Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.

Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. <b>Chapter 1</b> provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. <b>Chapter 2</b> describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. <b>Chapter 3</b> investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn²⁺ ion. The single channel recordings reveal a two step process, in which the Zn²⁺ ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. <b>Chapter 4</b> demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. <b>Chapter 5</b> explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]²⁺ binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.

Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 153-165).

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 269-270).

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.

<p>Sol-gel derived silica provides a bio-compatible material for the solid-phase entrapment of viable cells. A selection of <em>E. coli</em> cells containing unique promoter-linked GFP expression vectors were applied to fluorescence microwell plate assays, plate counting and various microscopy methods to assess changes in the entrapped bacteria and compatibility towards compound screening. Materials screening showed that a fastgelation sol-gel composition from sodium silicate precursor and PBS buffer provided a consistently greater fluorescence signal than non-entrapped cells. It is shown for the first time that entrapped cells are capable of dividing within pockets of the silica gel, and can di vide at a comparable rate to free cells. The entrapment of cells within a silica matrix does not induce the basal expression level of promoters tested here. Silica entrapment provides improved storage capabilities over non-entrapped cells in solution. A set of 12 related GFP-linked promoters were induced in solution and within silica when screened by two DNA gyrase inhibitors, providing similar expression profiles but greater signal-tonoise ratios in silica. The sol-gel derived material is amenable in an array format, and is a prospective material for the fabrication of sol-gel cell microarrays.</p> / Master of Science (MSc)

Hydrogenases are a family of enzyme that catalyses the bidirectional interconversion of H⁺ and H₂. There are two major classes of hydrogenases: the [NiFe(Se)]- and [FeFe]-hydrogenases. Both of these benefit from characteristics which would be advantageous to their use in technological devices for H₂ evolution and the generation of energy. These features are explored in detail in this thesis, with a particular emphasis placed on defining the conditions that limit the activity of hydrogenases when reducing H⁺ to produce H₂. Electrochemistry can be used as a direct measure of enzymatic activity; thus, Protein Film Electrochemistry, in which the protein is adsorbed directly onto the electrode, has been employed to probe catalysis by hydrogenases. Various characteristics of hydrogenases were probed. The catalytic bias for H₂ production was interrogated and the inhibition of H₂ evolution by H₂ itself (a major drawback to the use of some hydrogenases in technological devices to produce H₂) was quantified for a number of different hydrogenase. Aerobic inactivation of hydrogenases is also a substantial technological limitation; thus, inactivation of both H₂ production and H₂ oxidation by O₂ was studied in detail. This was compared to inhibition of hydrogenases by CO so as to elucidate the mechanism of binding of diatomic molecules and determine the factors limiting inactivation. This allows for a preliminary proposal for the genetic redesigning of hydrogenases for biotechnological purposes to be made. Finally, preliminary investigation of the binding of formaldehyde, potentially at a site integral to proton transfer, opens the field for further research into proton transfer pathways, the structural implications thereof and their importance in catalysis.

Understanding the factors influencing the ET characteristics of redox proteins confined at an electrochemical interface is of fundamental importance from both pure (fundamental science) and applied (biosensory) perspectives. This thesis reports on progress made in the emerging field of coupled electrochemical characterization and optical imaging in moving the analysis of redox-active films to molecular scales. More specifically the combination of cyclic voltammetry and wide-field Total Internal Reflection (TIRF) microscopy, here named ‘Fluorescent Cyclic Voltammetry’ (FCV), was applied to monitoring the response of surface-confined redox active proteins at submonolayer concentrations. The combined submicrometre spatial resolution and photon capture efficiency of an inverted TIRF configuration enabled the redox reactions of localized populations of proteins to be directly imaged at scales down to a few hundreds of molecules. This represents a 6-9 orders of magnitude enhancement in sensitivity with respect to classical current signals observed in bioelectrochemical analysis. Importantly, measurements of redox potentials at this scale could be achieved from both natural and artificially designed bioelectrochemical fluorescent switches and shed fundamental light on the thermodynamic and kinetic dispersion within a population of surface confined metalloproteins. The first three chapters of this thesis provide an overview of the relevant literature and a theoretical background to both the rapidly expanding fields of electroactive monolayers bioelectrochemistry and TIRF imaging. The initial design and construction of a robust electrochemically and optically addressable fluorescent switch, crucial to the applicability of FCV is reported in chapter 5. The generation of optically transparent, and chemically modifiable electrode surfaces suitable for FCV are also described. Chapter 6 describes the response of the surface confined azurin-based switch. Analysis of the spatially-resolved redox reaction of zeptomole samples in various conditions enables the mapping of thermodynamic dispersion across the sampled areas. In chapter 7 the newly developed FCV detection method was extended to investigate more complex bioelectrochemical systems containing multiple electron transferring redox centres and responding optically at different wavelengths. This approach provides a platform for spectral resolution of different electrochemical processes on the same sample. Finally in chapter 8 an electrochemical procedure is proposed for investigating the kinetic response of redox proteins using a fundamentally new methodology based on interfacial capacitance. In using variations in the surface chemistry to tune the rate of electron transfer, the approach was shown to be a robust and facile means of characterising redox active films in considerably more detail than possible through standard electrochemical methodologies. Ultimately, it can be applied to probe dispersion within protein populations and represents a powerful means of analysing molecular films more generally.

JMJD6 or PSR (phosphatidyl serine receptor) was initially proposed to be a membrane receptor involved in apoptotic cell clearance by recognition of apoptotic cells. However, sequence analyses implied the presence of a jelly roll or double stranded beta helix (DSBH) structural domain in PSR/JMJD6 and similarity with JmjC family of enzymes which are involved in chromatin regulation. Subsequently, PSR was renamed as JMJD6 and was reported to be a histone arginine demethylase. Previous work from our group has shown that JMJD6 is a lysine hydroxylase that interacts with nuclear proteins including CROP and U2AF65 which are involved in mRNA splicing. Peptide screening and cell based assays led to the conclusion that JMJD6 catalyses lysine hydroxylation of splicing regulatory proteins containing arginine serine rich domains (SR proteins) including U2AF65 and Luc7like-2. Studies were carried out to investigate the putative arginine demethylation activity of JMJD6 using MS analysis of histone peptides and luminescence-based assays. New substrates from SR proteins were identified by immunoprecipitation of JMJD6 expressed in human cell lines followed by LC-MS/MS analysis and MALDI-MS based assays of synthesised peptide substrates. Work then focussed on studying the mechanism of lysyl-hydroxylation from substrate and enzyme perspective. A crystal structure of seleno-methionine labelled JMJD6 was obtained and it provided insights into the JMJD6 active site and its substrate interactions. Based on this data, single point variants of JMJD6 were prepared and their substrate binding properties were studied by MALDI-MS and 2OG turnover assays. Collagen lysyl-hydroxylases are also 2OG dependent oxygenases. Efforts to investigate the stereochemistry of JMJD6 catalysed hydroxylation, employing NMR and amino acid analyses were carried out. These studies led to the interesting finding that the C-5 stereochemistry of hydroxylysine in LUC7L2 peptide is opposite (2S,5S-hydroxylysine) to that present in collagen (2S,5R-hydroxylysine). It was found that JMJD6 undergoes autocatalytic self-hydroxylation. Lysine residues from both recombinant JMJD6 and that from HeLa cells at endogenous level were identified to be hydroxylated by amino acid and LC-MS/MS analyses. JMJD6 has a strong tendency to form aggregates and gel electrophoresis always reveals multimeric bands of various JMJD6 constructs. Characterisation and identification of oligomeric states of JMJD6 was carried out using Electron Microscopy. Studies were initiated to identify possible inhibitors by screening a set of 2OG analogues. The results from this preliminary inhibition studies have identified the tricarboxylic acid (TCA) cycle intermediates, succinate and fumarate to be JMJD6 inhibitors and form a basis of further studies aimed at identifying selective inhibitors.