Biology subjects

Weresczczynski, J.

Publications and source records attributed to Weresczczynski, J..

6 recordsLinked to original sources

bioRxiv2020

Effects of H2A.B incorporation on nucleosome structures and dynamics

The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B and canonical H2A containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein/protein and protein/DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B containing nucleosomes have significantly broader free wells, and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription. SIGNIFICANCENature has evolved a plethora of mechanisms for altering the physical and chemical properties of chromatin fibers as a means of controlling gene expression. These epigenetic processes may serve to increase or decrease DNA accessibility, manage the recruitment of remodeling factors, or tune the stability of the nucleosomes that make up chromatin. Here, we have used both conventional and enhanced sampling molecular dynamics simulations to understand how one of these epigenetic mechanisms, the substitution of canonical H2A proteins with the H2A.B variant, exerts its influence on the structures and dynamics of the nucleosome. Results show at the molecular level how this variant alters inter-molecular interactions to increase DNA accessibility as a means of increasing genetic accessibility and gene transcription.

biophysics↗

bioRxiv2020

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility

Sub-nucleosomal complexes including hexasomes and tetrasomes have been identified as intermediates in nucleosome assembly and disassembly. Their formation is promoted by certain histone chaperones and ATP-dependent remodelers, as well as through transcription by RNA polymerase II. In addition, hexasomes appear to be maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition affects the structure and accessibility of the nucleosomal DNA, its influence on the histone tails is largely unknown. Previously, we found that the H3 tail accessibly is occluded in the context of the nucleosome due to interactions with DNA (Morrison et al, 2018). Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. Similar to what we previously found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling at the histone tails and ultimately help shape the chromatin landscape.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Lipopolysaccharide Simulations are Sensitive to Phosphate Charge and Ion Parameterization

The high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria make it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations are parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS as well as how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pKa values for LPS, and subsequently validated by 31P solid-state NMR experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields. In all, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters, while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.

biophysics↗

bioRxiv2019

Elucidating the Influence of Linker Histone Isoforms on Chromatosome Dynamics and Energetics

Linker histones are epigenetic regulators that bind to nucleosomes and alter chromatin structures and dynamics. Biophysical studies have revealed two binding modes in the linker histone/nucleosome complex, the chromatosome, where the linker histone is either centered on or askew from the dyad axis. Each has been posited to have distinct effects on chromatin, however the molecular and thermodynamic mechanisms that drive them and their dependence on linker histone compositions remain poorly understood. We present molecular dynamics simulations of chromatosomes with the globular domain of two linker histone variants, generic H1 (genGH1) and H1.0 (GH1.0), to determine how their differences influence chromatosome structures, energetics, and dynamics. Results show that both unbound linker histones adopt a single compact conformation. Upon binding, DNA flexibility is reduced, resulting in increased chromatosome compaction. While both variants enthalpically favor on-dyad binding, energetic benefits are significantly higher for GH1.0, suggesting that GH1.0 is more capable than genGH1 of overcoming the large entropic reduction required for on-dyad binding which helps rationalize experiments that have consistently demonstrated GH1.0 in on-dyad states but that show genGH1 in both locations. These simulations highlight the thermodynamic basis for different linker histone binding motifs, and details their physical and chemical effects on chromatosomes.

biophysics↗

bioRxiv2019

Empirical and Computational Comparison of Alternative Therapeutic Exon Skip Repairs for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a common and devastating genetic disease that is primarily caused by exon deletions that create a genetic frameshift in dystrophin. Exon skipping therapy seeks to correct this by masking an exon during the mRNA maturation process, which restores dystrophin expression, but creates an edited protein missing both the original defect and the therapeutically skipped region. Crucially, it is possible to correct many defects in alternative ways, by skipping an exon either before, or after the patients defect. This results in alternatively edited, hybrid proteins, of possibly different properties and therapeutic consequences. Here, we examined three such dystrophin exon skipped edits, comprising two pairs of alternative repairs of the same underlying DMD defect. We found that in both cases, one member of each alternative repair was more stable than the other by a variety of thermodynamic and biochemical measures. We also examined the origin of these differences by molecular dynamics simulations, which showed that these stability differences were the result of different types of structural perturbations. For example, in one edit there was partial unfolding at the edit site which caused domain-localized perturbations, while in another there was unfolding at the protein domain junctions distal to the edit site which increased molecular flexibility. These results demonstrate that alternative exon skip repairs of the same underlying defect can have very different consequences at the level of protein structure and stability, and furthermore that these can arise by different mechanisms, either locally, or by more subtle long-range perturbations.

biophysics↗

bioRxiv2018

BEES: Bayesian Ensemble Estimation from SAS, a SASSIE-web Module

1Many biomolecular complexes exist in a flexible ensemble of states in solution which are necessary to perform their biological function. Small angle scattering (SAS) measurements are a popular method for characterizing these flexible molecules due to their relative ease of use and ability to simultaneously probe the full ensemble of states. However, SAS data is typically low-dimensional and difficult to interpret without the assistance of additional structural models. In theory, experimental SAS curves can be reconstituted from a linear combination of theoretical models, although this procedure carries significant risk of overfitting the inherently low-dimensional SAS data. Previously, we developed a Bayesian-based method for fitting ensembles of model structures to experimental SAS data that rigorously avoids overfitting. However, we have found that these methods can be difficult to incorporate into typical SAS modeling workflows, especially for users that are not experts in computational modeling. To this end, we present the "Bayesian Ensemble Estimation from SAS" (BEES) program. Two forks of BEES are available, the primary one existing as module for the SASSIE webserver and a developmental version that is a standalone python program. BEES allows users to exhaustively sample ensemble models constructed from a library of theoretical states and to interactively analyze and compare each models performance. The fitting routine also allows for secondary data sets to be supplied, thereby simultaneously fitting models to both SAS data as well as orthogonal information. The flexible ensemble of K63-linked ubiquitin trimers is presented as an example of BEES capabilities.

biophysics↗