Thesis--Catholic University of America. / Bibliography: p. 25-27.

Lysozyme is a natural antimicrobial agent that is effective against many food spoilage and pathogenic microorganisms by disintegrating their cell walls. Immobilization of lysozyme has attractive applications for use in the food industry: (1) The enzyme could be readily separated from treated foods and beverages and re-used while the foods could still be claimed additive-free; (2) It could impart stable antimicrobial capability to the surface of food packaging polymers. In this study, a novel method is described for the preparation of a highly active immobilized lysozyme system. The method addressed three key issues in the covalent attachment of a biological active protein to an insoluble support: 1.) The protein should be attached to the matrix by the fewest possible bonds to minimize conformational change; 2.) The binding site(s) on the enzyme to the supports should be located as far as practical from its active center and be nonessential for its tertiary structure; 3.) The binding method should minimize the steric interference between the support and the immobilized enzyme. Using polystyrene resin beads as support matrix, peptide spacers of various lengths composed of 6-aminocaproic acid were synthesized with the solid phase peptide synthesis method. Then the amino terminals of the spacers were derivatized with bromoacetyl bromide and coupled to the protein's only histidine residue (His-15) that is nonessential for its lytic activity. Immobilized lysozyme with a spacer composed of three 6-aminocaproic acid units displayed the best lytic result against lyophilized M. lysodeikticus cells: 2736 U/g resin with a protein load of 2.21 mg/ resin. Retained activity was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher protein load yet the retained activity remained at about 14% level. A control consisted of random coupling of lysozyme to polystyrene beads without spacer gave an activity of 158 U/G with a protein load of 1.24 mg/g resin and 1.4% of retained activity, Properties of the immobilized lysozyme system were studied, including stability, effect of pH, surface characteristics of the support. A kinetics study of the system using Eadie-Hofstee plot demonstrated strong external diffusion effects, which resulted in deviation from classic Michaelis-Menton kinetic behavior. / Graduation date: 2004

A spontaneous deletion mutant of Bacillus phage φ29 (φ29Δ1) is characterized in the first part of this study. This mutant has a 1,112 base pair deletion, which covers the entire coding sequence of genes 14 and 15 including the early promoter, B2. While lysis is very delayed, the phage DNA synthesis and internal phage development appears to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in φ29Δ1. Results also suggest that genes 14 and 15 are dispensable for bacteriophage φ29 growth, and that the B2 promoter may also be despensable for the early functions of φ29. To further explore the function of gene 15, a DNA fragment of φ29 chromosome, encoding the entire sequence of this gene, has been cloned into the Escherichia coli expression vector pPLc245, under the control of the phage lambda major early leftward promoter, PL. Upon heat induction, a protein with an apparent size of 26 kdal was over-produced. This protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to the other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. This is the first lysozyme to be cloned and purified from a gram positive system. Bacteriophage φ29 lysozyme has been characterized in the last part of this study. Results show that this enzyme seems to more active than hen egg-white lysozyme against B. subtilis, E. coli, and M. lysodeikticus cells. Most of the characteristics of φ29 lysozyme appears to be similar to the P22 and T4 lysozymes, however, φ29 lysozyme seems to be about 2 times more thermostable than the other two lysozymes.

The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from solution samples and powder samples at varied hydration levels for pH 2, 3, 5, 7 and 10. The dependence of exchange of powder samples on the degree of hydration was the same at all pH, reaching rates of exchange equivalent to solution samples at 0.2 g H₂0/g protein (160 mol H₂0/mol protein), which corresponds to coverage of one-half the protein surface with monolayer water (Yang and Rupley, 1979). No additional hydrogen exchange was observed for protein powders equilibrated with higher water content. Considered in conjunction with other lysozyme hydration data (Rupley et al, 1983), this observation indicated that internal protein dynamics are not strongly coupled with surface properties. The use of powder samples offered control of water activity through regulation of water vapor pressure. The dependence of exchange rate on water activity was of low order, 2.9 average, and pH independent. This value, observed from 95 to 7 mol hydrogen remaining unexchanched/mol lysozyme, indicated that the rate determining step for protein hydrogen exchange is similar for all backbone amides and involves few water lilolecules. Powder samples were hydrated either by isopiestic equilibration against H₂S0₄ or NaOH solutions or by addition and mixing of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. Conformational change with hydration was ruled out as an explanation of this difference by a powder to solution jump experiment at pH 5, which proved the rank order of exchange was preserved. The difference can be explained by salt and pH effects expected to contribute to exchange of the nearly dry protein. Solution hydrogen exchange measured using the same lyophilized protein as the hydration experiments was in good agreement with published data. Rank order was proven the same for all pH by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pH and exhibited a complex character, in contrast to the dependence of exchange observed for positively charged polypeptide models.

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The work described in this thesis is broadly divided into two sections. The structural study on the lanthanide macrocyclic complexes was afforded by means of X-ray crystallography In this chapter, the molecules dota, the cationic enantiopure tetraamide europium and dysprosium complexes, the sodium complexes of the tetranaphthylamide and quinoyl derivative, the enantiopure gadolinium and europium complexes of the tetraamide series with esteratic sidechains, the lanathanum and ytterbium complexes of the dota derivative with benzyl phosphinate sidechains, and the tetracarboxyethyl series both as three uncomplexed stereoisomer and complexes of the RRRR stereoisomer with europium, gadolinium and terbium. These complexes exhibit quite a lot of structural diversity. Chapter five deals with experiments carried out at ultra low temperatures. A phase transition that the molecule benzil undergoes is investigated on the Fddd diffractometer, a study of the interesting 1,12-dicarbonyl borane was undertaken to obtain precise values for the carbonyl bond lengths and the unprecedented structure of its hydrate was revealed to be a carbene diol and not the expected carboxylic acid complex The standard for macromolecular tests for diffraction, chicken egg white lysozyme, was crystallised and used to optimise conditions for low-temperature data acquisition from macromolecular samples The work described in this Thesis was carried out in the Department of Chemistry, Durham University from October 1995 to January 1999, under the supervision of Professor J.A.K. Howard. All of the work is my own, unless stated to the contrary, and it has not been submitted previously for a degree at this or any other university.

Graduation date: 1995

Most proteins must fold into their native conformations to fulfil their biological functions. Failure of proteins to fold leads to cell pathology and a broad range of human diseases referred to as protein misfolding disease, e.g., Alzheimer’s disease, Parkinson’s disease, and type II diabetes. More than 40 proteins are known to be connected with misfolding diseases. These proteins share no sequence homology but all assemble into cross-b sheet containing insoluble fibrillar aggregates. Despite the pathological conditions that these proteins can induce, living organisms can take advantage of the inherent ability of these proteins to form such structures and to generate novel and diverse biological function, the functional amyloid.  This thesis examines different aspects of cross-b sheet containing aggregates. The first paper describes the humoral response to aggregated structures of insulin and the astrocytical biomarker S100B in patients suffering from Parkinson’s disease. We show that the patients have an increased immunreactivity towards insulin and S100B in Parkinson’s disease patients compared to a control group.  The second part of this work focuses on a functional amyloid. HAMLET (human a-lactalbumin made lethal for tumour cells) is a complex of a-lactalbumin and oleic acid, which kills tumour cells but not healthy differentiated cells. We wish to expand the concept of HAMLET to a structurally related protein and therefore create and characterize a complex of equine lysozyme and oleic acid (Paper II). We chose equine lysozyme because both proteins (equine lysozyme and a-lactalbumin) share common ancestors and are spatially related. The newly designed complex was named ELOA, for equine lysozyme with oleic acid. ELOA represents a functional oligomer due to its multimeric state and its ability to bind amyloid specific dyes. In the third paper, we investigate the interaction of the cytotoxic ELOA with live cells in real time to find a mechanistic model (Paper III).  It is known that HAMLET is not only tumouricidal but is also toxic towards many bacteria. Therefore in the last part of the thesis, we investigated the effects of ELOA on different bacterial strains and focused on its interplay Streptococcus pneumoniae (Paper IV).  These studies have added significantly to many aspects of protein folding and misfolding from its involvement in Parkinson’s disease to the newly gained functions and structural aspects of de novo produced ELOA.

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