Physical Chemistry Chemical Physics

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

Intercalation of small molecules between DNA base pairs affects DNA conformation, disrupting essential cellular processes including replication, transcription, and repair. We investigated conformational changes in 18-mer DNA upon intercalation of doxorubicin, SYBR Gold and YOYO-1 using extensive MD simulations. Two main patterns for the intercalation were identified: RISE-type intercalation occurs between adjacent base pairs and extends the DNA helix with decreased twist angles, while OPEN-type intercalation proceeds through base-pair opening without significant DNA extension. Kinetic analysis revealed that association rates for intercalation followed the order: first YO-moiety (mono-intercalation) > SYBR Gold > doxorubicin > YOYO-1 (bis-intercalation). Free energy landscape showed that forces at DNA termini reached up to 117 pN during stretching. Notably, base pairs adjacent to intercalators were protected from strand separation, accompanied by additional helical unwinding. MM-PBSA/GBSA analysis revealed that the driving force for intercalation is the stacking energy, and the binding affinity was highest for minor groove binding. Persistence length decreased with single molecule binding but recovered with two molecules due to their electrostatic repulsion. Mechanical properties of intercalated DNA showed position-dependence, demonstrating that multiple intercalation modes coexist in solution. The heterogeneous nature of intercalation explains why experimental measurements reflect ensemble averages rather than single binding configurations.

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

Reactive oxygen species generated during inflammation can oxidize viral envelope lipids, with outcomes ranging from modulated infectivity to viral inactivation. For SARS-CoV-2, the molecular mechanisms by which membrane lipid oxidation influences spike protein anchoring remain poorly understood. We use all-atom molecular dynamics (MD) simulations to quantify how graded oxidation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) affects the anchoring of the SARS-CoV-2 spike transmembrane (TM) region in an endoplasmic-reticulum-Golgi intermediate compartment (ERGIC)-like multicomponent membrane. Viral envelopes containing 0, 25, 50, 75, and 100% oxidized POPC (PoxnoPC) corresponding to 0 - 55% oxidation of all PO-type phospholipids were simulated with the spike TM helix and cytoplasmic tail embedded in a POPC/POPE/POPI/POPS/cholesterol mixture. Steered MD and umbrella sampling were used to calculate the potential of mean force (PMF) for extracting the TM+CT region along the membrane normal. Partial oxidation (25 - 75% POPC) produced reductions in the detachment barrier that were not statistically distinguishable from the native system within the sampling uncertainty, whereas full POPC oxidation lowered the anchoring free energy by about 23% (from 606 {+/-} 39 to 464 {+/-} 38 kJ mol-1), indicating that oxidation of roughly half of the glycerophospholipids can measurably weaken spike-membrane coupling. Despite this reduction, the remaining barrier (about 180kBT ) is still large, suggesting that oxidation alone may be insufficient for spontaneous spike detachment and likely acts synergistically with mechanical forces during fusion or immune engagement. Analysis of acyl-chain order parameters, area per lipid, membrane thickness, number-density profiles, and lateral lipid clustering reveals that POPC peroxidation decreases lipid order, thins and softens the bilayer, and disrupts cholesterol-stabilized clusters that refer to large cooperative lipid assemblies (>10 lipids) identified via RDF-based clustering. These oxidation-induced changes reduce hydrophobic matching around the TM helix and facilitate its extraction from the viral envelope. Our results provide a mechanistic link between lipid peroxidation, membrane nanostructure, and spike anchoring, supporting lipid oxidation for example during cold atmospheric plasma or ozone treatment as a physically grounded contributing antiviral mechanism against SARS-CoV-2.

Amyloid beta (A{beta}), one of the hallmark proteins of Alzheimers Disease (AD), aggregates into plaques that are strongly linked to cognitive decline and neuronal death. Reducing its aggregation propensity may provide a strategy to slow the progression of AD. While chirality modulation has emerged as an innovative approach to disrupt this process, research has primarily focused on alterations at the C position, often overlooking the impact of the second chiral center, such as the C{beta} atom of Threonine. Furthermore, the underlying mechanisms governing these chiral effects remain elusive. Given the intrinsically disordered nature of the A{beta} peptide, we employed temperature-replica exchange molecular dynamics (T-REMD) simulations to explore its rugged conformational landscape. We considered sequence mutations (A2T, A2V), N-terminal chirality inversion of the first six residues (A2V1-6D and WT1-6D), and alteration of the second chiral center (C{beta}) of Threonine (A2TC{beta}). By analyzing the effect size and population change induced by these mutations and chiral modulation, we concluded that the modulation at the N-termini is not confined locally but also exerts specific effects on the central hydrophobic core (CHC) region. Inspection of their free energy landscape and representative structures reveals that the protective or pathogenic effects of these variants correlate with their similarity to the wild type (WT) ensemble. Beyond these static thermodynamics analyses, a direct connection to phase transitions was made by estimating heat capacity as a function of temperature. Both analyses predict that A2TC{beta} may exert a pathogenic effect, in contrast to the protective nature of A2T. These findings offer a deeper understanding of the effects of site-specific mutations and chirality and shed light on the development of advanced therapeutic strategies for AD.

The apparent pKa of ionizable lipids in lipid nanoparticles (LNPs) is a key determinant of RNA encapsulation during formulation and endosomal release after cellular uptake. However, it is difficult to predict the effective pKa of a given ionizable lipid solely from its solution pKa, because it is sensitive to the membranes composition, as well as solution conditions such as the salt concentration. We developed a simple continuum electrostatics model, based on Gouy-Chapman theory, to predict the shift in effective pKa for ionizable lipids in lipid bilayers as a function of salt concentration and membrane composition. We derive equations for the surface potential and fraction of lipids charged, which are solved self-consistently as a function of solution pH to extract the titration curve and effective pKa. The model shows that the shift in effective pKa is largest when the concentration of titratable lipid is high, and the effect is diminished by increasing salt concentration. We provide a python implementation of the model and an interactive notebook that will allow users to further easily explore the predicted pKa shifts as a function of formulation variables.

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

Accurate ion force fields are essential for molecular dynamics simulations of biomolecular systems, particularly in combination with modern water models such as OPC. While OPC water improves the description of bulk water and biomolecules, the transferability of existing ion force fields to this model remains an open question. Here, we systematically assess the transferability of monovalent and divalent ion force field parameters (Li+, Na+, K+, Cs+, Mg2+,Ca2+, Sr2+, Ba2+, Cl- and Br-) to OPC water by comparing single-ion and ion-pairing properties with experimental data. Our analysis reveals that no single literature parameter set provides accurate results for all ions when directly transferred to OPC water. We hence introduce the MS/G-LB(OPC) force field, which combines Mamatkulov-Schwierz-Grotz cation parameters with Loche-Bonthuis anion parameters. MS/G-LB(OPC) reproduces hydration free energies, first-shell structural properties and activity derivatives at low salt concentrations. Our results demonstrate that transferring ion parameters to OPC can lead to significant and ion-specific deviations from experimental data, making careful validation essential. At the same time, the systematic transfer and combination of ion parameters from existing force fields can provide a practical and computationally efficient alternative to full reparameterization. MS/G-LB(OPC) is available at https://git.rz.uni-augsburg.de/cbio-gitpub/opc-ion-force-fields.

Molecular dynamics (MD) simulations are a powerful tool for investigating biomolecular dynamics underlying biological functions. However, the accessible spatiotemporal scales of conventional all-atom simulations remain limited by high computational costs. Coarse-graining reduces these costs by decreasing the number of interaction sites and enabling longer timesteps. In extreme cases, proteins are represented as single spherical particles; while such approximations facilitate cellular-scale simulations, they often sacrifice essential structural information, such as molecular shape and interaction anisotropy. Here, we present CGRig, a rigid-body protein model with residue-level interaction sites designed for long-time, large-scale simulations. In CGRig, each protein is treated as a single rigid-body embedding residue-level interaction sites. Its translational and rotational motions are described by the overdamped Langevin equation incorporating a shape-dependent friction matrix. Intermolecular interactions are calculated using G[o]-like native contact potentials, Debye-Huckel electrostatics, and volume exclusion. We validated that CGRig accurately reproduces the translational and rotational diffusion coefficients expected from the friction matrix for an isolated protein. For dimeric systems, the model successfully maintained native complex structures. Furthermore, two initially separated proteins converged into the correct complex with an association rate consistent with all-atom simulations. Notably, CGRig achieved a simulation performance exceeding 17 s/day for a 1,024-molecule system. These results demonstrate that CGRig provides an efficient framework for simulating protein assembly while retaining residue-level interaction specificity, making it a valuable tool for investigating large-scale biomolecular self-assembly.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

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.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Microplastic (MP) pollution, which is present in the ecosystem in vast quantities, adversely affects human health and the environment, making it imperative to develop methods for its mitigation. The challenge of detecting or capturing MPs could potentially be addressed using plastic-binding peptides (PBPs). The ideal PBP for MP remediation would not only bind strongly to plastic, but also have other properties such as high solubility in water or great binding specificity to a certain plastic. However, the scarcity or absence of known PBPs for common plastics along with the lack of methods that can discover PBPs with all of the desired properties precludes the development of peptide-based MP remediation strategies. In this study, we discovered short linear PBPs with high predicted water solubility and binding specificity by employing an in-silico discovery pipeline that combines deep learning and biophysical modeling. First, a long short-term memory (LSTM) network was trained on biophysical modeling data to predict peptide affinity to plastic. High affinity peptides were generated by pairing the trained LSTM with a Monte Carlo tree search (MCTS) algorithm. Molecular dynamics (MD) simulations showed that the PBPs discovered for polyethylene, the most common plastic, had 15% lower binding free energy than PBPs obtained using biophysical modeling alone. PBPs with both high affinity and high predicted solubility in water were found by including the CamSol solubility score in the MCTS peptide scoring function, increasing the average solubility score from 0.2 to 0.9, while only minimally decreasing affinity for polyethylene. The framework also discovered peptides with high binding specificity between polystyrene and polyethylene, two major constituents of MP pollution, using a competitive MCTS approach that optimized the difference in affinity between the two plastics. MD simulations showed that competitive MCTS increased the binding specificity of PBPs for polystyrene and identified peptides with relatively great preference for either of the two plastics. The framework can readily be applied to design PBPs for other types of plastic. Overall, the high-affinity PBPs with desirable properties discovered by marrying artificial intelligence and biophysics can be valuable for remediating MP pollution and protecting the health of humans and the environment.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biomolecular condensates formed by complex coacervation of highly charged proteins provide a powerful framework to understand how microscopic interactions give rise to macroscopic material properties. Atomistic molecular dynamics simulations provide detailed insights but remain limited in accesing the spatio-temporal scales relevant for condensate behavior. Here, we use the residue-level coarse-grained Mpipi-Recharged model to investigate condensates formed by ProT and positively charged partners, including histone H1, protamine, poly-lysine, and poly-arginine. Material properties, in this context, provide a stringent experimental benchamark for coarse-grained models. Our model reproduces salt-dependent phase behavior, protein binding affinities, and sequence-specific stability trends in agreement with in vitro experiments, despite the fact that material properties were not included in the model parametrization. We then establish a direct link between protein dynamics and macroscopic material properties by quantifying monomeric diffusion, conformational reconfiguration, and translational mobility within the dense phase, and relating these to condensate viscosity. By comparing dynamics across dense and dilute phases, we uncover a pronounced length scale-dependent behavior. While residue-level binding and unbinding events remain equally fast in both phases, protein reconfiguration time and self-diffusion are significantly slowed down within the condensates. This decoupling reveals how fast intermolecular interactions coexist with slow mesoscale condensate dynamics depending on the molecular length scale. Together, our results establish a predictive framework that links encoded sequence intermolecular forces and multiscale dynamics to the emergent material properties of complex biomolecular condensates.

Mitochondrial potassium channels, located in the inner mitochondrial membrane, play a crucial role in the cells life/death phenomenon. Activation of mitochondrial potassium channels by potassium channel openers may protect cells against ischemia-reperfusion injury. It is known that mitochondrial large conductance calcium-activated potassium channels interact with various mitochondrial proteins, including enzymes of the respiratory chain. Numerous studies indicate that the mitochondria, especially cytochrome c oxidase, play a crucial role as a chromatophore in the cellular response to red and near-infrared light. In this study, we employ the patch-clamp technique and single-channel recordings to investigate the regulation of glioblastoma mitochondrial large conductance calcium-activated potassium channel activity by infrared light. Specifically, we examined the effects of wavelengths 620 nm, 680 nm, 760 nm, and 820 nm in a redox-controlled environment. Our findings suggest that illuminating the inner mitochondrial membrane with these wavelengths may activate mitochondrial large conductance calcium-activated potassium channels. These results offer new insights into the regulation of mitochondrial potassium channels by cytochrome c oxidase, which may lead to the development of non-pharmacological interventions with potential cytoprotective benefits.

Osmotic pressure has been known to play essential roles in living systems from single cells to complex tissues. However, direct in-situ measurements of osmotic pressures in biosystems have remained challenging, especially in complicated heterogeneous systems in which osmotic pressure gradients could exist and induce directed forces. Bacterial biofilms -- organized communities of bacteria encased in a self-produced extracellular matrix -- are a major mode of bacterial life. It has, however, remained unexplored how the osmotic pressure is distributed in the biofilm and how this distribution contributes to biofilm growth and activity. Here, liposomal nano-sensors are developed for the in-situ mapping of osmotic pressures at an unprecedented microscale resolution in real time using Escherichia coli. biofilm as a model system that develops at the surface of a hydrogel containing the nutrients. The measurements reveal osmotic pressure gradients with a radially increasing trend from the inner regions to the outer regions of the biofilm, which is associated with biofilm formation, morphology, and metabolism. The gradients likely contribute to mechanical properties, internal stresses, and nutrient transport. The sensor readouts also show that there is an osmotic pressure difference between the biofilm and the adjacent medium, which may promote biofilm expansion through matrix swelling and bacteria growth via water and nutrient uptake from the surroundings. Our novel approach based on in-situ osmotic pressure mapping in a growing biofilm reveals a sophisticated spatial regulation of physical forces, which may inspire new models and approaches in the field of mechanobiology.

Light wavelengths modulate plant growth, metabolism, and physiology. Amaranthus, a C4 underutilized climate resilient crop with promising nutritional properties remained unexplored in terms of metabolite enrichment under monochromatic light wavelengths of visible spectrum. In current study, two cultivars of Amaranthus tricolor (green and red) were exposed to seven light regimes of photosynthetically active radiation (PAR; 400-700 nm): deep blue, blue, green, amber, red, deep red, far red, and their metabolic responses were captured using Gas Chromatography-Mass Spectrometry. The metabolic analysis revealed wavelength-specific reprogramming in the levels of organic acids, sugars, amino acids, fatty acids as well as phenolics. In both the green and red Amaranthus, branched-chain amino acids and phenylalanine, which are nutritionally essential, were significantly elevated under far-red light. While the phenolics such as caffeic acid and ferulic acid were elevated under green and deep blue light respectively in green Amaranthus, amber light wavelengths enhanced these phenolics in red Amaranthus. The study highlighted cultivar-specific metabolic rewiring triggered by specific wavelengths. Altogether, these findings provides insights into metabolic adaptation and demonstrate the ability of light wavelength to specifically enrich the targeted metabolite of nutritional relevance in Amaranthus. It offers strategies to improve the nutritional value of crops in controlled agriculture systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/714947v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1a4477dorg.highwire.dtl.DTLVardef@518550org.highwire.dtl.DTLVardef@7682dorg.highwire.dtl.DTLVardef@4876e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ubiquitin-like protein 3 (UBL3) is a post-translational modifier that sorts proteins into small extracellular vesicles and regulates the trafficking of disease-associated proteins such as -synuclein. The structural and dynamic features of the UBL domain that underlie these functions, however, remain poorly understood. Here we performed in silico structural dynamics analysis of the UBL3 UBL domain using an NMR structure ensemble combined with anisotropic network modeling (ANM) and perturbation response scanning (PRS). Principal component analysis and residue-wise fluctuation analysis consistently revealed high flexibility in the C-terminal region of UBL3. Comparative ANM analysis across 20 ubiquitin-like proteins (UBLs) further showed that C-terminal flexibility is a conserved yet variable property within the UBL family. PRS analysis demonstrated that residues forming the central -helix of the {beta}-grasp fold exert greater dynamic control over collective motions than {beta}-sheet residues. Notably, UBL3 displayed the highest helix/sheet PRS effectiveness ratio among all UBLs analyzed, highlighting the prominent dynamic contribution of helix residues in this domain. Together, these results provide a structural basis for understanding UBL3-dependent protein interactions and disease-related mechanisms, and suggest that helix-centered dynamic control in the UBL domain may represent a potential target for modulating UBL3 function.

Ligand binding has been shown to induce significant alterations in the conformational landscape of proteins. Traditional crystallography approaches have provided valuable input about the end states in ligand-binding reactions. However, dynamical relationships between ligand binding and backbone rearrangement often remain obscured by crystallographic structures. In the present study, we use time-resolved serial synchrotron crystallography (TR-SSX) to directly visualize indole binding in the cavity of T4 lysozyme L99A in microcrystals under controlled environmental conditions. By integrating fixed target crystallography with LAMA-based ligand delivery, we have been able to track the progression of ligand binding and backbone rearrangement. By utilizing an occupancy refinement protocol, we have been able to quantify structural populations. Our studies reveal that ligand binding for this protein cavity follows a diffusion-limited process that progressively rearranges the F -helix of the protein towards a dominant conformational state. These findings establish an observable link between ligand diffusion, occupancy evolution and conformational adaptation within a crystalline environment. More broadly, our work shows how TR-SSX can quantify ligand and conformational populations during binding, providing a framework to interpret structural adaptation in real time.