Molecular dynamics in multidimensional space explains how mutations affect the association path of neomycin to a riboswitch.

Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C[Formula: see text], U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin.

Molecular dynamics simulation of the effect of temperature on the conformation of ubiquitin protein.

CONTENT: Ubiquitin, a ubiquitous small protein found in all living organisms, is crucial for tagging proteins earmarked for degradation and holds pivotal importance in biomedicine. Protein functionality is intricately linked to its structure. To comprehend the impact of diverse temperatures on ubiquitin protein structure, our study delved into the energy landscape, hydrogen bonding, and overall structural stability of ubiquitin protein at varying temperatures. Through meticulous analysis of root mean square deviation and root mean square fluctuation, we validated the robustness of the simulation conditions employed. Within our simulated system, the bonding energy and electrostatic potential energy exhibited linear augmentation, while the van der Waals energy demonstrated a linear decline. Additionally, our findings highlighted that the α-Helix secondary structure of the ubiquitin protein gradually transitions toward helix destabilization under high-temperature conditions. The secondary structure of ubiquitin protein experiences distinct changes under varying temperatures. The outcomes of our molecular simulations offer a theoretical framework that enhances our comprehension of how temperature impacts the structural stability of ubiquitin protein. These insights contribute not only to a deeper understanding of iniquity's behavior but also hold broader implications in the realm of biomedicine and beyond. METHODS: All the MD simulations were performed using the GROMACS software with GROMOS96 force field and SPC for water. The ubiquitin protein was put in the center of a cubic box with a length of 8 nm, a setting that allowed > 0.8 nm in the minimal distance between the protein surface and the box wall. To remove the possible coordinate collision of the configurations, in the beginning, the steepest descent method was used until the maximum force between atoms was under 100 kJ/mol·nm with a 0.01 nm step size. Minimization was followed by 30 ps of position-restrained MD simulation. The protein was restrained to its initial position, and the solvent was freely equilibrated. The product phase was obtained with the whole system simulated for 10 ns without any restraint using an integral time step of 1 fs with different temperatures. The cutoff for short-range electronic interaction was set to 1.5 nm. The long-range interactions were treated with a particle-mesh Ewald (PME) method with a grid width of 1.2 nm.

Computational insights into the conformational transition of STING: Mechanistic, energetic considerations, and the influence of crucial mutations.

STING (stimulator of interferon genes) is a crucial protein in the innate immune system's response to viral and bacterial infections. In this study, we investigated the mechanistic and energetic mechanism of the conformational transition process of STING activated by cGAMP binding. We found that the STING connector region undergoes an energetically unfavorable rotation during this process, which is compensated by the favorable interaction between cGAMP and the STING ligand binding domain. We further studied several disease-causing mutations and found that the V155 M mutation facilitates a smoother transition in the STING connector region. However, the V147L mutation exhibits unfavorable conformational transition energy, suggesting it may hinder STING activation pathway that relies on connector region rotation. Despite being labeled as hyperactive, the widespread prevalence of V147L/V147I mutations across species implies a neutral character, indicating complexity in its role. Overall, our analysis deepens the understanding of STING activation within the connector region, and targeting this region with compounds may provide an alternative approach to interfering with STING's function.

Photophysics of a nucleic acid-protein crosslinking model strongly depends on solvation dynamics: an experimental and theoretical study.

We present a combined experimental and theoretical study of the photophysics of 5-benzyluracil (5BU) in methanol, which is a model system for interactions between nucleic acids and proteins. A molecular dynamics study of 5BU in solution through efficient DFT-based hybrid ab initio potentials revealed a remarkable conformational flexibility - allowing the population of two main conformers - as well as specific solute-solvent interactions, which both appear as relevant factors for the observed 5BU optical absorption properties. The simulated absorption spectrum, calculated on such an ensemble, enabled a molecular interpretation of the experimental UV-Vis lowest energy band, which is also involved in the induced photo-reactivity upon irradiation. In particular, the first two excited states (mainly involving the uracil moiety) both contribute to the 5BU lowest energy absorption. Moreover, as a key finding, the nature and brightness of such electronic transitions are strongly influenced by 5BU conformation and the microsolvation of its heteroatoms.

Transient interdomain interactions in free USP14 shape its conformational ensemble.

The deubiquitinase (DUB) ubiquitin-specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin-like (Ubl) domain separated by a 25-residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full-length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full-length USP14 by nuclear magnetic resonance spectroscopy and used small angle x-ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.

Conformational Effects on the Absorption Spectra of Phytochromes.

Bacteriophytochrome is a photoreceptor protein that contains the biliverdin (BV) chromophore as its active component. The spectra of BV upon mutation remain remarkably unchanged, as far as spectral positions are concerned. This points toward the minimal effect of electrostatic effects on the electronic structure of the chromophore. However, the relative intensities of the Q and Soret bands of the chromophore change dramatically upon mutation. In this work, we delve into the molecular origin of this unusual intensity modulation. Using extensive classical MD and QM/MM calculations, we show that due to mutation, the conformational population of the chromophore changes significantly. The noncovalent interactions, especially the stacking interactions, lead to extra stabilization of the cyclic form in the D207H mutated species as opposed to the open form in the wild-type BV. Thus, unlike the commonly observed direct electrostatic effect on the spectral shift, in the case of BV the difference observed is in varying intensities, and this in turn is driven by a conformational shift due to enhanced stacking interaction.

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

Synthesis, crystal structure and in-silico evaluation of arylsulfonamide Schiff bases for potential activity against colon cancer.

This report presents a comprehensive investigation into the synthesis and characterization of Schiff base compounds derived from benzenesulfonamide. The synthesis process, involved the reaction between N-cycloamino-2-sulfanilamide and various substituted o-salicylaldehydes, resulted in a set of compounds that were subjected to rigorous characterization using advanced spectral techniques, including 1H NMR, 13C NMR and FT-IR spectroscopy, and single-crystal X-ray diffraction. Furthermore, an in-depth assessment of the synthesized compounds was conducted through Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) analysis, in conjunction with docking studies, to elucidate their pharmacokinetic profiles and potential. Impressively, the ADMET analysis showcased encouraging drug-likeness properties of the newly synthesized Schiff bases. These computational findings were substantiated by molecular properties derived from density functional theory (DFT) calculations using the B3LYP/6-31G* method within the Jaguar Module of Schrödinger 2023-2 from Maestro (Schrodinger LLC, New York, USA). The exploration of frontier molecular orbitals (HOMO and LUMO) enabled the computation of global reactivity descriptors (GRDs), encompassing charge separation (Egap) and global softness (S). Notably, within this analysis, one Schiff base, namely, 4-bromo-2-{N-[2-(pyrrolidine-1-sulfonyl)phenyl]carboximidoyl}phenol, 20, emerged with the smallest charge separation (ΔEgap = 3.5780 eV), signifying heightened potential for biological properties. Conversely, 4-bromo-2-{N-[2-(piperidine-1-sulfonyl)phenyl]carboximidoyl}phenol, 17, exhibited the largest charge separation (ΔEgap = 4.9242 eV), implying a relatively lower propensity for biological activity. Moreover, the synthesized Schiff bases displayed remarkeable inhibition of tankyrase poly(ADP-ribose) polymerase enzymes, integral in colon cancer, surpassing the efficacy of a standard drug used for the same purpose. Additionally, their bioavailability scores aligned closely with established medications such as trifluridine and 5-fluorouracil. The exploration of molecular electrostatic potential through colour mapping delved into the electronic behaviour and reactivity tendencies intrinsic to this diverse range of molecules.

Exploring the free energy landscape of proteins using magnetic tweezers.

Proteins fold to their native states by searching through the free energy landscapes. As single-domain proteins are the basic building block of multiple-domain proteins or protein complexes composed of subunits, the free energy landscapes of single-domain proteins are of critical importance to understand the folding and unfolding processes of proteins. To explore the free energy landscapes of proteins over large conformational space, the stability of native structure is perturbed by biochemical or mechanical means, and the conformational transition process is measured. In single molecular manipulation experiments, stretching force is applied to proteins, and the folding and unfolding transitions are recorded by the extension time course. Due to the broad force range and long-time stability of magnetic tweezers, the free energy landscape over large conformational space can be obtained. In this article, we describe the magnetic tweezers instrument design, protein construct design and preparation, fluid chamber preparation, common-used measuring protocols including force-ramp and force-jump measurements, and data analysis methods to construct the free energy landscape. Single-domain cold shock protein is introduced as an example to build its free energy landscape by magnetic tweezers measurements.

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Asymmetric α-Fluoroalkyl-α-Amino Acids: Recent Advances in Their Synthesis and Applications.

Due to the specific properties provided by fluorine atoms to biomolecules, amino acids with fluorinated side chains are of great interest for medicinal chemistry and chemical biology. Among them, α-fluoroalkyl-α-amino acids constitute a unique class of compounds. In this review, we outline the strategies adopted for their syntheses in enantiopure or enantioenriched forms and their incorporation into peptides. We then describe the consequences of the introduction of fluorine atoms in these compounds for the modulation of their hydrophobicity and the control of their conformation. Emerging applications are presented in the areas of enzyme inhibition, medicinal chemistry, hydrolytic stability of peptides, antimicrobial peptides, PET, and 19F NMR probes.

Probing solution conformations of l-DOPA: Integration of experiment and simulation via vibrational optical activity.

l-DOPA plays a critical role as a precursor to dopamine and is a standard treatment for Parkinson's disease. Recent research has highlighted the potential therapeutic advantages of deuterated l-DOPA analogs having a longer biological half-life. For their spectroscopic characterization, the in-detail characterization of l-DOPA itself is necessary. This article presents a thorough examination of the vibrational spectra of l-DOPA, with a particular emphasis on chirally sensitive VOA techniques. We successfully obtained high-quality Raman and ROA spectra of l-DOPA in its cationic form, under low pH conditions, and at a high concentration of 100 mg/ml. These spectra cover a broad spectral range, allowing for precise comparisons with theoretical simulations. We also obtained IR and VCD spectra, but they faced limitations due to the narrow accessible spectral region. Exploration of l-DOPA's conformational landscape revealed its intrinsic flexibility, with multiple coexisting conformations. To characterize these conformations, we employed two methods: one involved potential energy surface scans with implicit solvation, and the other utilized molecular dynamics simulations with explicit solvation. Comparing ROA spectra from different conformer groups and applying spectral decomposition proved crucial in determining the correct conformer ratios. The use of explicit solvation significantly improved the quality of the final simulated spectral profiles. The accurate determination of conformer ratios, rather than solely relying on the number of averaged spectra, played a crucial role in simulation accuracy. In conclusion, our study offers valuable insights into the structure and conformational behavior of l-DOPA and represents a valuable resource for subsequent spectroscopic studies of its deuterated analogs.

Conformational Heterogeneity of ß-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of ß-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.

Does Inter-Residue Hydrogen Bonding in ß-(1→4)-Linked Disaccharides Influence Linkage Conformation in Aqueous Solution?

Two disaccharides, methyl ß-d-galactopyranosyl-(1→4)-α-d-glucopyranoside (1) and methyl ß-d-galactopyranosyl-(1→4)-3-deoxy-α-d-ribo-hexopyranoside (3), were prepared with selective 13C-enrichment to allow measurement of six trans-O-glycosidic J-couplings (2JCOC, 3JCOCH, and 3JCOCC) in each compound. Density functional theory (DFT) was used to parameterize Karplus-like equations that relate these J-couplings to either ϕ or ψ. MA'AT analysis was applied to both linkages to determine mean values of ϕ and ψ in each disaccharide and their associated circular standard deviations (CSDs). Results show that deoxygenation at C3 of 1 has little effect on both the mean values and librational motions of the linkage torsion angles. This finding implies that, if inter-residue hydrogen bonding between O3H and O5' of 1 is present in aqueous solution and persistent, it plays little if any role in dictating preferred linkage conformation. Hydrogen bonding may lower the energy of the preferred linkage geometry but does not determine it to any appreciable extent. Aqueous 1-µs MD simulation supports this conclusion and also indicates greater conformational flexibility in deoxydisaccharide 3 in terms of sampling several, conformationally distinct, higher-energy conformers in solution. The populations of these latter conformers are low (3-14%) and could not be validated by MA'AT analysis. If the MD model is correct, however, C3 deoxygenation does enable conformational sampling over a wider range of ϕ/ψ values, but linkage conformation in the predominant conformer is essentially identical in both 1 and 3.

scENCORE: leveraging single-cell epigenetic data to predict chromatin conformation using graph embedding.

Dynamic compartmentalization of eukaryotic DNA into active and repressed states enables diverse transcriptional programs to arise from a single genetic blueprint, whereas its dysregulation can be strongly linked to a broad spectrum of diseases. While single-cell Hi-C experiments allow for chromosome conformation profiling across many cells, they are still expensive and not widely available for most labs. Here, we propose an alternate approach, scENCORE, to computationally reconstruct chromatin compartments from the more affordable and widely accessible single-cell epigenetic data. First, scENCORE constructs a long-range epigenetic correlation graph to mimic chromatin interaction frequencies, where nodes and edges represent genome bins and their correlations. Then, it learns the node embeddings to cluster genome regions into A/B compartments and aligns different graphs to quantify chromatin conformation changes across conditions. Benchmarking using cell-type-matched Hi-C experiments demonstrates that scENCORE can robustly reconstruct A/B compartments in a cell-type-specific manner. Furthermore, our chromatin confirmation switching studies highlight substantial compartment-switching events that may introduce substantial regulatory and transcriptional changes in psychiatric disease. In summary, scENCORE allows accurate and cost-effective A/B compartment reconstruction to delineate higher-order chromatin structure heterogeneity in complex tissues.

Importance of Non-Covalent Interactions in Yeast Cell Wall Molecular Organization.

This review covers a group of non-covalently associated molecules, particularly proteins (NCAp), incorporated in the yeast cell wall (CW) with neither disulfide bridges with proteins covalently attached to polysaccharides nor other covalent bonds. Most NCAp, particularly Bgl2, are polysaccharide-remodeling enzymes. Either directly contacting their substrate or appearing as CW lipid-associated molecules, such as in vesicles, they represent the most movable enzymes and may play a central role in CW biogenesis. The absence of the covalent anchoring of NCAp allows them to be there where and when it is necessary. Another group of non-covalently attached to CW molecules are polyphosphates (polyP), the universal regulators of the activity of many enzymes. These anionic polymers are able to form complexes with metal ions and increase the diversity of non-covalent interactions through charged functional groups with both proteins and polysaccharides. The mechanism of regulation of polysaccharide-remodeling enzyme activity in the CW is unknown. We hypothesize that polyP content in the CW is regulated by another NCAp of the CW-acid phosphatase-which, along with post-translational modifications, may thus affect the activity, conformation and compartmentalization of Bgl2 and, possibly, some other polysaccharide-remodeling enzymes.

Rapid generation of high-quality structure figures for publication with PyMOL-PUB.

MOTIVATION: The advancement of structural biology has increased the requirements for researchers to quickly and efficiently visualize molecular structures in silico. Meanwhile, it is also time-consuming for structural biologists to create publication-standard figures, as no useful tools can directly generate figures from structure data. Although manual editing can ensure that figures meet the standards required for publication, it requires a deep understanding of software operations and/or program call commands. Therefore, providing interfaces based on established software instead of manual editing becomes a significant necessity. RESULTS: We developed PyMOL-PUB, based on the original design of PyMOL, to effectively create publication-quality figures from molecular structure data. It provides functions including structural alignment methods, functional coloring schemes, conformation adjustments, and layout plotting strategies. These functions allow users to easily generate high-quality figures, demonstrate structural differences, illustrate inter-molecular interactions, and predict performances of biomacromolecules. AVAILABILITY AND IMPLEMENTATION: Our tool is publicly available at https://github.com/BGI-SynBio/PyMOL-PUB.

Characterization of Complexes and Supramolecular Structures by Electron Microscopy.

Recent advancements in cryo-electron microscopy (cryo-TEM) have enabled the determination of structures of macromolecular complexes at near-atomic resolution, establishing it as a pivotal tool in Structural Biology. This high resolution allows for the detection of ligands and substrates under physiological conditions. Enhancements in detectors and imaging devices, like phase plates, improve signal quality, facilitating the reconstruction of even smaller macromolecular complexes. The 100-kDa barrier has been surpassed, presenting new opportunities for pharmacological research and expanding the scope of crystallographic analyses in the pharmaceutical industry. Cryo-TEM produces vast data sets from minimal samples, and refined classification methods can identify different conformational states of macromolecular complexes, offering deeper insights into the functional characteristics of macromolecular systems. Additionally, cryo-TEM is paving the way for time-resolved microscopy, with rapid freezing techniques capturing snapshots of vital structural changes in biological complexes. Finally, in Structural Cell Biology, advanced cryo-TEM, through tomographic procedures, is revealing conformational changes related to the specific subcellular localization of macromolecular systems and their interactions within cells.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.