A Probabilistic Model of RNA Conformational Space

The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling procedure. Both are only partly solved problems. Here, we focus on the problem of conformational sampling. The current state of the art solution is based on fragment assembly methods, which construct plausible conformations by stringing together short fragments obtained from experimental structures. However, the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows efficient sampling of RNA conformations in continuous space, and with associated probabilities. We show that the model captures several key features of RNA structure, such as its rotameric nature and the distribution of the helix lengths. Furthermore, the model readily generates native-like 3-D conformations for 9 out of 10 test structures, solely using coarse-grained base-pairing information. In conclusion, the method provides a theoretical and practical solution for a major bottleneck on the way to routine prediction and simulation of RNA structure and dynamics in atomic detail.     

G-quadruplex structures of human telomere DNA examined by single molecule FRET and BrG-substitution

The human telomere is known to form the G-quadruplex structure to inhibit the activity of telomerase. Its detailed structure has been of great interest. Recently, two kinds of the parallel-antiparallel hybrid structures have been specified in K(+) solution. However, the G-quadruplex structure is generally thought to be in equilibrium among different structures. Here, we describe the single-pair fluorescence resonance energy transfer (sp-FRET) experiments on telomere samples with bromoguanine (BrG)-substitutions, which control the G-quadruplex structures, at different positions and one without any substitution. The observed FRET distributions were decomposed into five components and the relative population of these components depended on the BrG-substitution positions. In order to consistently explain the variety of conformations, we proposed a novel structural model, the so-called triple-strand-core model. On the basis of this model, the components of the FRET distributions were attributed to the mixed-chair hybrid structures, which were reported recently, and chair-type antiparallel structures, which can be predicted from this model. The FRET efficiencies of these structures were explained in terms of partially broken structures due to steric hindrance and inappropriate capping. This basic model also consistently explains experimental results reported previously. Furthermore, using this model, the folding pathway of the hybrid structures and T-loop formation can be predicted.     

Solution NMR structure of a D,L-alternating oligonorleucine as a model of beta-helix

beta-Helix structures are of particular interest due to their capacity to form transmembrane channels with different transport properties. However, the relatively large number of beta-helices configurations does not allow a direct conformational analysis of beta-helical oligopeptides. A synthetic alternating D,L-oligopeptide with twelve norleucines (XIIMe) has been used as a model to get insight in the conformational features of beta-helix structures. The spatial configuration of XIIMe in solution has been determined by NMR. An extensive set of distances (nuclear Overhauser effect) and dihedral (J coupling constants) constraints have been included in molecular dynamics calculations. The NMR experimental data and theoretical calculations clearly indicate that the XIIMe adopts a single beta(4.4)-helix-type conformation in nonpolar solvents.     

Solution structure of a D,L-alternating oligonorleucine as a model of double-stranded antiparallel beta-helix

Conformational characteristics of alternating D,L linear peptides are of particular interest because of their capacity to form transmembrane channels with different transport properties, as some natural antibiotics do. Single- and double-stranded beta-helical structures are common for alternating D,L peptides. The stability of the beta-helix depends on several structural factors, such as the backbone peptide length, type and position of side chains, and nature of terminal groups. The NMR and molecular dynamics solution conformation of a synthetic alternating D,L-oligopeptide with 15 norleucines (XVMe) has been used as a model to get insight in to the conformational features of double-stranded beta-helix structures. The NH chemical shift values (delta(NH)) and long-range nuclear Overhauser effects (NOE) cross peaks, in particular interstrand connectivities, clearly point to an antiparallel double-stranded beta-helix for the XVMe major conformation in solution. An extensive set of distances (from NOE cross peaks) and H-bonds (from delta(NH)) has been included in the molecular dynamics calculations. The experimental NMR data and theoretical calculations clearly indicate that the most probable conformation of XVMe in solution is a double-strand antiparallel beta(5.6) increasing decreasing-helix structure.     

Computer modeling of actinomycin D interactions with double-helical DNA

We have performed molecular mechanical calculations on intercalation complexes of actinomycin D with a series of base-paired hexanucleoside pentaphosphates; d(GCGCGC)2, d(GCCGGC)2, d(GCATGC)2, d(GCTAGC)2 and d(ATGCAT)2. Our results are in good agreement with previous experimental work on sequence selectivity. The results provide a rationalization for the strong preference of actinomycin D to intercalate on the 3' side of guanine residues, consistent with previously proposed models. Finally, the computed structures for d(ATGCAT)2-actinomycin D complexes have been compared with two-dimensional nuclear magnetic resonance nuclear Overhauser effect experimental results. To our knowledge, this is the first extensive comparison of molecular mechanical model structures for a drug-DNA complex with experimental solution phase data. We find generally good agreement between our computational models and the experimental solution phase structures.     

Structural studies on the 30 S ribosomal subunit from Escherichia coli

Small-angle X-ray scattering curves computed for various 30 S subunit structures have been compared with the experimental scattering curve. The curve from the 30 S subunit is best approximated by that calculated for a 1:3.6:3.6 ellipsoidal structure. The rather prolate ellipsoidal model suggested by recent electron microscope studies gives a scattering curve considerably different from the 30 S curve, suggesting that the electron microscope model is not that found in solution. Analysis of the more extended portions of the experimental scattering curve suggests some internal structure. A model is proposed that contains RNA and protein in positions such that the calculated scattering curve shows more extensive, yet similar internal structure. Resultant constraints on the structure of the 30 S subunit in solution are given.     

Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.     

Crystallographic molecular replacement using an in silico‐generated search model of SARS‐CoV‐2 ORF8

The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre‐existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for MR. The ORF8 protein of SARS‐CoV‐2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β‐sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.     

Large-scale networks of hydration water molecules around bovine beta-trypsin revealed by cryogenic X-ray crystal structure analysis.

The hydration structure of bovine beta-trypsin was investigated in cryogenic X-ray diffraction experiments. Three crystal forms of the enzyme inhibited by benzamidine with different molecular packing were selected to deduce the hydration structure for the entire surface of the enzyme. The crystal structures in all three of the crystal forms were refined at the resolution of 1.8 A at 100 K and 293 K. The number of hydration water molecules around the enzyme at 100 K was 1.5 to two times larger than that at 293 K, indicating that the motion of hydration water was quenched by cooling. In particular, the increase in the number of hydration water molecules was prominent on flat and electrostatically neutral surface areas. The water-to-protein mass ratio and the radius of gyration of a structural model of hydrated trypsin at 100 K was consistent with the results obtained by other experimental techniques for proteins in solution. Hydration water molecules formed aggregates of various shapes and dimensions, and some of the aggregates even covered hydrophobic residues by forming oligomeric arrangements. In addition, the aggregates brought about large-scale networks of hydrogen bonds. The networks covered a large proportion of the surface of trypsin like a patchwork, and mechanically linked several secondary structures of the enzyme. By merging the hydration structures of the three crystal forms at 100 K, a distribution function of hydration water molecules was introduced to approximate the static hydration structure of trypsin in solution. The function showed that the negatively charged active site of trypsin tended to be easily exposed to bulk solvent. This result is of interest with respect to the solvent shielding effect and the recognition of a positively charged substrate by trypsin.     

Diffusion limited immunochemical sensing

The time-dependent surface coverage of antigen-antibody complexes for a sensor in which antigens are bound to surface immobilized antibodies is determined analytically. Assuming a reversible first order reaction between the antigens and antibodies, a model is derived describing the dynamical response of the sensor. The surface coverage is related explicitly to the antigen concentration which is of special interest in experimental situations. The stationary state and short time behaviour are determined explicitly. Several illustrations of the full solution are provided.     

A theoretical study of glucose mutarotation in aqueous solution

In this work the mechanism of glucose mutarotation is investigated in aqueous solution considering the most likely pathways proposed from experimental work. Two mechanisms are studied. The first involves an intramolecular proton transfer as proposed by textbooks of organic chemistry, and the second uses one solvent water molecule to assist proton transfer. Both mechanisms are studied in the gas phase and in aqueous solution with the help of a polarizable continuum model, which is adopted to introduce the electrostatic nonspecific influence of bulk solvent. The structures are fully characterized through the calculation of the corresponding vibrational frequencies. The rate coefficients for each mechanism are calculated following transition-state theory in both the gas phase and in aqueous solution. Values computed for the water-assisted pathway in the continuum solvent agree best with the experimental results.     

"Bridge" structures between nucleic acid molecules fixed in the structure of a liquid crystal

The binding of daunomycin and copper ions to poly(I).poly(C) molecules fixed in a particle of a liquid-crystalline dispersion was studied. A thermodynamic model of adsorption was developed, which makes it possible to describe the formation of complexes of a particular kind, "bridges" that connect adjacent nucleic acid molecules fixed in a liquid crystal. The bridges represent chelate complexes, which incorporate the molecules of the antibiotic daunomycin and copper ions. Equations describing the dependence of the concentration of these bridges in solution on the concentration of their constituents were derived. The family of dependences of experimental amplitudes of bands in CD spectra typical of "bridge" structures on the concentration of copper ions represents a set of S-shaped curves, and, as the concentration of daunomycin in solution increases, the level of saturation of these curves increases. The analysis of experimental data with the use of this model suggests that the structures of this type compete with daunomycin molecules for the binding sites on poly(I).poly(C). By using this model, the energies of formation of bridge structures were calculated.     

Molecular modeling of protein function regions

Experimental protein structures often provide extensive insight into the mode and specificity of small molecule binding, and this information is useful for understanding protein function and for the design of drugs. We have performed an analysis of the reliability with which ligand-binding information can be deduced from computer model structures, as opposed to experimentally derived ones. Models produced as part of the CASP experiments are used. The accuracy of contacts between protein model atoms and experimentally determined ligand atom positions is the main criterion. Only comparative models are included (i.e., models based on a sequence relationship between the protein of interest and a known structure). We find that, as expected, contact errors increase with decreasing sequence identity used as a basis for modeling. Analysis of the causes of errors shows that sequence alignment errors between model and experimental template have the most deleterious effect. In general, good, but not perfect, insight into ligand binding can be obtained from models based on a sequence relationship, providing there are no alignment errors in the model. The results support a structural genomics strategy based on experimental sampling of structure space so that all protein domains can be modeled on the basis of 30% or higher sequence identity.     

Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic alpha-helical antimicrobial peptides

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic alpha-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly alpha-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.     

Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

BackgroundIn protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.MethodsWe designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.ResultsYeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.ConclusionsLoop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.General significanceNo single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.     

Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures

DNA nanotechnology enables the programmed synthesis of intricate nanometer-scale structures for diverse applications in materials and biological science. Precise control over the 3D solution shape and mechanical flexibility of target designs is important to achieve desired functionality. Because experimental validation of designed nanostructures is time-consuming and cost-intensive, predictive physical models of nanostructure shape and flexibility have the capacity to enhance dramatically the design process. Here, we significantly extend and experimentally validate a computational modeling framework for DNA origami previously presented as CanDo [Castro,C.E., Kilchherr,F., Kim,D.-N., Shiao,E.L., Wauer,T., Wortmann,P., Bathe,M., Dietz,H. (2011) A primer to scaffolded DNA origami. Nat. Meth., 8, 221-229.]. 3D solution shape and flexibility are predicted from basepair connectivity maps now accounting for nicks in the DNA double helix, entropic elasticity of single-stranded DNA, and distant crossovers required to model wireframe structures, in addition to previous modeling (Castro,C.E., et al.) that accounted only for the canonical twist, bend and stretch stiffness of double-helical DNA domains. Systematic experimental validation of nanostructure flexibility mediated by internal crossover density probed using a 32-helix DNA bundle demonstrates for the first time that our model not only predicts the 3D solution shape of complex DNA nanostructures but also their mechanical flexibility. Thus, our model represents an important advance in the quantitative understanding of DNA-based nanostructure shape and flexibility, and we anticipate that this model will increase significantly the number and variety of synthetic nanostructures designed using nucleic acids.     

Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic α-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly α-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.     

Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.