The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites

Halogen bonds in biological macromolecules

Includes bibliographical references.2016 Fall.The purpose of this dissertation is to study how halogen bonds (X-bonds) affect the stability of biological macromolecules and to develop a set of empirical mathematical equations that can provide insight into the anisotropic nature of covalently bound halogens. To achieve this end, we first conducted a detailed analysis of the Protein Data Bank (PDB) to determine the prevalence of X-bonding in biological macromolecules, which allowed us to study the geometrical trends associated with X-bonding. Quantum mechanical (QM) calculations were also applied to determine how the strength of X-bonds interaction could be "tuned." The next chapter used QM calculations to help parameterize an equation that can model the anisotropic size and charge of covalently bound chlorine, bromine and iodine. The energies obtained from this equation were validated on experimentally determined X-bond data by differential scanning calorimetry (DSC) in DNA holiday junctions and were found to nearly duplicate the energies obtained in the solution state experiments. In the final chapter, we engineer X-bonds into the structure of T4 lysozyme to studying structural and thermodynamic effects of X-bonds on protein. X-bonds were introduced into the enzyme via site-specific non-canonical amino acid incorporation and then the structure and stability of the protein were assayed via X-ray crystallography and DSC, respectively. The culmination of this work has elucidated many concepts that need to be considered when trying to engineer new biologically based materials with halogens

Public Conceptions of Scientific Consensus

Despite decades of concerted efforts to communicate to the public on important scientific issues pertaining to the environment and public health, gaps between public acceptance and the scientific consensus on these issues remain stubborn. One strategy for dealing with this shortcoming has been to focus on the existence of the scientific consensus. Recent science communication research has added support to this general idea, though the interpretation of these studies and their generalizability remains a matter of contention. In this paper, we describe results of a large qualitative interview study on different models of scientific consensus and the relationship between such models and trust of science, finding that familiarity with scientific consensus is rarer than might be expected. These results suggest that consensus messaging strategies may not be effective

Structure–Energy Relationships of Halogen Bonds in Proteins

The structures and stabilities of proteins are defined by a series of weak noncovalent electrostatic, van der Waals, and hydrogen bond (HB) interactions. In this study, we have designed and engineered halogen bonds (XBs) site-specifically to study their structure–energy relationship in a model protein, T4 lysozyme. The evidence for XBs is the displacement of the aromatic side chain toward an oxygen acceptor, at distances that are equal to or less than the sums of their respective van der Waals radii, when the hydroxyl substituent of the wild-type tyrosine is replaced by a halogen. In addition, thermal melting studies show that the iodine XB rescues the stabilization energy from an otherwise destabilizing substitution (at an equivalent noninteracting site), indicating that the interaction is also present in solution. Quantum chemical calculations show that the XB complements an HB at this site and that solvent structure must also be considered in trying to design molecular interactions such as XBs into biological systems. A bromine substitution also shows displacement of the side chain, but the distances and geometries do not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomolecular design

Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds

The construction of more stable proteins is important in biomolecular engineering, particularly in the design of biologics-based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid <i>m</i>-chlorotyrosine (^<i>m</i>ClY) increases both the thermal stability (increasing the melting temperature by ∼1 °C and the melting enthalpy by 3 kcal/mol) and the enzymatic activity at elevated temperatures (15% higher than that of the parent enzyme at 40 °C) of this classic enzyme. The chlorine of ^<i>m</i>ClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chemical calculations to be significantly enhanced by polarization via an intramolecular hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side chain, resulting in a distinctive synergistic HB-enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function associated with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics

Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds

The construction of more stable proteins is important in biomolecular engineering, particularly in the design of biologics-based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid <i>m</i>-chlorotyrosine (^<i>m</i>ClY) increases both the thermal stability (increasing the melting temperature by ∼1 °C and the melting enthalpy by 3 kcal/mol) and the enzymatic activity at elevated temperatures (15% higher than that of the parent enzyme at 40 °C) of this classic enzyme. The chlorine of ^<i>m</i>ClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chemical calculations to be significantly enhanced by polarization via an intramolecular hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side chain, resulting in a distinctive synergistic HB-enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function associated with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study

INTRODUCTION Asthma is a heterogeneous disease with poorly defined phenotypes. Severe asthmatics often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS Baseline urine was collected prospectively from healthy participants (n=100), mild-to-moderate asthmatics (n=87) and severe asthmatics (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from severe asthmatics (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS Ninety metabolites were identified, with 40 significantly altered (p<0.05, FDR<0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and mild-to-moderate asthmatics differed significantly from severe asthmatics (p=2.6×10-20), OCS-treated asthmatics differed significantly from non-treated (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the necessity to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma