets, suggesting that our framework captures features relevant to pathogenic variation. Together, ICed-ENM and the associated mutation-scanning pipeline provide a scalable and mechanistically interpretable strategy to identify mutation-sensitive regions and substitutions, offering deeper insight into how sequence variation reshapes functional conformational landscapes.