Conformational Adaptation Research Articles

Dynamic conformational response of von Willebrand factor to varying shear stress: A hybrid computational approach

This computational study examines the mechanobiological responses of von Willebrand Factor (vWF) to different shear stress levels, emphasizing the unique structural dynamics of its various domains. We introduce a novel coarse-grained model, representing each cluster of six amino acids as a bead, to synthesize the Lattice Boltzmann method with Brownian dynamics. This approach captures the vWF molecule’s conformational adaptability across a spectrum of shear rates, ranging from 5 to 5e + 7s−1, encompassing normal physiological flows to those resembling the high-shear environment of arterial stenosis.Our results reveal domain-specific responses of vWF, particularly in the A2 and A3 domains, which demonstrate significant elongation under elevated shear—reflecting their pivotal roles in clotting processes. Moreover, the study underscores the critical influence of random thermal forces on the molecule’s conformation, with simulations including these forces showing substantially greater conformational variability compared to those without.As shear rates increase, we observe a notable reorganization in the spatial arrangement of vWF domains. This reconfiguration is most evident in the increased separation between the A1 and D’D3 domains under high shear conditions, potentially enhancing the accessibility of platelets to key binding sites on vWF. These findings are paramount for a deeper understanding of vWF’s mechanical behavior in blood clotting and thrombosis, particularly the delicate balance between facilitating platelet adhesion and avoiding excessive stretching that could lead to thrombosis.Our work provides a foundation for future studies investigating vWF behavior in complex flow geometries and its interactions with other blood components. Such insights pave the way for more informed therapeutic strategies targeting vWF function in vascular health and disease, potentially leading to improved treatments for thrombotic disorders.

The solution structure of macrocyclic glecaprevir and its conformational adaptation mechanism towards antitumor targets

The realm of drug development and supramolecular chemistry has been revolutionized by the emergence of macrocyclic compounds, which have emerged as formidable contenders. Glecaprevir is an 18-membered macrocycle used for the treatment of chronic hepatitis C virus infection. The inherent structural flexibility of glecaprevir's side chain enables it to adopt diverse solution conformations, thereby influencing its binding modes with biological proteins and unlocking new potentials for the treatment of other diseases. The present study delved into the solution structural characteristics of glecaprevir through a combination of experimental UV–Vis and electronic circular dichroism spectra, complemented by theoretical calculations. Our findings reveal that glecaprevir consistently exhibits spectral characteristics in both protic and aprotic solvents due to its stable conformational distribution. DFT-assisted structural analyses were conducted to explore the weak noncovalent interactions within the stable conformations of glecaprevir. The molecular docking analysis conducted on a diverse range of conformers of glecaprevir revealed its exceptional selectivity against proteins associated with breast cancer, colorectal cancer, lung cancer, and lymphoma. These remarkable findings suggest that glecaprevir holds immense potential as a lead compound for drug design targeting these specific types of cancers. Furthermore, the conformationally adapted mechanism is indicated by the alteration of intramolecular hydrogen bonds subsequent to molecular docking.

Pyrrole-based acyclic hexapodal aldehyde for anion recognition

A novel acyclic hexapodal host (4a) featuring with multiple formyl groups, pyrrole cores, and ester linkages has been prepared and characterized through 1H NMR, 13C NMR, high-resolution mass spectrometry and single-crystal X-ray diffraction techniques. This versatile host molecule exhibits robust anion recognition for CN−, F−, SO42−, HSO4−, H2PO4−, and HP2O73− with the binding constants spanning 0.18 × 105 M−1 to 7.72 × 105 M−1. Interactions with these anions were elucidated through UV–visible and 1H NMR titrations, complemented by DFT calculations. At low guest concentrations (5- to 100-fold excess), a 1:1 complexation is predominant. However, at high concentrations (1000-fold excess), the host's flexibility promotes the assembly of other host-guest complexes. Comparative studies with related pyrrole-based hexapodal ester 4b and tetrapodal aldehyde 4c highlight the importance of peripheral formyl groups, pyrrolic NH groups, the number of pendant arms, and conformational adaptability in anion recognition. These insights underscore the potential of acyclic multipodal hosts in advancing supramolecular chemistry and anion recognition.

Adaptive Reaktion bei der Lösung von flexiblen Molekülen: Oligohydrate von 4‐Hydroxy‐2‐butanon

AbstractDurch Wasser hervorgerufene Strukturveränderungen spielen eine zentrale Rolle in der Chemie und Biologie, sind aber auf molekularer Ebene nach wie vor schwer vorherzusagen, zu messen und zu kontrollieren. Hier untersuchen wir in der Gasphase die größenabhängige Wasseraggregation am flexiblen Molekül 4‐Hydroxy‐2‐butanon und modellieren die Konformationsanpassungsfähigkeit flexibler Substrate an Wirtswassergerüste, sowie die Präferenzen für sequentielles Tröpfchenwachstum. Das Experiment wurde mittels breitbandiger Rotationsspektroskopie durchgeführt und mit quantenchemischen Berechnungen nachvollzogen. Experimentell wurden vom Di‐ bis zum Pentahydrat (4‐Hydroxy‐2‐butanon‐(H2O)n=2–5) zwei verschiedene Isomere beobachtet, einschließlich der 18O‐Isotopologen für die Di‐ und Trihydrate. Um Wassermoleküle effektiv unterzubringen, verändert sich interessanterweise das schwere Atomgerüst von 4‐Hydroxy‐2‐butanon in jedem beobachteten Isomer und entspricht nicht der stabilen Konformation des freien Monomers. Alle Solvate gehen von der Alkoholgruppe (Protonendonor) aus, behalten aber die Carbonylgruppe als sekundären Bindungspunkt bei. Die Wassergerüste ähneln stark denen in reinen Wasserclustern und balancieren zwischen der Fähigkeit von 4‐Hydroxy‐2‐butanon, die Orientierung und Position der Wassermoleküle zu steuern, und der Fähigkeit von Wasser, die Konformation des Monomers zu modulieren. Die vorliegende Arbeit liefert somit eine genaue molekulare Beschreibung, wie sich torsionsflexible Moleküle im Verlauf der fortschreitenden Solvatation dynamisch an Wasser anpassen.

Adaptive Response to Solvation in Flexible Molecules: Oligo Hydrates of 4-Hydroxy-2-butanone.

Structural changes induced by water play a pivotal role in chemistry and biology but remain challenging to predict, measure, and control at molecular level. Here we explore size-governed gas-phase water aggregation in the flexible molecule 4-hydroxy-2-butanone, modeling the conformational adaptability of flexible substrates to host water scaffolds and the preference for sequential droplet growth. The experiment was conducted using broadband rotational spectroscopy, rationalized with quantum chemical calculations. Two different isomers were observed experimentally from the di- to the pentahydrates (4-hydroxy-2-butanone-(H2O)n=2-5), including the 18O isotopologues for the di- and trihydrates. Interestingly, to accommodate water molecules effectively, the heavy atom skeleton of 4-hydroxy-2-butanone reshapes in every observed isomer and does not correspond to the stable conformer of the free monomer. All solvates initiate from the alcohol group (proton donor) but retain the carbonyl group as secondary binding point. The water scaffolds closely resemble those found in the pure water clusters, balancing between the capability of 4-hydroxy-2-butanone for steering the orientation and position of the water molecules and the ability of water to modulate the monomer's conformation. The present work thus provides an accurate molecular description on how torsionally flexible molecules dynamically adapt to water along progressing solvation.

Mixing water, sugar, and lipid: Conformations of isolated and micro-hydrated glycolipids in the gas phase.

Both sugars and lipids are important biomolecular building blocks with exceptional conformational flexibility and adaptability to their environment. Glycolipids bring together these two molecular components in the same assembly and combine the complexity of their conformational landscapes. In the present study, we have used selective double resonance vibrational spectroscopy, in combination with a computational approach, to explore the conformational preferences of two glycolipid models (3-0-acyl catechol and guaiacol α-D-glucopyranosides), either fully isolated in the gas phase or controlled interaction with a single water molecule. We could identify the preferred conformation and structures of the isolated and micro-hydrated species and evidence of the presence of a strong water pocket, which may influence the conformational flexibility of such systems, even in less controlled environments.

Integrin mechanosensing relies on a pivot-clip mechanism to reinforce cell adhesion

Cells intricately sense mechanical forces from their surroundings, driving biophysical and biochemical activities. This mechanosensing phenomenon occurs at the cell-matrix interface, where mechanical forces resulting from cellular motion, such as migration or matrix stretching, are exchanged through surface receptors, primarily integrins, and their corresponding matrix ligands. A pivotal player in this interaction is the α5β1 integrin and fibronectin (FN) bond, known for its role in establishing cell adhesion sites for migration. However, upregulation of the α5β1-FN bond is associated with uncontrolled cell metastasis. This bond operates through catch bond dynamics, wherein the bond lifetime paradoxically increases with greater force. The mechanism sustaining the characteristic catch bond dynamics of α5β1-FN remains unclear. Leveraging molecular dynamics simulations, our approach unveils a pivot-clip mechanism. Two key binding sites on FN, namely the synergy site and the RGD (Arg-Gly-Asp) motif, act as active points for structural changes in α5β1 integrin. Conformational adaptations at these sites are induced by a series of hydrogen bond formations and breaks at the synergy site. We disrupt these adaptations through a double mutation on FN, known to reduce cell adhesion. A whole-cell finite-element model is employed to elucidate how the synergy site may promote dynamic α5β1-FN binding, resisting cell contraction. In summary, our study integrates molecular- and cellular-level modeling to propose that FN’s synergy site reinforces cell adhesion through enhanced binding dynamics and a mechanosensitive pivot-clip mechanism. This work sheds light on the interplay between mechanical forces and cell-matrix interactions, contributing to our understanding of cellular behaviors in physiological and pathological contexts.

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers.

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

Exploring Binding Pockets in the Conformational States of the SARS-CoV-2 Spike Trimers for the Screening of Allosteric Inhibitors Using Molecular Simulations and Ensemble-Based Ligand Docking.

Understanding mechanisms of allosteric regulation remains elusive for the SARS-CoV-2 spike protein, despite the increasing interest and effort in discovering allosteric inhibitors of the viral activity and interactions with the host receptor ACE2. The challenges of discovering allosteric modulators of the SARS-CoV-2 spike proteins are associated with the diversity of cryptic allosteric sites and complex molecular mechanisms that can be employed by allosteric ligands, including the alteration of the conformational equilibrium of spike protein and preferential stabilization of specific functional states. In the current study, we combine conformational dynamics analysis of distinct forms of the full-length spike protein trimers and machine-learning-based binding pocket detection with the ensemble-based ligand docking and binding free energy analysis to characterize the potential allosteric binding sites and determine structural and energetic determinants of allosteric inhibition for a series of experimentally validated allosteric molecules. The results demonstrate a good agreement between computational and experimental binding affinities, providing support to the predicted binding modes and suggesting key interactions formed by the allosteric ligands to elicit the experimentally observed inhibition. We establish structural and energetic determinants of allosteric binding for the experimentally known allosteric molecules, indicating a potential mechanism of allosteric modulation by targeting the hinges of the inter-protomer movements and blocking conformational changes between the closed and open spike trimer forms. The results of this study demonstrate that combining ensemble-based ligand docking with conformational states of spike protein and rigorous binding energy analysis enables robust characterization of the ligand binding modes, the identification of allosteric binding hotspots, and the prediction of binding affinities for validated allosteric modulators, which is consistent with the experimental data. This study suggested that the conformational adaptability of the protein allosteric sites and the diversity of ligand bound conformations are both in play to enable efficient targeting of allosteric binding sites and interfere with the conformational changes.

Thermodynamics of a hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9

Hyperthermostable enzymes are highly desirable biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme exhibited remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new β-sheets at 80 °C, making the core of the hydrolase fold more stable. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, which resided at the interface between the large and cap domains. To further investigate the structural adaptation in extreme conditions, the intramolecular interactions of the native structure were investigated. EstD9 revealed 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters, which is higher than the previously reported thermostable Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to both fundamental and applied research involving protein engineering of industrial thermostable enzymes.

Comprehensive Analysis of Coupled Proline Cis-Trans States in Bradykinin Using ωBP-REMD Simulations.

It is well-known that proline (Pro) cis-trans isomerization plays a decisive role in the folding and stabilization of proteins. The conformational coupling between isomerization states of different Pro residues in proteins during conformational adaptation processes is not well understood. In the present work, we investigate the coupled cis-trans isomerization of three Pro residues using bradykinin (BK), a partially unstructured nonapeptide hormone, as a model system. We use a recently developed enhanced-sampling molecular dynamics method (ω-bias potential replica exchange molecular dynamics; ωBP-REMD) that allows us to exhaustively sample all combinations of Pro isomer states and obtain converged probability densities of all eight state combinations within 885 ns ωBP-REMD simulations. In agreement with experiment, the all-trans state is seen to be the preferred isomer of zwitterionic aqueous BK. In about a third of its structures, this state presents the characteristic C-terminal β-turn conformation; however, other isomer combinations also contribute significantly to the structural ensemble. Unbiased probabilities can be projected onto the peptide bond dihedral angles of the three Pro residues. This unveils the interdependence of the individual Pro isomerization states, i.e., a possible coupling of the different Pro isomers. The cis/trans equilibrium of a Pro residue can change by up to 2.5 kcal·mol-1, depending on the isomerization state of other Pro residues. For example, for Pro7, the simulations indicate that its cis state becomes favored compared to its trans state when Pro2 is switched from the trans state to the cis state. Our findings demonstrate the efficiency of the ωBP-REMD methodology and suggest that the coupling of Pro isomerization states may play an even more decisive role in larger folded proteins subject to more conformational restraints.

Solid-State Emissive Pillar[6]arene Derivative Having Alternate Methylene and Nitrogen Bridges.

Macrocyclic arenes show conformational adaptability, which allows host-guest complexations with the size-matched guest molecules. However, their emission properties are often poor in the solid states due to the self-absorption. Herein, we newly synthesized pillar[6]arene derivatives having alternate methylene and nitrogen bridging structures. Solvatochromic study reveals that the nitrogen-embedding into the cyclic structures can strengthen the intramolecular charge transfer (CT) nature compared to that of the linear nitrogen-bridged precursor. Owing to the large Stokes shift in the solid state, one of the nitrogen-embedded pillar[6]arenes shows high absolute photoluminescence quantum yield (ΦPL=0.36). Furthermore, it displays a turn-off sensing ability toward nitrobenzene (NB) vapor; a fluorescence quenching is observed when exposed to the NB vapor. From the structural analysis before and after the exposure of NB vapor, the amorphous nitrogen-embedded pillar[6]arene efficiently co-crystallize with NB and formed non-emissive intermolecular CT complexes with NB.

Multistate B- to A- transition in protein-DNA Binding – How well is it described by current AMBER force fields?

When DNA interacts with a protein, its structure often undergoes a significant conformational adaptation, usually involving a transition from B-DNA towards the A-DNA form. This is not a two-state, but rather a multistate transition. The A- and B- forms differ mainly in sugar pucker (north/south) and glycosidic torsion χ (anti/high-anti). The combination of A-like pucker and B-like χ (and vice versa) represents the nature of the intermediate states between the pure A- and B- forms. Here we study how the A/B equilibrium and the A/B intermediate states at protein-DNA interfaces are modeled by current AMBER force fields. Eight diverse protein-DNA complexes and their naked (unbound) DNAs were simulated with OL15 and bsc1 force fields and an experimental combination OL15χOL3. We found that while the geometries of the A-like intermediate states agree well with the native X-ray geometries, their populations (stabilities) are significantly underestimated. Different force fields predict different propensities for A-like states growing in the order OL15 < bsc1 < OL15χOL3, yet all underestimate A-like form populations. Interestingly, the force fields seem to predict the correct sequence-dependent A-form propensity, as they predict larger populations of the A-like form in unbound DNA in those steps that acquire A-like conformations in protein-DNA complexes. The instability of A-like geometries in current force fields significantly alters the geometry of simulated protein-DNA complexes and destabilizes the binding motif, suggesting that refinement is required to improve description of protein-DNA interactions in AMBER force fields. Communicated by Ramaswamy H. Sarma

Conformational mechanics and adaptability of tandem repeat proteins: Insights from elastic network models

Conformational mechanics and adaptability of tandem repeat proteins: Insights from elastic network models

GPCR-IPL score: multilevel featurization of GPCR-ligand interaction patterns and prediction of ligand functions from selectivity to biased activation.

G-protein-coupled receptors (GPCRs) mediate diverse cell signaling cascades after recognizing extracellular ligands. Despite the successful history of known GPCR drugs, a lack of mechanistic insight into GPCR challenges both the deorphanization of some GPCRs and optimization of the structure-activity relationship of their ligands. Notably, replacing a small substituent on a GPCR ligand can significantly alter extracellular GPCR-ligand interaction patterns and motion of transmembrane helices in turn to occur post-binding events of the ligand. In this study, we designed 3D multilevel features to describe the extracellular interaction patterns. Subsequently, these 3D features were utilized to predict the post-binding events that result from conformational dynamics from the extracellular to intracellular areas. To understand the adaptability of GPCR ligands, we collected the conformational information of flexible residues during binding and performed molecular featurization on a broad range of GPCR-ligand complexes. As a result, we developed GPCR-ligand interaction patterns, binding pockets, and ligand features as score (GPCR-IPL score) for predicting the functional selectivity of GPCR ligands (agonism versus antagonism), using the multilevel features of (1) zoomed-out 'residue level' (for flexible transmembrane helices of GPCRs), (2) zoomed-in 'pocket level' (for sophisticated mode of action) and (3) 'atom level' (for the conformational adaptability of GPCR ligands). GPCR-IPL score demonstrated reliable performance, achieving area under the receiver operating characteristic of 0.938 and area under the precision-recall curve of 0.907 (available in gpcr-ipl-score.onrender.com). Furthermore, we used the molecular features to predict the biased activation of downstream signaling (Gi/o, Gq/11, Gs and β-arrestin) as well as the functional selectivity. The resulting models are interpreted and applied to out-of-set validation with three scenarios including the identification of a new MRGPRX antagonist.

Elevated fucose content enhances the cryoprotective performance of anionic polysaccharides

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18–35.7 mol%), uronic acids (UA) (13.5–26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

Structural basis and selectivity of sulfatinib binding to FGFR and CSF-1R

Acquired drug resistance poses a challenge for single-target FGFR inhibitors, leading to the development of dual- or multi-target FGFR inhibitors. Sulfatinib is a multi-target kinase inhibitor for treating neuroendocrine tumors, selectively targeting FGFR1/CSF-1R. To elucidate the molecular mechanisms behind its binding and kinase selectivity, we determined the crystal structures of sulfatinib with FGFR1/CSF-1R. The results reveal common structural features and distinct conformational adaptability of sulfatinib in response to FGFR1/CSF-1R binding. Further biochemical and structural analyses disclose sensitivity of sulfatinib to FGFR/CSF-1R gatekeeper mutations. The insensitivity of sulfatinib to FGFR gatekeeper mutations highlights the indispensable interactions with the hydrophobic pocket for FGFR selectivity, whereas the rotatory flexibility may enable sulfatinib to overcome CSF-1RT663I. This study not only sheds light on the structural basis governing sulfatinib’s FGFR/CSF-1R inhibition, but also provides valuable insights into the rational design of dual- or multi-target FGFR inhibitors with selectivity for CSF-1R and sensitivity to gatekeeper mutations.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Conformational adaptation and large amplitude motions of 1-phenyl-2,2,2-trifluoroethanol with two water molecules: a rotational spectroscopic and ab initio investigation.

The 1 : 2 adduct of 1-phenyl-2,2,2-trifluoroethanol (PhTFE), a chiral fluoroalcohol, with two water molecules (PhTFE⋯2H2O) was investigated via chirped pulse Fourier-transform microwave (CP-FTMW) spectroscopy and theoretical calculations. A systematic search of the PhTFE⋯2H2O conformational landscape identified 38 stable minima at the B3LYP-D3BJ/def2-TZVPPD level of theory, 27 of which are within an energy window of 10 kJ mol-1 after applying zero-point energy corrections. Rotational spectra of a single PhTFE⋯2H2O conformer along with eight deuterated and three oxygen-18 isotopologues were assigned. Interestingly, the observed PhTFE⋯2H2O conformer contains PhTFE II, the second most stable monomer conformer, and the most stable PhTFE I dihydrate is ca. 4 kJ mol-1 higher in energy. In contrast, PhTFE I⋯H2O was identified experimentally and theoretically as the most stable 1 : 1 conformer. Furthermore, the observed dihydrate structure experiences large amplitude motions connecting three theoretical minima which differ only in which water oxygen lone pairs are involved in the hydrogen-bonds, i.e., the free OH pointing directions. Additionally, the ortho and para-H2O tunnelling splittings were detected and attributed to the interchange water hydrogen atoms which interact with the aromatic part of PhTFE but not for the water interacting with PhTFE hydroxy group. Extensive theoretical modelling was carried out to gain insight into the associated large amplitude motions including tunnelling, supported by the experimental isotopic and tunnelling splitting data.

Exploring conformational landscapes and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variant complexes with the ACE2 receptor using AlphaFold2-based structural ensembles and molecular dynamics simulations.

In this study, we combined AlphaFold-based approaches for atomistic modeling of multiple protein states and microsecond molecular simulations to accurately characterize conformational ensembles evolution and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variants BA.1, BA.2, BA.2.75, BA.3, BA.4/BA.5 and BQ.1.1. We employed and validated several different adaptations of the AlphaFold methodology for modeling of conformational ensembles including the introduced randomized full sequence scanning for manipulation of sequence variations to systematically explore conformational dynamics of Omicron spike protein complexes with the ACE2 receptor. Microsecond atomistic molecular dynamics (MD) simulations provide a detailed characterization of the conformational landscapes and thermodynamic stability of the Omicron variant complexes. By integrating the predictions of conformational ensembles from different AlphaFold adaptations and applying statistical confidence metrics we can expand characterization of the conformational ensembles and identify functional protein conformations that determine the equilibrium dynamics for the Omicron spike complexes with the ACE2. Conformational ensembles of the Omicron RBD-ACE2 complexes obtained using AlphaFold-based approaches for modeling protein states and MD simulations are employed for accurate comparative prediction of the binding energetics revealing an excellent agreement with the experimental data. In particular, the results demonstrated that AlphaFold-generated extended conformational ensembles can produce accurate binding energies for the Omicron RBD-ACE2 complexes. The results of this study suggested complementarities and potential synergies between AlphaFold predictions of protein conformational ensembles and MD simulations showing that integrating information from both methods can potentially yield a more adequate characterization of the conformational landscapes for the Omicron RBD-ACE2 complexes. This study provides insights in the interplay between conformational dynamics and binding, showing that evolution of Omicron variants through acquisition of convergent mutational sites may leverage conformational adaptability and dynamic couplings between key binding energy hotspots to optimize ACE2 binding affinity and enable immune evasion.