Biophysics and Physicobiology

Formation of the {beta}-amyloid (A{beta}) plaques is a pathological hallmark of Alzheimers disease (AD), and is believed to be a primary cause of dementia in elderly individuals. In the present work, we simulated the conformational evolution of A{beta}42 dimers in solution and in membrane-like environment to explore the folding of A{beta}42 along fibrillation. The molecular dynamics (MD) simulation was steered by experimental internuclear distance restraints obtained using solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Our results revealed that several hydrophobic and polar motifs within the A{beta}42 sequence played key roles in the early-stage nucleation process of fibrillation and those motifs are also the stabilizing agents in the mature fibrils judged by the energy contribution. Our results also indicated that the peptide association with membrane bilayers could modulate the structural evolution pathways towards fibrillation. These findings contributed to a better understanding of the molecular level structural polymorphisms inherent to A{beta}42 fibrils. Further, the current work demonstrated that the combination of MD simulations with ssNMR-based experimental restraints provided a reliable method for studying structural changes of A{beta}. HighlightO_LIUsing solid-state NMR restraints guided molecular dynamic simulation, {beta}-amyloid dimers displayed consistent {beta}-strand-prone regions, which are major stabilizing segments for mature fibrils. C_LIO_LI{beta}-amyloid dimers evolved differently with or without interacting with the lipid bilayers. C_LIO_LIExperimental restraints guided simulation provided molecular level insights about early-stage interactions along the progress of {beta}-amyloid fibrillation C_LI

When three cyanobacterial proteins--KaiA, KaiB, and KaiC--are incubated with ATP in vitro, the phosphorylation level of KaiC exhibits stable circadian oscillations. Biochemical and structural analyses have shown that KaiCs ATPase activity is crucial for these oscillations, leading to the hypothesis that ATP-consuming dynamics function as a molecular clock, determining the oscillation period of individual molecules. Moreover, these molecular clocks synchronize with one another, resulting in collective oscillations at the ensemble level. In this study, we develop a theoretical model to test this molecular clockwork hypothesis. Our model clarifies the relationship between the oscillation period and ATPase activity, explaining the significant changes in the period induced by amino-acid substitutions near the CI-CII domain boundary of the KaiC hexamer. Furthermore, the model addresses the physical basis for temperature compensation concerning both the oscillation period and ATPase activity. Thus, the molecular clockwork perspective provides a framework for understanding the atomic design behind collective oscillations.

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.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are are a family of voltage-gated, cyclic-nucleotide modulated Na+/K+ channels that regulate spontaneous rhythmic electrical activity in both the heart and the brain. Understanding differences in the responsiveness to cyclic adenosine monophosphate (cAMP) modulation between HCN isoforms would offer insight into the specific binding interactions that drive channel activation. Using all-atom molecular dynamics (MD) simulations and the free-energy perturbation (FEP) approach, we determined the absolute binding free energy of cAMP to the the cyclicnucleotide-binding domain (CNBD) of HCN isoforms 1-4. By studying the free-energy of ligand binding to the various isoforms of HCN, our study advances the understanding of HCN channel activation and modulation mechanisms. Overall, our work offers insight into explaining differences in channel sensitivity across the isoforms of HCN.

The HIV-1 Vpu protein aids viral adaptation by influencing host cell pathways via protein interactions. While Vpu is mainly found in plasma and endomembranes, we recently discovered a soluble form that forms a stable, equimolar complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially affecting Vpus cellular trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y mutant with substitutions in Vpus helix 1. All Vpus variants were labeled at residue L42C, and Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100-600 nM) revealed dissociation constants (Kd) and binding energies ({Delta}G) for heterocomplexes. FL Vpu-Ca2+-CaM showed high stability (Kd [~]40 nM,{Delta} G [~]10.1 kcal/mol), while the truncated C-terminal region and V22A/W23Y mutant formed less stable complexes with Ca2+-CaM (Kd[~]200 nM and 800 nM,{Delta} G [~]9 kcal/mol and [~]8.3 kcal/mol). This, a binding hot spot in Vpus CaM-binding motif in helix 1 was identified, which may control the stability of Vpu-Ca2+-CaM complex and Vpus insertion in the membrane: We hypothesize that upon delivery to the membrane, the hydrophobic helix 1 of Vpu dissociates from Ca2+-CaM and inserts in the lipid bilayer; thereafter, CaM dissociates from Vpu facilitated by the reduced Vpu-Ca2+-CaM complex stability. The findings from this study advance our understanding of HIV-1 Vpu interactions with cellular components and may aid the development of antivirals.

Nitrogen is essential for all life forms, and microorganisms prefer ammonium as a nitrogen source. Due to the low affinity of glutamine synthetase (GS) for ammonium, E. coli must maintain high intracellular ammonium (NH4+) concentrations to sustain its rapid growth. Under ammonium limitation, E. coli imports ammonium through the transporter AmtB and incorporates it into glutamine by using GS. On the basis of structural and mutagenesis information, mechanisms have been proposed for the transport of ammonia (NH3) and protons by AmtB through spatially (partly) separate routes. These mechanisms do not explain the required coupling between proton and ammonia transports. How does the membrane potential push the ammonia inward so as to attain high concentrations near GS? We here compare six candidate kinetic models of E. coli ammonium transport and assimilation in terms of how they reproduce experimental data from the literature: three variants of the electro-binding model in which the membrane potential affects AmtB-NH4+ binding, and three variants of the electro-flipping model in which it influences the conformational flip of the transporter. The computer simulations decide that the electro-binding models are 28 times more plausible than the electro-flipping models and suggest that the transmembrane electric potential affects AmtB-NH4+ binding from the cytoplasmic side. The addition of kinetic and thermodynamic features to existing structural information plus our requirement of an explanation of the coupling, suggest a new spatiotemporal mechanism of coupling of ammonia and proton flows in AmtB. Further simulations show that GS and AmtB regulation is coordinated via both the uridylyltransferase/uridylyl-removing enzyme (UTase) and 2-oxoglutarate binding, allowing the cell to minimize futile cycling while maintaining rapid growth. The free energy cost of transport-related futile cycling exceeded that of the GS reaction itself. Moreover, AmtB enabled robust growth under varying ammonium concentrations and pH levels, albeit at a cost of futile cycling that became substantial at low ammonium. These findings highlight the crucial roles of GS and AmtB in E. colis adaptations and provide new insights into the trade-off mechanism between nutrient acquisition and energy efficiency.

Molecular dynamics simulations provide a "computational microscope" by which molecular phenomena can be studied at atomic resolution. However, such simulations are often expensive, usually due to a combination of system size and timescale. Various enhanced sampling methods have been proposed to overcome these challenges. Despite their effectiveness, many suffer from artifacts from energetic biases guiding the simulations, or lack of effective progress coordinates. Proteins normal modes uniquely defined by their 3D fold capture their intrinsic dynamics and could provide unbiased guidance, but how to combine these modes with molecular dynamics to generate continuous, energetically unbiased pathways has been challenging. In this study, we demonstrate that conformations generated along from normal modes using adaptive anisotropic network model provide a physical, intuitive, and generalizable progress coordinate for weighted ensemble simulations, providing a boost in efficiency and a means to generate pathways for any protein system without prior knowledge.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

The NAD+ dependent deacetylase sirtuin-1 (SIRT1) is known to elicit cellular defenses against aging, cancer, and other aberrant pathologies. Previous studies have identified an intrinsically disordered region of SIRT1 comprised of N-terminal residues 1-52, herein referred to as motif A, which activates SIRT1 activity, likely through intramolecular interactions. Additionally, phosphorylation of N-terminal residues Ser27 and Ser47 has been shown to be important for regulating SIRT1 activity and stability. The lack of in vitro characterization of these effects hampers our further understanding of the role of motif A in SIRT1 regulation. In this study, we elucidate the role phosphorylation plays in motif As structure as well as its regulatory effects on SIRT1 activity against Ac-p65. We find that phosphomimetic mutation at Ser27 significantly increases the activation effect of motif A towards SIRT1. This result is supported by molecular dynamics simulations of the phosphomimetics, which reveal stabilization of different transient structures for motif A depending on whether Ser27 and Ser47 have been modified. A key finding suggested by this study is that phosphorylation of S27 appears to activate SIRT1 by causing motif A, which is intrinsically disordered in the WT, to fold into an ordered structure. This conclusion is based on both the experimental findings and simulation results. These findings contribute to our understanding of SIRT1 regulation, specifically the role played by phosphorylation within the N-terminal disordered region.

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

Invertebrate vision relies on bistable visual pigments flipping upon photon absorption between rhodopsin and metarhodopsin states. In living butterflies, the UV-VIS absorption spectra of rhodopsin and metarhodopsin, respectively with 11-cis and all-trans isomers of 3-hydroxy-retinal (A3) chromophore, can be conveniently recorded from the eyeshine, the light reflected from the compound eye after passing twice through the light-guiding rhabdoms. * Here, a microscope coupled with a broadband LED source and a microspectrometer was used to record photorelaxations reported in eyeshine reflection spectra. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra that are analogous to absorbance difference and sum, respectively. Both types of spectra were subjected to singular value decomposition and to fitting of templated visual pigment absorption spectra. * The compound eye of the high brown fritillary Fabriciana adippe was exposed to a series of second-long broadband light pulses, causing photorelaxations with time constants between 40 and 120 ms that led to 80% metarhodopsin in equilibrium. The transient and baseline spectra were fitted with pigment templates, estimating the alpha peak wavelength 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The metarhodopsin to rhodopsin alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength at 530 nm. The second isosbestic wavelength indicates that rhodopsin beta (UV) peak absorbs more strongly than metarhodopsin below 405 nm. * Baseline spectra, which were not explicitly analysed in previous studies, enable concatenation of exposures, monitor long-term changes of pigment, and enhance the estimation of beta peak parameters. * The method can be directly used in many butterflies and could be adapted to other insects, particularly fruitflies, facilitating studies of the relation between the visual pigment spectra and the opsin sequences. Spectroscopic results can be complemented with physiologically measured photoreceptor spectral sensitivity datasets and analysed with the same global fitting procedure.

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.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Single-crystal X-ray diffraction remains one of the most direct and reliable techniques for clarifying the three-dimensional structures of small molecules; however, its wider use in developing research settings has historically been limited by access to advanced instrumentation. Here, we consider the performance of the in-house diffractometer, Turkish Light Source, for small-molecule structure determination using three rhodanine-derivative compounds. Diffraction data were collected, processed, and followed by full-matrix least-squares refinement as a user-friendly pipeline. The compounds were successfully resolved in the triclinic space group P-1 and refined to chemically reasonable models, although notable differences in data quality and refinement parameters were observed. Compounds 1 and 2 produced the most robust and internally coherent structure, whereas compound 3 displayed refinement tribulations. These might be attributed to the intrinsic structural disorder of c-5b, analogous to polymorphic perversity in higher Z' phase, likely due to the presence of dissymmetric molecules within the asymmetric unit (Z' = 2), rather than empirical limitations. Anisotropic displacement parameters were systematically computed by atom-resolved Ueq factors and anisotropy index. The combined analyses reveal that structural ambiguity of c-5b is largely governed by localized maxima in atomic displacement (up to 0.29 [A]2 in Ueq with 6.67 anisotropy) rather than by global disorder, caused by the fluorinated aryl moiety of c-5b. These findings indicate that the in-house SCXRD system, when coupled with our user-friendly downstream pipeline, can yield reliable structural data for small molecules. Brief video tutorials and detailed SOPs have been provided in the Tutorials folder, including CrysAlisPro and Olex2 tutorials, as well as are easily accessible for users.

I-motif (iM) DNA structures, formed by cytosine-rich sequences, are increasingly acknowledged for their involvement in gene regulation, maintenance of genomic stability, and their emerging potential as therapeutic targets, particularly in cancer. Despite their biological relevance, the discovery of selective small-molecule probes that can specifically recognize and interact with iM DNA remains an ongoing challenge. In this study, we have used TMPyP4 and screened for its ability to bind various iM DNA constructs, including HRAS1, HRAS2, VEGF, CMYC, CKIT and H-Telo. Structure-activity relationship analyses revealed that specific substitution patterns conferred selectivity towards HRAS2 iM target. Comprehensive spectroscopic investigations, including UV-Vis absorption, steady-state and time-resolved fluorescence, and fluorescence anisotropy, uncovered key photophysical signatures of binding, including significant hypochromic and bathochromic shifts, enhanced fluorescence emission, and prolonged fluorescence lifetimes. Circular dichroism (CD),thermal denaturation (UV-melting) and thermodynamic investigations confirmed that TMPyP4 effectively stabilized the HRAS2 iM structures without disrupting their native topologies. Meanwhile, FT-IR spectroscopy revealed local structural rearrangements upon TMPyP4 binding, offering further evidence of molecular interaction. Collectively, these findings provide valuable insights into the molecular recognition of iM DNA by TMPyP4 and highlight its promise as both selective HRAS2 iM-binding agent and responsive fluorescent probe. This work lays a strong foundation for the development of novel tools for studying iM structures in biological systems and for designing future therapeutics targeting iM DNA in cancer and related diseases.

Signaling through the insulin receptor (IR) and the type 1 insulin-like growth factor receptor (IGF1R) is modulated by secreted hormones and growth factor ligands (e.g. insulin and insulin-like growth factor 1, IGF1). Impaired signaling in these receptors often leads to diabetes and oncogenic diseases. The discovery of entirely novel viral insulin/IGF-like peptides (VILPs) that can stimulate receptors from the insulin family has raised questions about their structures and binding modes to receptors. These peptides exist in a single-chain (sc) or a double-chain (dc) configuration with folds likely similar to IGF1 and insulin, respectively. The interactions of VILPs with the human receptors are beginning to be mapped but little is known about their interactions with the receptors in fish-the host organism for viruses known to carry these peptide sequences. We have previously reported [Chuard et al., Cell Rep. 2025 44(8):116149] structural models of several VILPs from the Iridoviridae virus family bound to their cognate receptors in Zebrafish (Zeb). In this work, we conducted all-atom molecular dynamics (MD) simulations of these peptides and their receptor-bound complexes along with free energy calculations to assess the energetic contributions of VILP residues for their binding to Zebrafish receptors. Most of the observed Zeb insulin/Zeb {micro}IR and Zeb IGF1/Zeb {micro}IGF1R site 1 interactions are consistent with previously known interactions of human peptides with their receptors, highlighting similarities in their binding modes. However, we also report some non-conserved residues in VILPs that establish significant and unique interactions with residues in Zeb receptors. Furthermore, we identified residues in each VILP which can be potentially mutated into conserved insulin/IGF1 residues to possibly enhance the binding affinity of these peptides.

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.

Conformational plasticity of RNAs plays important roles in recognizing RNA-binding proteins, and is often modulated by their binding partners. Here, we investigate RNA conformational preferences in a non-redundant dataset of 263 protein-RNA complexes to characterize the structural landscape associated with protein recognition. RNA dinucleotide segments are analyzed using seven backbone torsion angles ({delta}1, {varepsilon}1, {zeta}1, 2, {beta}2, {gamma}2, and {delta}2), two glycosidic torsion angles ({chi}1 and {chi}2) and the pseudo-torsion angle . Focusing on dinucleotide steps present in both interface and non-interface regions, we performed density-based clustering using selected backbone torsion angles to identify recurrent conformational states. We identify 28 distinct RNA dinucleotide conformers containing at least ten members each. Among these, eight conformers represent previously unreported nucleotide conformers (NtCs), including the transitional and the non-canonical states AB06, AB07, BB21, BB22, OP32, OP33, IC08 and IC09. Several of these conformers are preferentially enriched at protein-binding interfaces, suggesting their involvement in local conformational adaptation during protein-RNA recognition. The newly identified conformers span transitional A-B geometries, distorted B-like states, open conformations and compact intercalated structures, highlighting the remarkable structural plasticity of RNA in ribonucleoprotein complexes. Overall, this study expands the current understanding of RNA conformational space and provides a refined RNA dinucleotide conformer library for protein-RNA complexes. These findings will facilitate the identification of novel RNA structural motifs and improved RNA structural modeling, docking protein-RNA complexes and deep learning-based prediction frameworks for describing RNA tertiary structures.

Structure-based drug design has become increasingly important in the pharmaceutical industry for accelerating the discovery of effective drug candidates. In particular, ligand binding free energy serves as a critical metric for predicting drug efficacy during the key stages of hit discovery and lead optimization. Continuous progresses have been made in the prediction of ligand binding free energies, but direct comparisons of different methods using the same force field remain challenging due to their unique implementations into different simulation engines. In this study, we present a direct comparison of four popular methodologies (Amber-TI, GROMACS-NETI, OpenMM-FEP, and BLaDE-MSLD) for calculating relative binding free energies ({Delta}{Delta}Gbind) with the same Amber protein and ligand force fields using MolCube Alchemical Free Energy Simulator (MolCube-AFES), which provides an input generation workflow to support {Delta}{Delta}Gbind calculations of all four methods. We used 80 alchemical transformations (among the JACS benchmark set by Wang et al.) and two additional applications to compare the predicted {Delta}{Delta}Gbind from the four methods against experimental measurements. All four methods reproduced experimentally observed trends with most transformations within {+/-}2 kcal/mol from experiments and show broadly comparable accuracy with no statistically significant performance differences across the benchmark dataset. These results demonstrate that MolCube-AFES enables controlled, cross platform benchmarking and show that all four different alchemical free energy methods deliver statistically equivalent accuracy, with method selection guided by workflow requirements such as throughput, portability, and perturbation network design rather than expected differences in performances.