Density functional theory based calculations of the vibrational properties of chlorophyll-a

Abstract Chlorophyll-a plays a fundamental role in the solar energy conversion processes that occur in oxygen evolving organisms, such as plants algae and cyanobacteria. To study the chlorophyll-a species at the heart of these solar conversion processes FTIR difference spectroscopy has been a valuable tool. However, FTIR difference spectra are only partially understood. To gain a more detailed understanding of FTIR difference spectra one of our goals is to calculate the vibrational properties of the chlorophyll-a systems found in plants and bacteria, and compare this to the properties found for isolated chlorophyll molecules in the gas phase and in solvents. As a first approach to this problem, we have calculated the vibrational properties of several chlorophyll-a model molecular systems using hybrid density functional theory at the B3LYP/6-31G(d) level. In particular, attention is focused on the infrared active vibrational modes of the carbonyl groups of chlorophyll-a, since these are the species that give rise to intense bands in infrared absorbance and absorbance difference spectra. The different chlorophyll-a models studied differ only in the number of carbonyl groups included in the model. In this way, it is possible to asses how the different C O modes couple. This is an important goal for a detailed understanding of the FTIR difference spectra. It also provides a very stringent test of the applicability of various computational approaches. Knowledge of how the different C O modes in monomeric chlorophyll species couple is also an important prerequisite for studies of multimeric or aggregated chlorophyll species, because in these aggregated species the carbonyl groups of the chlorophylls provide axial ligands to other chlorophylls. The infrared absorbance spectra and ''cation minus neutral'' infrared absorbance difference spectra of a model chlorophyll-a molecular system that contains the 13 1 -keto carbonyl group and the 17 3 -ester carbonyl group are calculated. The calculated spectra agree well with the corresponding experimentally determined spectra for pyrochlorophyll-a in polar solvents. When the vibrational properties of model chlorophyll-a molecular systems that contain both the 13 1 -keto and 13 3 -ester carbonyl group are calculated it is found that there is a strong coupling between the two carbonyl modes for the neutral species. In addition, for the chlorophyll-a cation, it is found that the calculated 13 1 -keto carbonyl mode frequency is higher than that of the 13 3 -ester carbonyl mode (although the two modes are no longer coupled). These calculated results do not agree with experiment. At the computationally more expensive 6-31+G(d) level, the calculations did give a more accurate description of the C O modes of neutral chlorophyll-a. However, there was still a considerable coupling between the 13 1 -keto and 13 3 -ester carbonyl modes. Calculations at the 6-31G(d) level do provide an accurate description of the experimentally determined behavior of the C C modes of chlorophyll-a, however. Finally, infrared absorbance spectra were calculated for chlorophyll-a model molecular systems that were fully 2 H, 15 N and 13 C labeled. In spite of the complicated coupling between the 13 1 -keto and 13 3 -ester carbonyl modes, it is found that calculated isotope induced vibrational frequency band shifts do closely match experiment. The calculations described here form a foundation on which to base more detailed calculations of chlorophylls in solvent or protein environments, or calculations of multimeric chlorophyll species. Such calculations will be undertaken as computational power increases.

Epigenetic and Transcriptional Dysregulation in T cells of Patients with Atopic Dermatitis

Rationale: Atopic dermatitis (AD) is linked to genetic and environmental risk factors. The effect of these factors on molecular and transcriptional events is not well understood. Immunologically, AD involves skin barrier defects and CD4+ T cells that produce inflammatory cytokines and amplify epidermal dysfunction Our objective was to investigate epigenetic mechanisms that may account for genetic susceptibility in CD4+ T cells. Methods: We measured chromatin accessibility (ATAC-seq), NFKB1 binding (ChIP-seq), and gene expression (RNA-seq) in anti-CD3/CD28 stimulated CD4+ T cells from 6 subjects with active moderate-to-severe AD and 6 age-matched non-allergic controls. Results: AD genetic risk loci were enriched for open chromatin regions in stimulated CD4+ T cells. The majority of ATAC-seq peaks were shared between matched AD-control pairs, consistent with those sections of chromatin being equally available. In contrast, NFKB DNA binding motifs were enriched in AD-dependent open chromatin. NFKB1 ChIP-seq identified genomic regions that were more strongly bound in AD cases, more strongly bound in controls, or shared between cases and controls. Chromatin that was strongly accessible and bound by NFKB1 in AD was enriched for AD genetic risk variants. Using whole genome sequencing data, we identified genotype-dependent accessible chromatin at AD risk loci corresponding to 32 genes with genotype-dependent expression in stimulated CD4+ T cells. Conclusions: The response of CD4+ T cells to stimulation is AD-specific and results in differential chromatin accessibility and transcription factor binding. These differences in transcriptional regulation result in epigenetic and transcriptional dysregulation in CD4+ T cells of patients with AD

Epigenetic and transcriptional dysregulation in CD4+ T cells in patients with atopic dermatitis

Atopic dermatitis (AD) is one of the most common skin disorders among children. Disease etiology involves genetic and environmental factors, with 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated the potential epigenetic mechanisms responsible for the genetic susceptibility of CD4+ T cells. To understand the differences in gene regulatory activity in peripheral blood T cells in AD, we measured chromatin accessibility (an assay based on transposase-accessible chromatin sequencing, ATAC-seq), nuclear factor kappa B subunit 1 (NFKB1) binding (chromatin immunoprecipitation with sequencing, ChIP-seq), and gene expression levels (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD, as well as in age-matched non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of the AD risk loci overlapping AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF-κB DNA-binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD- or control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF-κB DNA-binding motifs. Surprisingly, control-specific NFKB1 ChIP-seq peaks were not enriched for NFKB1 motifs, but instead contained motifs for other classes of human transcription factors, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of altered genotype-dependent chromatin accessibility at 36 AD risk variant loci (30% of AD risk loci) that might lead to genotype-dependent gene expression. Based on these findings, we propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease- and genotype-dependent chromatin accessibility alterations involving NFKB1 binding

Solar Energy Conversion in Plants and Bacteria Studied Using FTIR Difference Spectroscopy and Quantum Chemical Computational Methodologies

This dissertation presents a study of the molecular mechanism underlying the highly efficient solar energy conversion processes that occur in the Photosystem I (PS I) reaction centers in plants and bacteria. The primary electron donor P700 is at the heart of solar energy conversion process in PS I and the aim is to obtain a better understanding of the electronic and structural organization of P700 in the ground and excited states. Static Fourier Transform Infra-Red (FTIR) difference spectroscopy (DS) in combination with site directed mutagenesis and Density Functional Theory (DFT) based vibrational frequency simulations were used to investigate how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. (P700+-P700) FTIR DS at 77K were obtained from a series of mutants from the cyanobacterium Synechocystis sp. 6803 (S. 6803) where the amino acid residues near the C=O groups of the two chlorophylls of P700 where specifically changed. (P700+-P700) FTIR DS was also obtained for a set of mutants from C. reinhardtii where the axial ligand to A0-, the primary electron acceptor in PS I was modified. The FTIR DS obtained from these mutants provides information on the axial ligands, the hydrogen bonding status as well as the polarity of the environment of specific functional groups that are part of the chlorophyll molecules that constitute P700. Assignment of the FTIR bands to vibrational modes in specific types of environment is very difficult. In order to assist the assignment of the difference bands in experimental spectra DFT based vibrational mode frequency calculations were undertaken for Chl-a and Chl-a+ model molecular systems under different set of conditions; in the gas phase, in solvents using the Polarizable Continuum Model (PCM), in the presence of explicit solvent molecules using QM/MM methods, and in the presence of axial ligands and hydrogen bonds. DFT methods were also used to calculate the charge, spin and redox properties of Chl-a/Chl-a’ dimer models that are representative of P700, the primary electron donor in PS I

Box size effects are negligible for solvation free energies of neutral solutes.

Hydration free energy calculations in explicit solvent have become an integral part of binding free energy calculations and a valuable test of force fields. Most of these simulations follow the conventional norm of keeping edge length of the periodic solvent box larger than twice the Lennard-Jones (LJ) cutoff distance, with the rationale that this should be sufficient to keep the interactions between copies of the solute to a minimum. However, for charged solutes, hydration free energies can exhibit substantial box size-dependence even at typical box sizes. Here, we examine whether similar size-dependence affects hydration of neutral molecules. Thus, we focused on two strongly polar molecules with large dipole moments, where any size-dependence should be most pronounced, and determined how their hydration free energies vary as a function of simulation box size. In addition to testing a variety of simulation box sizes, we also tested two LJ cut-off distances, 0.65 and 1.0 nm. We show from these simulations that the calculated hydration free energy is independent of the box-size as well as the LJ cut-off distance, suggesting that typical hydration free energy calculations of neutral compounds indeed need not be particularly concerned with finite-size effects as long as standard good practices are followed

Predicting the excess solubility of acetanilide, acetaminophen, phenacetin, benzocaine, and caffeine in binary water/ethanol mixtures via molecular simulation.

We present a general framework to predict the excess solubility of small molecular solids (such as pharmaceutical solids) in binary solvents via molecular simulation free energy calculations at infinite dilution with conventional molecular models. The present study used molecular dynamics with the General AMBER Force Field to predict the excess solubility of acetanilide, acetaminophen, phenacetin, benzocaine, and caffeine in binary water/ethanol solvents. The simulations are able to predict the existence of solubility enhancement and the results are in good agreement with available experimental data. The accuracy of the predictions in addition to the generality of the method suggests that molecular simulations may be a valuable design tool for solvent selection in drug development processes

TET1 regulates responses to house dust mite by altering chromatin accessibility, DNA methylation, and gene expression in airway epithelial cells

BACKGROUND: Previous studies have identified TET1 as a potential key regulator of genes linked to asthma. TET1 has been shown to transcriptionally respond to house dust mite extract, an allergen known to directly cause allergic asthma development, and regulate the expression of genes involved in asthma. How TET1 regulates expression of these genes, however, is unknown. TET1 is a DNA demethylase; therefore, most prior research on TET1-based gene regulation has focused on how TET1 affects methylation. However, TET1 can also interact directly with transcription factors and histone modifiers to regulate gene expression. Understanding how TET1 regulates expression to contribute to allergic responses and asthma development thus requires a comprehensive approach. To this end, we measured mRNA expression, DNA methylation, chromatin accessibility and histone modifications in control and TET1 knockdown human bronchial epithelial cells treated or untreated with house dust mite extract. RESULTS: Throughout our analyses, we detected strong similarities between the effects of TET1 knockdown alone and the effects of HDM treatment alone. One especially striking pattern was that both TET1 knockdown and HDM treatment generally led to decreased chromatin accessibility at largely the same genomic loci. Transcription factor enrichment analyses indicated that altered chromatin accessibility following the loss of TET1 may affect, or be affected by, CTCF and CEBP binding. TET1 loss also led to changes in DNA methylation, but these changes were generally in regions where accessibility was not changing. CONCLUSIONS: TET1 regulates gene expression through different mechanisms (DNA methylation and chromatin accessibility) in different parts of the genome in the airway epithelial cells, which mediates inflammatory responses to allergen. Collectively, our data suggest novel molecular mechanisms through which TET1 regulates critical pathways following allergen challenges and contributes to the development of asthma

Runx1 shapes the chromatin landscape via a cascade of direct and indirect targets.

Runt-related transcription factor 1 (Runx1) can act as both an activator and a repressor. Here we show that CRISPR-mediated deletion of Runx1 in mouse metanephric mesenchyme-derived mK4 cells results in large-scale genome-wide changes to chromatin accessibility and gene expression. Open chromatin regions near down-regulated loci enriched for Runx sites in mK4 cells lose chromatin accessibility in Runx1 knockout cells, despite remaining Runx2-bound. Unexpectedly, regions near upregulated genes are depleted of Runx sites and are instead enriched for Zeb transcription factor binding sites. Re-expressing Zeb2 in Runx1 knockout cells restores suppression, and CRISPR mediated deletion of Zeb1 and Zeb2 phenocopies the gained expression and chromatin accessibility changes seen in Runx1KO due in part to subsequent activation of factors like Grhl2. These data confirm that Runx1 activity is uniquely needed to maintain open chromatin at many loci, and demonstrate that Zeb proteins are required and sufficient to maintain Runx1-dependent genome-scale repression

CASCADE: high-throughput characterization of regulatory complex binding altered by non-coding variants

Summary: Non-coding DNA variants (NCVs) impact gene expression by altering binding sites for regulatory complexes. New high-throughput methods are needed to characterize the impact of NCVs on regulatory complexes. We developed CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements), an array-based high-throughput method to profile cofactor (COF) recruitment. CASCADE identifies DNA-bound transcription factor-cofactor (TF-COF) complexes in nuclear extracts and quantifies the impact of NCVs on their binding. We demonstrate CASCADE sensitivity in characterizing condition-specific recruitment of COFs p300 and RBBP5 (MLL subunit) to the CXCL10 promoter in lipopolysaccharide (LPS)-stimulated human macrophages and quantify the impact of all possible NCVs. To demonstrate applicability to NCV screens, we profile TF-COF binding to ∼1,700 single-nucleotide polymorphism quantitative trait loci (SNP-QTLs) in human macrophages and identify perturbed ETS domain-containing complexes. CASCADE will facilitate high-throughput testing of molecular mechanisms of NCVs for diverse biological applications