Diffuse X-Ray Scattering for Ensemble Modeling of Crystalline Proteins

Selectivity and transport in aquaporins from molecular simulation studies.

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

Diffuse x-ray scattering (DXS) and computer simulation techniques were employed to investigate the defect structure produced in Si by low keV ion and MeV electron irradiations. DXS measurements were performed for keV Ga and He implants, demonstrating the ability of the technique to provide both bulk and near-surface measurements at defect concentrations of about 1000 ppm. A rigorous analysis of these results is complicated due to the complex nature of the ion damage in Si. A computer simulation framework is developed to aid in the analysis of this data. In this technique, defects are simulated and their strain fields are calculated by simply relaxing the atoms around the defect to their equilibrium positions. The diffuse scattering is then calculated from the strain field, and the results are compared to the experimental measurements. Computer simulations are presented here only for the case of electron irradiation damage and compared to published measurements.1 Application of the technique to more complicated structures is planned and should pose no serious problems in the computational framework already developed.

To gain insight into crystalline protein dynamics, we performed molecular-dynamics (MD) simulations of a periodic 2 × 2 × 2 supercell of staphylococcal nuclease. We used the resulting MD trajectories to simulate X-ray diffraction and to study collective motions. The agreement of simulated X-ray diffraction with the data is comparable to previous MD simulation studies. We studied collective motions by analyzing statistically the covariance of alpha-carbon position displacements. The covariance decreases exponentially with the distance between atoms, which is consistent with a liquidlike motions (LLM) model, in which the protein behaves like a soft material. To gain finer insight into the collective motions, we examined the covariance behavior within a protein molecule (intraprotein) and between different protein molecules (interprotein). The interprotein atom pairs, which dominate the overall statistics, exhibit LLM behavior; however, the intraprotein pairs exhibit behavior that is consistent with a superposition of LLM and rigid-body motions (RBM). Our results indicate that LLM behavior of global dynamics is present in MD simulations of a protein crystal. They also show that RBM behavior is detectable in the simulations but that it is subsumed by the LLM behavior. Finally, the results provide clues about how correlated motions of atom pairs both within and across proteins might manifest in diffraction data. Overall, our findings increase our understanding of the connection between molecular motions and diffraction data and therefore advance efforts to extract information about functionally important motions from crystallography experiments.

Molecular dynamics simulation of the mixed anion glasses Li 4SiO 4Li 3BO 3

The term molecular mechanics (MM) refers to the use of simple potential-energy functions (e.g., harmonic oscillator or Coulombic potentials) to model molecular systems. Molecular mechanics approaches are widely applied in molecular structure refinement, molecular dynamics (MD) simulations, Monte Carlo (MC) simulations, and ligand-docking simulations. Typically, molecular mechanics models consist of spherical atoms connected by springs which represent bonds. Internal forces experienced in the model structure are described using simple mathematical functions. For example, Hooke’s law is commonly used to describe bonded interactions, and the nonbonded atoms might be treated as inelastic hard spheres or may interact according to a Lennard-Jones potential. Using these simple models, a molecular dynamics simulation numerically solves Newton’s equations of motion, thus allowing structural fluctuations to be observed with respect to time. Dynamic simulation methods are widely used to obtain information on the time evolution of conformations of proteins and other biological macromolecules1–4 and also kinetic and thermodynamic information. Simulations can provide fine detail concerning the motions of individual particles as a function of time. They can be utilized to quantify the properties of a system at a precision and on a time scale that is otherwise inaccessible, and simulation is, therefore, a valuable tool in extending our understanding of model systems. Theoretical consideration of a system additionally allows one to investigate the specific contributions to a property through “computational alchemy”,5 that is, modifying the simulation in a way that is nonphysical but nonetheless allows a model’s characteristics to be probed. One particular example is the artificial conversion of the energy function from that representing one system to that of another during a simulation. This is an important technique in free-energy calculations.6 Thus, molecular dynamics simulations, along with a range of complementary computational approaches, have become valuable tools for investigating the basis of protein structure and function. This review offers an outline of the origin of molecular dynamics simulation for protein systems and how it has developed into a robust and trusted tool. This review then covers more recent advances in theory and an illustrative selection of practical studies in which it played a central role. The range of studies in which MD has played a considerable or pivotal role is immense, and this review cannot do justice to them; MD simulations of biomedical importance were recently reviewed.4 Particular emphasis will be placed on the study of dynamic aspects of protein recognition, an area where molecular dynamics has scope to provide broad and far-ranging insights. This review concludes with a brief discussion of the future potential offered to advancement of the biological and biochemical sciences and the remaining issues that must be overcome to allow the full extent of this potential to be realized. 1.1. Historical Background MD methods were originally conceived within the theoretical physics community during the 1950s. In 1957, Alder and Wainwright7 performed the earliest MD simulation using the so-called hard-sphere model, in which the atoms interacted only through perfect collisions. Rahman8 subsequently applied a smooth, continuous potential to mimic real atomic interactions. During the 1970s, as computers became more widespread, MD simulations were developed for more complex systems, culminating in 1976 with the first simulation of a protein9,10 using an empirical energy function constructed using physics-based first-principles assumptions. MD simulations are now widely and routinely applied and especially popular in the fields of materials science11,12 and biophysics. As will be discussed later in this review, a variety of experimental conditions may be simulated with modern theories and algorithms. The initial simulations only considered single molecules in vacuo. Over time, more realistic or at least biologically relevant simulations could be performed. This trend is continuing today. The initial protein MD simulation, of the small bovine pancreatic trypsin inhibitor (BPTI), covered only 9.2 ps of simulation time. Modern simulations routinely have so-called equilibration periods much longer than that, and production simulations of tens of nanoseconds are routine, with the first microsecond MD simulation being reported in 1998.13 In addition, the original BPTI simulation included only about 500 atoms rather than the 104-106 atoms that are common today. While much of this advancement results from an immense increase in availability of computing power, major theoretical and methodological developments also contribute significantly. The number of publications regarding MD theory and application of MD to biological systems is growing at an extraordinary pace. A single review cannot do justice to the recent applications of MD. Using data from ISI Web of Science, the authors estimate that during 2005 at least 800 articles will be published that discuss molecular dynamics and proteins. The historical counts are shown in Figure 1. Open in a separate window Figure 1 Articles matching ISI Web of Science query “TS=(protein) AND TS=(molecular dynamics)”.

The microstructures of pure water and aqueous NaCl solutions over a wide range of salt concentrations (0-4 m) under ambient conditions are characterized by X-ray scattering and molecular dynamics (MD) simulations. MD simulations are performed with the rigid SPC water model as a solvent, while the ions are treated as charged Lennard-Jones particles. Simulated data show that the first peaks in the O...O and O...H pair correlation functions clearly decrease in height with increasing salt concentration. Simultaneously, the location of the second O...O peak, the signature of the so-called tetrahedral structure of water, gradually disappears. Consequently, the degree of hydrogen bonding in liquid water decreases when compared to pure fluid. MD results also show that the hydration number around the cation decreases as the salt concentration increases, which is most likely because some water molecules in the first hydration shell are occasionally substituted by chlorine. In addition, the fraction of contact ion pairs increases and that of solvent-separated ion pairs decreases. Experimental data are analyzed to deduce the structure factors and the pair correlation functions of each system. X-ray results clearly show a perturbation of the association structure of the solvent and highlight the appearance of new interactions between ions and water. A model of intermolecular arrangement via MD results is then proposed to describe the local order in each system, as deduced from X-ray scattering data.

Vicenin-2 reduces inflammation and apoptosis to relieve skin photoaging via suppressing GSK3β.

To elucidate the mechanism of Schizonepeta tenuifolia-Diosmetin in acne treatment using an integrated approach combining network pharmacology, molecular dynamics simulation, and experimental validation. Acne-related expression datasets were acquired from GEO, with differentially expressed genes (DEGs) identified using R. Regulatory patterns were visualized via volcano plots and heatmaps. Enrichment analyses (GO and KEGG) revealed significant biological processes and pathways. Candidate targets were screened using LASSO regression and PPI network analysis, followed by drug discovery via the Coremine Medical database. Molecular docking and dynamics simulations assessed compound-target binding stability. To evaluate the in vitro effects of diosmetin, this study employed the HaCaT keratinocyte model. Cell viability and inflammatory cytokine expression levels following drug treatment were assessed using the CCK-8 assay and ELISA, respectively. Western blot analysis was used to investigate the effects of diosmetin proteins such as NF-κB, Nrf2, and KLK7. Immunofluorescence was used to detect ROS levels and mitochondrial morphological changes. GO and KEGG enrichment analyses revealed that DEGs were significantly enriched in pathways related to mitochondrial function and ROS. PPI network analysis further identified potential hub genes. Molecular docking and dynamics simulations suggested that diosmetin, an active component of S. tenuifolia, binds strongly to KLK7. Diosmetin (Dios) restored KLK7 levels, reduced ROS accumulation, and improved mitochondrial dysfunction induced by acne. Furthermore, the study found that diosmetin significantly reduced ear swelling and alleviated inflammation, providing preliminary preclinical evidence for its treatment of acne. The pathogenesis of acne is related to inflammation and oxidative stress. The anti-acne effect of S. tenuifolia is mainly mediated by diosmetin, which can alleviate oxidative stress and reduce inflammatory responses. This study provides supportive evidence for the potential application of diosmetin in acne treatment.

The lack of reliable predictive modeling methods and robust experimental techniques has hindered the rational design of hierarchical materials with desired structure–property–performance attributes suitable for extreme environments. With this context in mind, we explore the utility of ReaxFF reactive molecular dynamics (MD) simulations in combination with in-operando wide-angle X-ray scattering (WAXS) and X-ray pair distribution function (PDF) analyses. To demonstrate the method, we consider kaolinite, a natural hierarchical material, as the candidate and determine thermally induced chemical and structural transformations when heated from 298 to 1673 K. We first compare the key structural features from the PDF data and WAXS peaks obtained experimentally to those calculated from MD simulations. Upon observing excellent agreement, we proceed to elucidate the underlying chemical reaction mechanisms associated with dehydroxylation and sintering, identify intermediate and transition states, and also estimate energy barriers of individual reactions and their effects on the structural organization of kaolinite obtained using MD simulations. On heating from 298 to 873 K, dehydroxylation reactions lead to the transformation of crystalline kaolinite with octahedrally coordinated aluminum atoms to semicrystalline metakaolin with ∼90% tetrahedrally coordinated aluminum atoms. Sintering reactions and the subsequent emergence of mullite (a high-temperature phase of kaolinite) are observed on heating metakaolin from 1055 to 1673 K. We also find that heating rates have a significant effect on the onset temperature of dehydroxylation and sintering reactions. A rapid heating rate leads to early dehydroxylation (425 K) and sintering (1055 K), whereas a 10 times slower heating rate delays dehydroxylation (622 K) and sintering reactions (1100 K). An outcome of our method is a regime map that illustrates the degree of agreement between the experimental data and simulation results in describing the thermally induced onset of atomic-level reorganizations in materials. Herein, quantitative agreement between simulation predictions and experimental data is noted at lower temperatures (T < 1000 K), and minor deviations between these methods is noted for T > 1000 K. The remarkable agreement between the methods observed in our study reiterates the reliability of the combined ReaxFF approach in predicting material properties under chemically reactive and extreme conditions.

Molecular dynamics (MD) simulations were employed to investigate the structure, dynamics, and local base-pair step deformability of the free 16S ribosomal helix 44 from Thermus thermophilus and of a canonical A-RNA double helix. While helix 44 is bent in the crystal structure of the small ribosomal subunit, the simulated helix 44 is intrinsically straight. It shows, however, substantial instantaneous bends that are isotropic. The spontaneous motions seen in simulations achieve large degrees of bending seen in the X-ray structure and would be entirely sufficient to allow the dynamics of the upper part of helix 44 evidenced by cryo-electron microscopic studies. Analysis of local base-pair step deformability reveals a patch of flexible steps in the upper part of helix 44 and in the area proximal to the bulge bases, suggesting that the upper part of helix 44 has enhanced flexibility. The simulations identify two conformational substates of the second bulge area (bottom part of the helix) with distinct base pairing. In agreement with nuclear magnetic resonance (NMR) and X-ray studies, a flipped out conformational substate of conserved 1492A is seen in the first bulge area. Molecular dynamics (MD) simulations reveal a number of reversible alpha-gamma backbone flips that correspond to transitions between two known A-RNA backbone families. The flipped substates do not cumulate along the trajectory and lead to a modest transient reduction of helical twist with no significant influence on the overall geometry of the duplexes. Despite their considerable flexibility, the simulated structures are very stable with no indication of substantial force field inaccuracies.

Molecular dynamics (MD) simulation was carried out for the molten NaNO3, NaNO2 and their eutectic mixture systems to compare their simulated structures with those determined experimentally by X-ray diffraction. As for the pure NaNO3 and NaNO2, parameters for repulsion potential were adjusted so that the structure factors of their simulated systems coincide reasonably with the corresponding experimental ones. The MD simulation of the mixture was carried out with the parameters determined for the component salts. The structure factor of the simulated mixture system successfully reproduced the experimental one, which shows validity of the proposed pair potential. The assignment of most of the peaks in the experimental correlation function was performed with the aid of simulated results. The simulated data has been used to determine the diffusion coefficients of the component ions.

Quorum sensing (QS) regulates bacterial functions like virulence and biofilm formation, mediated by proteins such as LasI and QscR in Pseudomonas aeruginosa. This study investigates the structural dynamics of LasI and QscR proteins in complex with Sulfamerazine and Sulfaperin, using AiiA lactonase as a negative control, through molecular dynamics (MD) simulations to identify potential QS modulators. Molecular docking and MD simulations assessed binding affinity and structural dynamics, analyzing parameters like docking scores, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), solvent-accessible surface area (SASA), radius of gyration (Rg), principal component analysis (PCA), and covariance analysis. Sulfamerazine exhibited the highest binding affinity for LasI based on docking scores, indicating strong ligand-protein interactions. MD simulations revealed stability in the LasI-Sulfamerazine complex, with lower RMSD compared to LasI-Sulfaperin and QscR complexes. RMSF analysis indicated greater flexibility in ligand-binding regions of LasI-Sulfaperin and QscR complexes, suggesting weaker binding. SASA showed a decrease in solvent-accessible surface area for the LasI-Sulfamerazine complex, supporting a compact structure. Rg values confirmed this, with the LasI-Sulfamerazine complex being more compact (~2.00 nm) than QscR-ligand complexes (2.10-2.30 nm). PCA revealed significant conformational changes in the LasI-Sulfamerazine complex, with PC1 explaining 57.26% variance. Covariance analysis indicated stronger residue coupling in the LasI-Sulfamerazine complex, suggesting higher rigidity, while LasI-Sulfaperin and QscR complexes exhibited flexible dynamics. AiiA lactonase was used as a negative control due to its established quorum quenching activity, which hydrolyzes AHL molecules and disrupts QS signaling. Unlike LasI and QscR, AiiA does not rely on small molecule binding for activation. However, a known LasI or QscR inhibitor would have served as a more appropriate positive control, which will be considered in future studies. These findings suggest the LasI-Sulfamerazine complex's stability and rigidity make Sulfamerazine a promising QS modulator. Computational analyses highlight its potential to disrupt bacterial communication. Further experimental validation is needed to confirm its therapeutic implications.

The efforts of this thesis are directed towards understanding the physical driving forces that mediate membrane protein self-assembly processes within the native environment of membrane proteins, the bilayer milieu. While many defined determinants contribute to the energetics of membrane protein self-assembly, such as electrostatics, non-polar side-chain packing, hydrogen bonding, and solvophobic effects, the roles that these driving forces play have not been well characterized. The inability to describe the role of these physical driving forces in processes involving membrane proteins comes from the lack of quantitative thermodynamic measurements concerning membrane protein self-assembly equilibria within the lipid bilayer environment. These thermodynamic measurements would describe the free energy of self-assembly (∆G°) and the breakdown of this term into enthalpic (∆H°) and entropic components (∆S°). The studies within this thesis bridge the gaps in our knowledge of membrane protein self-assembly by quantifying the contributions of non-polar side-chain packing in the dimerization reaction of the large poly-topic membrane protein CLC-ec1 within lipid bilayers. An emphasis on describing non-polar side-chain packing was chosen due to membrane proteins commonly exhibiting the packing of transmembrane helices and surfaces lined with non-polar residues within lipid bilayers. Furthermore, non-polar side-chain packing is thought to be a significant contributor to membrane protein self-assembly, but its quantification has been limited. An emphasis is placed within the thesis on quantifying non-polar side-chain packing interactions in terms of van der Waals (VDW) interaction energies. Chapter 1 of this thesis describes the pertinent background of the driving forces thought to be involved in membrane protein self-assembly and provides an extensive review of the membrane protein CLC-ec1. The studies within this thesis employed a novel method, called the subunit capture method, to measure CLC-ec1 dimerization within membranes. Chapter 2 describes the subunit capture method and other methods commonly used throughout the thesis. Chapter 3 describes the first study, which characterizes the impact of removing non-polar side-chain contacts on the stoichiometry of the CLC-ec1 homodimer. Computational analysis of the dimerization interface of CLC-ec1 characterized non-polar side-chain packing by measuring the VDW interaction energies of side-chains within a structure of CLC-ec1 determined by x-ray crystallography. Subsequent experiments targeted non-polar side-chains exhibiting favorable VDW interaction energies by substituting 4-5 of the native residues at a time to the smaller residue alanine, depleting the favorable VDW interactions at the dimerization interface. The CLC-ec1 multiple alanine constructs revealed that the stoichiometry of CLC-ec1 is dependent upon the non-polar side-chains at the dimerization interface, as each construct becomes entirely monomeric in lipid bilayers. The studies also found that changes in CLC-ec1 stoichiometry occur in a solvent-dependent manner as three of the four CLC-ec1 multiple alanine constructs remain stable dimers in detergent micelles. Lastly, the study exposes a molecular hot-spot at residue 194, characterizing the substitution to alanine at that position as important for dimer stoichiometry in both detergents and lipids. The contents of Chapter 4 present the findings from a second study that sought answers to two questions. The study begins by posing the first question: Are protein-protein interactions driving the dimerization of CLC-ec1? To answer this question, we performed long all-atom molecular dynamics (MD) simulations to quantify the interactions of CLC-ec1 in a dimeric and monomeric state while embedded in a lipid bilayer. Through the quantification of VDW interaction energies, the measurements revealed that the interactions within each state are similar in magnitude. The findings from the MD simulations present VDW interactions as important to CLC-ec1 dimerization but not as a determinant that excessively stabilizes the CLC-ec1 dimer complex. The results from the analysis of the MD simulations allowed us to form the second question of the study: Will removing side-chain packing interactions at the dimerization interface destabilize the CLC-ec1 homodimer? The second question was thoroughly investigated by measuring the changes in dimerization free energy of CLC-ec1 constructs that contained single substitutions to alanine that generate cavities at the dimerization interface. The various changes in CLC-ec1 dimer stability due to the alanine substitutions were compared to the MD computational analysis to find no correlation between the two datasets. We conclude that while the CLC-ec1 single alanine substitutions destabilize the dimer complex, the removal of non-polar packing interactions from the dimer state of CLC-ec1 does not explain the measured changes in stability. The final chapter in this thesis, Chapter 5, presents a discussion of how the results obtained within this thesis fit into our current understanding of membrane protein self-assembly. Included in the discussion, we reason that CLC-ec1 may not have excessively favorable non-polar packing interactions at the CLC-ec1 dimerization interface in order to retain a flexible interface for function -related activities. Finally, Chapter 5 will conclude by posing a few future directions of study to answer new questions we have encountered while performing the work presented in this thesis.

The low-density lipoprotein receptor (LDLR) regulates cholesterol uptake by mediating the hepatic clearance of plasma low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type-9 (PCSK9) attenuates LDLR function by binding to the LDLR, leading to its lysosomal degradation and preventing the total depletion of circulating LDL-C. However, pathogenic PCSK9 variants can reduce LDLR availability, significantly increase plasma LDL-C levels. Despite this understanding, the detailed molecular mechanism of LDLR-PCSK9 interaction remains unclear due to the incomplete LDLR structure. This study uses molecular dynamics (MD) simulations to predict LDLR structural dynamics upon binding to PCSK9. Furthermore, PCSK9 variants, E498A and R499G, that were identified in Malaysian FH patients were investigated for their mutational effects. Throughout the simulations, PCSK9 remained stable, while LDLR explored a larger conformational space. The LDLR-PCSK9 wild-type (WT) complex showed minimal changes, while the LDLR-PCSK9(R499G) complex exhibited pronounced conformational rearrangement. The MM/GBSA analysis revealed that the LDLR-PCSK9(E498A) complex had the highest binding affinity (- 63.81kcal/mol), followed by the WT complex (- 33.07kcal/mol), and LDLR-PCSK9(R499G) (- 24.21kcal/mol). These findings offer novel insights into the dynamic interactions between LDLR and PCSK9, highlighting the role of structural flexibility in their relationship. Further MD simulation studies with the complete LDLR structure as well as experimental validation are needed to elucidate the molecular mechanisms underlying LDLR-PCSK9-mediated cholesterol homeostasis. The initial structure of the wild-type (WT) LDLR-PCSK9 complex was obtained from PDB ID 3P5C, and the PCSK9 mutant structures (E498A and R499G) were modeled using the SPDBV program. MD simulations for each complex-LDLR-PCSK9 WT, LDLR-PCSK9(E498A), and LDLR-PCSK9(R499G)-were conducted using the GROMACS package with the CHARMM36m force field. The simulations were performed at 310.15K with 2-fs timesteps under the isothermal-isobaric (NPT) ensemble, with each run lasting 500ns. Including triplicates, the total duration of MD simulation time for all complexes amounted to 3.5μs.