Archaea S‐layer nanotube from a “black smoker” in complex with cyclo‐octasulfur (S8) rings

Elemental sulfur exists primarily as an ring and serves as terminal electron acceptor for a variety of sulfur‐fermenting bacteria. Hyperthermophilic archaea from black smoker vents are an exciting research tool to advance our knowledge of sulfur respiration under extreme conditions. Here, we use a hybrid method approach to demonstrate that the proteinaceous cavities of the S‐layer nanotube of the hyperthermophilic archaeon Staphylothermus marinus act as a storage reservoir for cyclo‐octasulfur . Fully atomistic molecular dynamics (MD) simulations were performed and the method of multiconfigurational thermodynamic integration was employed to compute the absolute free energy for transferring a ring of elemental sulfur from an aqueous bath into the largest hydrophobic cavity of a fragment of archaeal tetrabrachion. Comparisons with earlier MD studies of the free energy of hydration as a function of water occupancy in the same cavity of archaeal tetrabrachion show that the sulfur ring is energetically favored over water.     

Inferring a weighted elastic network from partial unfolding with coarse‐grained simulations

A number of studies have demonstrated that simple elastic network models can reproduce experimental B‐factors, providing insights into the structure–function properties of proteins. Here, we report a study on how to improve an elastic network model and explore its performance by predicting the experimental B‐factors. Elastic network models are built on the experimental coordinates, and they only take the pairs of atoms within a given cutoff distance r_c into account. These models describe the interactions by elastic springs with the same force constant. We have developed a method based on numerical simulations with a simple coarse‐grained force field, to attribute weights to these spring constants. This method considers the time that two atoms remain connected in the network during partial unfolding, establishing a means of measuring the strength of each link. We examined two different coarse‐grained force fields and explored the computation of these weights by unfolding the native structures. Proteins 2014; 82:119–129. © 2013 Wiley Periodicals, Inc.     

Systematic evaluation of CS‐Rosetta for membrane protein structure prediction with sparse NOE restraints

We critically test and validate the CS‐Rosetta methodology for de novo structure prediction of ‐helical membrane proteins (MPs) from NMR data, such as chemical shifts and NOE distance restraints. By systematically reducing the number and types of NOE restraints, we focus on determining the regime in which MP structures can be reliably predicted and pinpoint the boundaries of the approach. Five MPs of known structure were used as test systems, phototaxis sensory rhodopsin II (pSRII), a subdomain of pSRII, disulfide binding protein B (DsbB), microsomal prostaglandin E2 synthase‐1 (mPGES‐1), and translocator protein (TSPO). For pSRII and DsbB, where NMR and X‐ray structures are available, resolution‐adapted structural recombination (RASREC) CS‐Rosetta yields structures that are as close to the X‐ray structure as the published NMR structures if all available NMR data are used to guide structure prediction. For mPGES‐1 and Bacillus cereus TSPO, where only X‐ray crystal structures are available, highly accurate structures are obtained using simulated NMR data. One main advantage of RASREC CS‐Rosetta is its robustness with respect to even a drastic reduction of the number of NOEs. Close‐to‐native structures were obtained with one randomly picked long‐range NOEs for every 14, 31, 38, and 8 residues for full‐length pSRII, the pSRII subdomain, TSPO, and DsbB, respectively, in addition to using chemical shifts. For mPGES‐1, atomically accurate structures could be predicted even from chemical shifts alone. Our results show that atomic level accuracy for helical membrane proteins is achievable with CS‐Rosetta using very sparse NOE restraint sets to guide structure prediction. Proteins 2017; 85:812–826. © 2016 Wiley Periodicals, Inc.     

Introducing folding stability into the score function for computational design of RNA‐binding peptides boosts the probability of success

A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL^Lys3) of human for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL^Lys3. Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL^Lys3. Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. Proteins 2016; 84:700–711. © 2016 Wiley Periodicals, Inc.     

NMR‐based conformation and dynamics of a tetrasaccharide‐repeating sulfated fucan substituted by different counterions

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [‐3)‐α‐l ‐Fucp‐4( )‐(1‐3)‐α‐l ‐Fucp‐2,4‐di( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐]_n. Conformation (of rings and chains) and dynamics of this tetrasaccharide‐repeating sulfated fucan substituted by Na⁺, Ca²⁺, and Li⁺ as counterions have been examined through experiments of liquid‐state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)‐based data have confirmed that all composing units occur as ¹C₄ chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin‐relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium‐based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well‐defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.     

Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool‐seq data to generate a de novo genome assembly for mining exons, upon which Pool‐seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual‐based single nucleotide polymorphisms [SNPs]) and from mapping the Pool‐seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (F_ST) between the two introduced populations exceeds that of the naturally sympatric populations (F_ST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ ( ≈ 0.002 and  ≈ 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high‐quality reference assembly from a divergent species. We conclude that the Pool‐seq‐only approach can be suitable for detecting and quantifying genome‐wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.     

Nucleotide‐dependent structural fluctuations and regulation of microtubule‐binding affinity of KIF1A

Molecular motors such as kinesin regulate affinity to a rail protein during the ATP hydrolysis cycle. The regulation mechanism, however, is yet to be determined. To understand this mechanism, we investigated the structural fluctuations of the motor head of the single‐headed kinesin called KIF1A in different nucleotide states using molecular dynamics simulations of a Gō‐like model. We found that the helix at the microtubule (MT) binding site intermittently exhibits a large structural fluctuation when MT is absent. Frequency of this fluctuation changes systematically according to the nucleotide states and correlates strongly with the experimentally observed binding affinity to MT. We also showed that thermal fluctuation enhances the correlation and the interaction with the nucleotide suppresses the fluctuation of the helix . These results suggest that KIF1A regulates affinity to MT by changing the flexibility of the helix during the ATP hydrolysis process: the binding site becomes more flexible in the strong binding state than in the weak binding state. Proteins 2015; 83:809–819. © 2015 Wiley Periodicals, Inc.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

The crystal structure of Z‐(Aib)10‐OH at 0.65 Å resolution: three complete turns of 310‐helix

The synthetic peptide Z‐(Aib)₁₀‐OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub‐atomic resolution, where the bonding electron density becomes visible between the non‐hydrogen atoms. Crystals belong to the centrosymmetric space group P . Both molecules in the asymmetric unit form regular 3₁₀‐helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib‐homopeptides. These other peptides are C‐terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C‐terminal protection group to form a hydrogen bond to the residue i‐3, the sense of the helix is reversed in the last residue. Here, the C‐terminal OH‐groups form hydrogen bonds to the residues i‐3, in part mediated by water molecules. This makes Z‐(Aib)₁₀‐OH an Aib‐homopeptide with three complete 3₁₀‐helical turns in spite of the shorter length it has compared with Z‐(Aib)₁₁‐OtBu, the only homopeptide to date with three complete turns.     

Roles of conserved tryptophans in trimerization of HIV‐1 membrane‐proximal external regions: Implications for virucidal design via alchemical free‐energy molecular simulations

The Dual‐Action Virolytic Entry Inhibitors, or “DAVEI's,” are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15‐residue fragment from the membrane‐proximal external region (MPER) of HIV‐1 gp41. DAVEI's trigger rupture of HIV‐1 virions, and the interaction site between DAVEI MPER and HIV‐1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free‐energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER₃. We found that a W666A mutation had a large positive for all three protomers, while W672A had a large positive for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER₃ stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER₃. Our findings are consistent with our previous experimental study of W‐to‐A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV‐1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.     

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z‐axis parallel and x‐axis perpendicular to the C₆ symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.     

Molprobity's ultimate rotamer‐library distributions for model validation

Here we describe the updated MolProbity rotamer‐library distributions derived from an order‐of‐magnitude larger and more stringently quality‐filtered dataset of about 8000 (vs. 500) protein chains, and we explain the resulting changes and improvements to model validation as seen by users. To include only side‐chains with satisfactory justification for their given conformation, we added residue‐specific filters for electron‐density value and model‐to‐density fit. The combined new protocol retains a million residues of data, while cleaning up false‐positive noise in the multi‐ datapoint distributions. It enables unambiguous characterization of conformational clusters nearly 1000‐fold less frequent than the most common ones. We describe examples of local interactions that favor these rare conformations, including the role of authentic covalent bond‐angle deviations in enabling presumably strained side‐chain conformations. Further, along with favored and outlier, an allowed category (0.3–2.0% occurrence in reference data) has been added, analogous to Ramachandran validation categories. The new rotamer distributions are used for current rotamer validation in MolProbity and PHENIX, and for rotamer choice in PHENIX model‐building and refinement. The multi‐dimensional distributions and Top8000 reference dataset are freely available on GitHub. These rotamers are termed “ultimate” because data sampling and quality are now fully adequate for this task, and also because we believe the future of conformational validation should integrate side‐chain with backbone criteria. Proteins 2016; 84:1177–1189. © 2016 Wiley Periodicals, Inc.     

Exploration of conformational changes in lactose permease upon sugar binding and proton transfer through coarse‐grained simulations

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H⁺ symport process. Lactose/H⁺ symport is a highly complex process that involves sugar translocation, H⁺ transfer, and large‐scale protein conformational changes. The complete picture of lactose/H⁺ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse‐grained force field that couples the united‐atom protein models with the coarse‐grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a ‐d ‐galactopyranosyl‐1‐thio‐ ‐d ‐galactopyranoside (TDG) molecule to a wild‐type LacY. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X‐ray experiment. Transitions from inward‐facing to outward‐facing conformations upon TDG binding and protonation of Glu269 have been achieved from ～5.5 µs simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with double electron–electron resonance and thiol cross‐linking experiments. Our analysis suggests that the conformational changes of LacY are a cumulative consequence of interdomain H‐bonds breaking at the periplasmic side, interdomain salt‐bridge formation at the cytoplasmic side, and the TDG orientational changes during the transition.     

Graphene oxide/silver nanohybrid: Optimization,antibacterial activity and its impregnation on bacterial cellulose as a potential wound dressing based on GO‐Ag nanocomposite‐coated BC

Recently, bacterial cellulose (BC) based wound dressing have raised significant interests in medical fields. However, to our best knowledge, it is apparent that the BC itself has no antibacterial activity. In this study, we optimized graphene oxide‐silver (GO‐Ag) nanohybrid synthesis using Response Surface Methodology and impregnate it to BC and carefully investigate their antibacterial activities against both the Gram‐negative bacteria Escherichia coli and the Gram‐positive bacteria Staphylococcus aureus. We discover that, compared to silver nanoparticles, GO‐Ag nanohybrid with an optimal GO suspension's pH and ratio is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose. The GO‐Ag nanohybrid is more toxic to E. coli than that to S. aureus. The antibacterial and mechanical properties of BC/GO‐Ag composite are further investigated.     

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide‐dependent mechanism

Protein tyrosine (Tyr) nitration is a post‐translational modification yielding 3‐nitrotyrosine (NO₂–Tyr). Formation of NO₂–Tyr is generally considered as a marker of nitro‐oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O₂ transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO₂–Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO₂–Tyr25 and NO₂–Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and by alternative means to nitrate reductase, probably via a ˙NO synthase‐like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires  + H₂O₂ and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to . Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO₂–Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.     

Norovirus RNA‐dependent RNA polymerase: A computational study of metal‐binding preferences

Norovirus (NV) RNA‐dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer‐template duplex was investigated using X‐ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back‐tracked state of the enzyme, in which the ‐end of the primer occupies the position expected for the post‐incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.     

Antibody side chain conformations are position‐dependent

Side chain prediction is an integral component of computational antibody design and structure prediction. Current antibody modelling tools use backbone‐dependent rotamer libraries with conformations taken from general proteins. Here we present our antibody‐specific rotamer library, where rotamers are binned according to their immunogenetics (IMGT) position, rather than their local backbone geometry. We find that for some amino acid types at certain positions, only a restricted number of side chain conformations are ever observed. Using this information, we are able to reduce the breadth of the rotamer sampling space. Based on our rotamer library, we built a side chain predictor, position‐dependent antibody rotamer swapper (PEARS). On a blind test set of 95 antibody model structures, PEARS had the highest average χ₁ and accuracy (78.7% and 64.8%) compared to three leading backbone‐dependent side chain predictors. Our use of IMGT position, rather than backbone ϕ/ψ, meant that PEARS was more robust to errors in the backbone of the model structure. PEARS also achieved the lowest number of side chain–side chain clashes. PEARS is freely available as a web application at http://opig.stats.ox.ac.uk/webapps/pears .     

Modeling physical activity data using L0‐penalized expectile regression

In recent years accelerometers have become widely used to objectively assess physical activity. Usually intensity ranges are assigned to the measured accelerometer counts by simple cut points, disregarding the underlying activity pattern. Under the assumption that physical activity can be seen as distinct sequence of distinguishable activities, the use of hidden Markov models (HMM) has been proposed to improve the modeling of accelerometer data. As further improvement we propose to use expectile regression utilizing a Whittaker smoother with an L₀‐penalty to better capture the intensity levels underlying the observed counts. Different expectile asymmetries beyond the mean allow the distinction of monotonous and more variable activities as expectiles effectively model the complete distribution of the counts. This new approach is investigated in a simulation study, where we simulated 1,000 days of accelerometer data with 1 and 5 s epochs, based on collected labeled data to resemble real‐life data as closely as possible. The expectile regression is compared to HMMs and the commonly used cut point method with regard to misclassification rate, number of identified bouts and identified levels as well as the proportion of the estimate being in the range of of the true activity level. In summary, expectile regression utilizing a Whittaker smoother with an L₀‐penalty outperforms HMMs and the cut point method and is hence a promising approach to model accelerometer data.     

Excess post‐hypoxic oxygen consumption in Atlantic cod Gadus morhua

Atlantic cod Gadus morhua experienced oxygen deficit () when exposed to oxygen levels below their critical level (c. 73% of p_crit) and subsequent excess post‐hypoxic oxygen consumption (C_EPHO) upon return to normoxic conditions, indicative of an oxygen debt. The mean ± s.e . C_EPHO: was 6·9 ± 1·5, suggesting that resorting to anaerobic energy production in severe hypoxia is energetically expensive.