Polypeptide motions are dominated by peptide group oscillations resulting from dihedral angle correlations between nearest neighbors

To identify basic local backbone motions in unfolded chains, simulations are performed for a variety of peptide systems using three popular force fields and for implicit and explicit solvent models. A dominant "crankshaft-like" motion is found that involves only a localized oscillation of the plane of the peptide group. This motion results in a strong anticorrelated motion of the phi angle of the ith residue (phi(i)) and the psi angle of the residue i - 1 (psi(i-1)) on the 0.1 ps time scale. Only a slight correlation is found between the motions of the two backbone dihedral angles of the same residue. Aside from the special cases of glycine and proline, no correlations are found between backbone dihedral angles that are separated by more than one torsion angle. These short time, correlated motions are found both in equilibrium fluctuations and during the transit process between Ramachandran basins, e.g., from the beta to the alpha region. A residue's complete transit from one Ramachandran basin to another, however, occurs in a manner independent of its neighbors' conformational transitions. These properties appear to be intrinsic because they are robust across different force fields, solvent models, nonbonded interaction routines, and most amino acids.     

Interpretation of Solution X-Ray Scattering by Explicit-Solvent Molecular Dynamics

Small- and wide-angle x-ray scattering (SWAXS) and molecular dynamics (MD) simulations are complementary approaches that probe conformational transitions of biomolecules in solution, even in a time-resolved manner. However, the structural interpretation of the scattering signals is challenging, while MD simulations frequently suffer from incomplete sampling or from a force-field bias. To combine the advantages of both techniques, we present a method that incorporates solution scattering data as a differentiable energetic restraint into explicit-solvent MD simulations, termed SWAXS-driven MD, with the aim to direct the simulation into conformations satisfying the experimental data. Because the calculations fully rely on explicit solvent, no fitting parameters associated with the solvation layer or excluded solvent are required, and the calculations remain valid at wide angles. The complementarity of SWAXS and MD is illustrated using three biological examples, namely a periplasmic binding protein, aspartate carbamoyltransferase, and a nuclear exportin. The examples suggest that SWAXS-driven MD is capable of refining structures against SWAXS data without foreknowledge of possible reaction paths. In turn, the SWAXS data accelerates conformational transitions in MD simulations and reduces the force-field bias.     

Nucleic acids: theory and computer simulation, Y2K

Molecular dynamics simulations on DNA and RNA that include solvent are now being performed under realistic environmental conditions of water activity and salt. Improvements to force-fields and treatments of long-range interactions have significantly increased the reliability of simulations. New studies of sequence effects, axis bending, solvation and conformational transitions have appeared.     

Acoustic homogeneity in the piranha Serrasalmus maculatus

Different studies suggest some social calls could be used in fish identification if their specificity is unambiguously assessed. Sounds of different populations of piranhas Serrasalmus maculatus Kner, 1858 were recorded to determine their homogeneity between rivers inside a single basin (Araguari and Grande River, upper Paraná River basin) and between separated basins (Amazon and Paraná basins). All fish from the different populations produced sounds with similar acoustic features. Consequently, the populations were not discernible based on individual sound characteristics. This high homogeneity between sounds from different populations indicates their usefulness for conservation projects using passive acoustic monitoring in piranhas. Moreover, it supports the use of acoustic features as complementary key characteristics in taxonomic studies.     

Proteins at Work: A COMBINED SMALL ANGLE X-RAY SCATTERING AND THEORETICAL DETERMINATION OF THE MULTIPLE STRUCTURES INVOLVED ON THE PROTEIN KINASE FUNCTIONAL LANDSCAPE*

C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.     

Antisymmetry,directional asymmetry,and dynamic morphogenesis

Fluctuating asymmetry is the most commonly used measure of developmental instability. Some authors have claimed that antisymmetry and directional asymmetry may have a significant genetic basis, thereby rendering these forms of asymmetry useless for studies of developmental instability. Using a modified Rashevsky-Turing reaction-diffusion model of morphogenesis, we show that both antisymmetry and directional asymmetry can arise from symmetry-breaking phase transitions. Concentrations of morphogen on right and left sides can be induced to undergo transitions from phase-locked periodicity, to phase-lagged periodicity, to chaos, by simply changing the levels of feedback and inhibition in the model. The chaotic attractor has two basins of attraction-right sidedominance and left side dominance. With minor disturbance, a developmental trajectory settles into one basin or the other. With increasing disturbance, the trajectory can jump from basin to basin. The changes that lead to phase transitions and chaos are those expected to occur with either genetic change or stress. If we assume that the morphogen influences the behavior of cell populations, then a transition from phase-locked periodicity to chaos in the morphogen produces a corresponding transition from fluctuating asymmetry to antisymmetry in both morphogen concentrations and cell populations. Directional asymmetry is easily modeled by introducing a bias in the conditions of the simulation. We discuss the implications of this model for researchers using fluctuating asymmetry as an indicator of stress.     

Genetic divergence among invasive and native populations of Plagioscion squamosissimus (Perciformes, Sciaenidae) in Neotropical regions

The genetic divergence among invasive and native populations of Plagioscion squamosissimus from four Neotropical hydrographic basins was assessed using the hypervariable domain of the mitochondrial DNA (mtDNA) control region. Plagioscion squamosissimus is native to the neighbouring hydrographic basins of the Parnaíba and Amazon Rivers, and the latter includes the Araguaia-Tocantins drainage, but it is invasive in other basins due to introductions. The mtDNA nucleotide polymorphism supported the hypothesis that the Amazon and Parnaíba populations constitute the same species and are separated into two independent evolutionary lineages. Absence of nucleotide polymorphism was observed within and among P. squamosissimus populations invasive to the uppper and middle Paraná River basins. Nucleotide divergence was null or low comparing the Paraná invasive populations with the populations native to the Parnaíba River basin, whereas it was significantly high compared to Tocantins populations. These results ascertain that P. squamosissimus populations invasive to the upper Paraná River basin and to the middle Paraná River basin downstream of the Itaipu dam are derived from the Parnaíba River basin. The genetic data presented are potentially useful to assist further studies on P. squamosissimus taxonomic and geographic distribution, development of ecological guidelines for managing populations invasive to the upper Paraná River basin and for preservation of native fish diversity.     

A fluid salt-bridging cluster and the stabilization of p53

p53 is a homotetrameric tumor suppressor protein that is found to be mutated in most human cancers. Some of these mutations, particularly mutations to R337, fall in the tetramerization domain and cause defects in tetramer formation leading to loss of function. Mutation to His at this site has been found to destabilize the tetramer in a pH-dependent fashion. In structures of the tetramerization domain determined by crystallography, R337 from one monomer makes a salt bridge with D352 from another monomer, apparently helping to stabilize the tetramer. Here we present molecular dynamics simulations of wild-type p53 and the R337His mutant at several different pH and salt conditions. We find that the 337-352 salt bridge is joined by two other charged side chains, R333 and E349. These four residues do not settle into a fixed pattern of salt bridging, but continue to exchange salt-bridging partners on the nanosecond time scale throughout the simulation. This unusual system of fluid salt bridging may explain the previous finding from alanine scanning experiments that R333 contributes significantly to protein stability, even though in the crystal structure it is extended outward into solvent. This extended conformation of R333 appears to be the result of a specific crystal contact and, this contact being absent in the simulation, R333 turns inward to join its interaction partners. When R337 is mutated to His but remains positively charged, it maintains the original interaction with D352, but the newly observed interaction with E349 is weakened, accounting for the reduced stability of R337H even under mildly acidic conditions. When this His is deprotonated, the interaction with D352 is also lost, accounting for the further destabilization observed under mildly alkaline conditions. Simulations were carried out using both explicit and implicit solvent models, and both displayed similar behavior of the fluid salt-bridging cluster, suggesting that implicit solvent models can capture at least the qualitative features of this phenomenon as well as explicit solvent. Simulations under strongly acidic conditions in implicit solvent displayed the beginnings of the unfolding process, a destabilization of the hydrophobic dimer-dimer interface. Computational alanine scanning using the molecular mechanics Poisson-Boltzmann surface area method showed significant correlation to experimental unfolding data for charged and polar residues, but much weaker correlation for hydrophobic residues.     

Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential

The conformation is described of the sialyl alpha(2----3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----N)Asn and NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----N)Asn is presented and for each glycosidic linkage, except for the alpha(2----6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1----4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2----3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2----6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2----6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta structural element. This provides an explanation for the shift effects induced by alpha(2----6) attachment of NeuAc to the N-acetyllactosamine unit.     

Quaternary relaxations in sol-gel encapsulated hemoglobin studied via NIR and UV spectroscopy

In this work, we study the kinetics of the R --> T transition in hemoglobin using a combination of near-infrared and near-ultraviolet spectroscopy. We use a sol-gel encapsulation protocol to decelerate the conformational transitions and to avoid spectral perturbations arising from ligand migration and recombination. We monitor two spectroscopic markers: band III in the near-IR, which is a fine probe of the heme pocket conformation, and the tryptophan band in the near-UV, which probes the formation of the Trpbeta37-Aspalpha94 hydrogen bond, characteristic of the T structure, at the critical alpha1beta2 subunit interface. The time evolution of these two bands is monitored after deoxygenation of encapsulated oxyhemoglobin, obtained by diffusion of a reducing agent into the porous silica matrix. Characteristic spectral shifts are observed: comparison with myoglobin enables us to assign them to quaternary structure relaxations. Band III spectral relaxation is clearly nonexponential, and analysis with the Maximum Entropy Method enables us to identify three processes. On the other hand, near-UV spectral relaxation follows an exponential decay with a time constant closely corresponding to the second process observed in the near IR. Very interestingly, the rates of all processes markedly depend on the viscosity of the co-encapsulated solvent, following a power law. Our results reveal correlations between heme pocket relaxations, induced by the R --> T transition, and structural event(s) occurring at the alpha1beta2 interface and highlight their solvent dependence. The power law viscosity dependence of relaxation rates suggests that the observed protein relaxations are "slaved" to the co-encapsulated solvent. The stepwise character of the quaternary transition is also evidenced.     

Spatial and temporal patterns in maternal energetic traits of yellow perch (Perca flavescens) in Lake Erie

1. For many fish species, survival during early life stages is linked to the size and energetic condition of females prior to reproduction. For example, females in good energetic condition are often more fecund and produce larger eggs and offspring than those in poor condition. 2. We measured the characteristics of female yellow perch (Perca flavescens) that may influence annual population fluctuations. From 2005 to 2007, we measured spatial variation in female reproductive traits, such as age, length, mass and energy density (J g^?1) of somatic tissues and ovaries among four spawning aggregations of yellow perch in western and central Lake Erie. 3. Maternal traits, such as somatic energy density and spawner age distribution, differed between the western and central basin, whereas reproductive traits, such as fecundity and ovarian energy density, differed across years. 4. To understand the implications of observed differences in demographic rates (growth and mortality rates) between basins, we developed a deterministic model to simulate the total egg production in the western and central basins under different scenarios of fishing mortality. 5. High growth rates and low mortality rates combined to produce higher modelled estimates of total egg production in the central than in the western basin, and a larger proportion of eggs were produced by old age classes in the central basin than in the western basin. 6. Our results demonstrate that changing harvest levels for populations with different demographic rates can influence total reproductive output through complex interactions between age‐specific mortality, growth and size‐specific fecundity, which has implications for the population dynamics of yellow perch and related species across a broad geographic range.     

Comparison of Generalised Born/Surface Area with Periodic Boundary Simulations to Study Protein Unfolding

Simulations comparing the rapid unfolding behaviour of the model protein barnase under explicit and implicit solvent systems have been undertaken in order to validate a faster implicit solvent method for studying proteins which are kinetically stable in silico. A comparison is made between all-atom explicitly solvated simulations of barnase undertaken using Particle Mesh Ewald electrostatic interactions with all-atom implicit solvent simulations undertaken using the generalised born/surface area (GBSA) method with a long non-bonded cut-off. The two explicit solvent unfolding trajectories appear to explore slightly different pathways showing the importance of having statistically valid ensembles which are not accessible from a single trajectory. The 500?K GBSA trajectory is unsuitable for exploring intermediate structures on the unfolding pathway of barnase, as the protein almost immediately jumps to a predominately random coil conformation. However, dropping the temperature to 400?K gives rise to trajectories where the protein is unable to climb out of the energy well containing the first intermediate state, in a reasonable timescale. A similar pattern to the explicit solvent unfolding trajectories is seen in 450?K GBSA runs, with the intermediate states differing between trajectories. The development of computer simulation methods suitable for application to more kinetically stable proteins will offer insight into the atomic detail of the conformational changes associated with protein unfolding diseases.     

Spatial and temporal dynamics of a freshwater eukaryotic plankton community revealed via 18S rRNA gene metabarcoding

The Brazilian Environmental Ministry (MMA) released a report in 2016 listing 163 non-native species into Brazilian inland waters. Reservoirs are among the freshwater habitats most frequently associated with the release of non-native species. Therefore, the aim of the present study was to evaluate the occurrence and distribution of the non-native species listed by the MMA, in large Brazilian reservoirs (≥ 30 km²). Further, we have tested the hypothesis that beta diversity of non-native species within reservoirs from the same basin is lower than those among reservoirs from distinct basins. A literature search was performed for 70 Brazilian reservoirs, resulting in the records of 91 non-native species. Reservoirs from the Paraná basin showed the highest number of occurrences, with 33 non-native species recorded only in the Itaipu reservoir. Beta diversity of non-native species showed higher variability among reservoirs from different basins than those within the same basin. Some basins were also distinguished by their composition of non-native species, as supported by the IndVal index. Non-native species were widespread along Brazilian reservoirs, and their distribution can be even higher than reported.     

Modeling loop reorganization free energies of acetylcholinesterase: a comparison of explicit and implicit solvent models

The treatment of hydration effects in protein dynamics simulations varies in model complexity and spans the range from the computationally intensive microscopic evaluation to simple dielectric screening of charge-charge interactions. This paper compares different solvent models applied to the problem of estimating the free-energy difference between two loop conformations in acetylcholinesterase. Molecular dynamics (MD) simulations were used to sample potential energy surfaces of the two basins with solvent treated by means of explicit and implicit methods. Implicit solvent methods studied include the generalized Born (GB) model, atomic solvation potential (ASP), and the distance-dependent dieletric constant. By using the linear response approximation (LRA), the explicit solvent calculations determined a free-energy difference that is in excellent agreement with the experimental estimate, while rescoring the protein conformations with GB or the Poisson equation showed inconsistent and inferior results. While the approach of rescoring conformations from explicit water simulations with implicit solvent models is popular among many applications, it perturbs the energy landscape by changing the solvent contribution to microstates without conformational relaxation, thus leading to non-optimal solvation free energies. Calculations applying MD with a GB solvent model produced results of comparable accuracy as observed with LRA, yet the electrostatic free-energy terms were significantly different due to optimization on a potential energy surface favored by an implicit solvent reaction field. The simpler methods of ASP and the distance-dependent scaling of the dielectric constant both produced considerable distortions in the protein internal free-energy terms and are consequently unreliable.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative cytogenetics and molecular phylogeography in the group astyanax altiparanae--Astyanax aff. bimaculatus (Teleostei, Characidae)

The genus Astyanax comprises small characin fish of the neotropical region. The so-called 'yellow-tailed characins' compose one of the most widely distributed Astyanax groups. A. altiparanae and A. aff. bimaculatus, are evolutionarily closely related and commonly found in several Brazilian hydrographic basins. In the present work, chromosomal data of specimens of A. altiparanae and A. aff. bimaculatus from 4 hydrographic basins in the states of S?o Paulo (Upper Tietê, Paranapanema, Ribeira de Iguape) and Rio de Janeiro (Guapimirim) are shown. All the populations showed 50 chromosomes, with different karyotypic formula. Although only a single Ag-NOR bearing chromosome pair was observed, all populations possess multiple cistrons of 18S rDNA. FISH with the 5S rDNA probe showed single signals at the interstitial position of one metacentric chromosome pair. C-bands are distributed in the terminal and interstitial regions of several chromosomes. However, the As-51 satDNA are frugally located in a few chromosomes of fishes from Upper Tietê, Paranapanema and Guapimirim Rivers, being absent in individuals of A. aff. bimaculatus from Ribeira de Iguape River basin. Beside these 4 populations, molecular phylogeography studies were also performed in individuals from Middle and Lower Tietê River basin and from 2 additional collection sites in the Paranapanema and Ribeira de Iguape River basins. The phylogeographic analysis using 2 mtDNA regions (totalizing 1.314 bp of ND2 and ATPase6/8 genes) of 8 populations of the group of 'yellow-tailed characins' from 3 major hydrographic basins showed structuring of populations, suggesting a correlation between chromosomal (nuclear) and molecular (mitochondrial) data.     

Molecular differentiation of species of the genus Zungaro (Siluriformes, Pimelodidae) from the Amazon and Paraná-Paraguay River basins in Brazil

Fish species of the Zungaro genus (Siluriformes, Pimelodidae) are amongst the largest migratory fish in Latin America and have considerable economic importance for commercial fishing in Brazil. However, natural populations of this large catfish are experiencing a severe decline. There are significant taxonomical inconsistencies for this fish. Two geographically separated species of the fish were initially described, one endemic in the Amazon and another in the Paraná-Paraguay River basins. A taxonomic review had recently proposed that there is only one Zungaro species in Brazil, based on morphological data. We made a molecular study of Zungaro populations in an attempt to solve taxonomical inconsistencies and to analyze genetic diversity in natural populations of this genus. We analyzed two regions of the mitochondrial DNA (the control region and the ATPase 6 gene region) of individuals sampled from the Paraná-Paraguay River and Amazon River basins. Analyses based on p-distances and maximum likelihood phylogenetic models showed a genetic difference between populations corresponding to different species. Genetic differentiation between Zungaro populations was at the same level as that observed between other Siluriformes species, using the same DNA sequences. We conclude that Zungaro species of the Paraná-Paraguay River basin do not belong to the same species found in the Amazon basin. This finding has a significant implication for conservation of this fish, given that populations are disappearing at a high rate in the Paraná-Paraguay River basin, mainly due to impoundments.     

High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.     

THE DUAL ROLE OF SELECTION AND EVOLUTIONARY HISTORY AS REFLECTED IN GENETIC CORRELATIONS

The patterns of genetic correlations between a series of eye and antenna characters were compared among two sets of spring-dwelling and cave-dwelling populations of Gammarus minus. The two sets of populations originate from different drainages and represent two separate invasions of cave habitats from surface-dwelling populations. Matrix correlations, using permutation tests, indicated significant correlations both between populations in the same basin and from the same habitat. The technique of biplot, which allows for the simultaneous consideration of relationships between different genetic correlations and different populations, was used to further analyze the correlation structure. A rank-3 biplot indicated that spring and cave populations were largely differentiated by eye-antennal correlations, whereas basins were differentiated by both eye-antennal and antennal-antennal correlations. Eye-antennal correlations, which are likely to be subject to selection, were most similar within habitats, which are likely to have similar selective regimes.