Halina Rose Novak

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Marine Rhodobacteraceae l‐haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water

The putative l‐haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an l‐haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 μm·min−1·mg−1 of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate‐bound forms were determined. The active site of DehRhb had significant differences from previously studied l‐haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other l‐haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 Å from its position in other l‐haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the l‐haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst.

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae.

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over‐expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6‐dichlorohexane, 1‐bromooctane, 1,3‐dibromopropane and 1‐bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di‐halogenated substrates.

Mechanisms of Thermal Stability Adopted by Thermophilic Proteins and Their Use in White Biotechnology

Considerable interest has been generated in the mechanism which nature utilises to increase the stability of enzymes found in thermophilic and hyperthermophilic species. This has been the subject of many reviews, and our understanding has been enhanced by the increasing number of high-resolution thermostable enzyme structures that have been determined. Different species of bacteria and Archaea have used different mechanisms to achieve stability. A comparative approach has been used to carry out a detailed study of specific enzymes from a range of organisms in order to understand acquired stability at a structural level. This chapter will discuss the rules to increase protein thermostability that have been obtained from protein structural studies that are currently available. It will also examine other ways to stabilise existing proteins by lessons learnt from nature and by protein immobilisation.

Marine enzymes with applications for biosynthesis of fine chemicals

Abstract: This chapter will discuss the application of enzymes to carry out biotransformation reactions for the synthesis of building blocks of new drugs within the fine chemicals industry. It will concentrate on the marine environment to discover novel enzymes that have applications in this important area of substainable chemistry. Marine enzymes that have been cloned and isolated from bacteria, archaea, macro algae and viruses will be used to illustrate specific examples and applications. These enzyme activities include haloperoxidases, dehalogenases, alcohol dehydrogenases, L-aminoacylases, proteases, esterases and lipases. The biochemical and structural studies on these marine enzymes will be described in relation to their mechanism of action and evolutionary diversity with regards to related enzymes.