No description available.

In recent years there have been two cycloterpenals, molecules consisting of a cyclohexadienal core, isolated from nature. Cyclocitral, the condensation product of citral, has been isolated from the North Sea bryozoans Flustra foliacea. In the human eyes, cycloretinal has been isolated and is a toxic by product of the vision cycle. This retinal dimer is believed to contribute to age related macular degeneration, the leading cause of blindness in the elderly. In 1992, it was discovered that if [beta]-ionylideneacetaldehyde was incubated with beta-lactoglobulin ([beta]-LG), the principal whey protein in dairy milk, that it would mediate the formation of cyclo-[beta]-ional. No follow up studies were performed on this protein mediated reaction or what biological activities these cycloterpenals may possess. This dissertation investigates the biological roles of substituted cyclohexadienes including cycloterpenals and cyclohexadiene enaminonitriles. To mimic the protein mediated reaction, we developed a synthetic procedure to produce a wide array of cycloterpenal by utilizing L-proline. Over 100 cycloterpenals were synthesized and screened for their biological activities against an array of cell based screens. The phenotypic effects of these cycloterpenals were screened against a PC12 assay where dramatic effects were observed on neurite outgrowth. During the synthesis of starting materials for the production of our cycloterpenal library, it was discovered that if excess base was added to the Horner-Wadsworth-Emmons reaction between a methyl-ketone and diethyl-(cyanomethyl)-phosphonate, conversion of the [alpha]-[beta]-unsaturated nitrile into an enaminonitrile was observed. This new synthetic procedure was optimized to generate a library of enaminonitriles as well as their quinazoline derivatives. The work within also includes the investigation of the [beta]-LG mediated reaction formation of cycloterpenals with natural and unnatural [beta]-methyl aldehydes. We were able to demonstrate that [beta]-LG could mediate the conversion of [alpha],[beta]-unsaturated aldehydes into their corresponding cycloterpenal. In vitro analysis was also performed with store bought milks and the [beta]-LG present was able to mediate the formation of cyclocitral. An in vivo experiment was also performed by utilizing New Zealand White rabbits to demonstrate the formation of cycloretinal within the blood stream by feeding a source of [beta]-LG with retinal. Interestingly, in human blood, [beta]-LG is present at concentrations of 0.7-1.2 g/dL. The protein has been identified within drusen pigments and lipofuscin granules that accumulate in the retina of macular degeneration patients. As humans do not produce beta-lactoglobulin, the source of this protein is from milk and milk products. With these experiments, we clearly demonstrate that under the appropriate conditions, cycloretinal can be produced with [beta]-LG. We have clearly established a direct link between [beta]-LG chemistry and age-related macular degeneration.

No description available.

Mixed solutions of β-lactoglobulin and anionic polysaccharides, specifically pectin, were formed into associative complexes through pH reduction from neutral conditions. Thermal treatment of these associative complexes was investigated as a function of biopolymer composition, heating conditions, pH, and ionic strength. Thermal treatment of β-lactoglobulin-pectin complexes at pH 4.5 – 5.0 was found to create protein-based particulates of consistent and narrow size distribution (diameter ~ 150 – 400 nm). These particulates were relatively stable to further pH adjustment and to high levels of salt (200 NaCl). Particle characteristics were maintained after re-suspending them in aqueous solutions after they have been either frozen or lyophilized. Thermal analysis of β- lactoglobulin-pectin complexes using calorimetry (DSC) and turbidity-temperature scanning indicated that the denaturation of β-lactoglobulin was unaffected by pectin, but protein aggregation was limited by the presence of pectin. Biopolymer particles formed using two different methods were compared: Type 1 – forming β-lactoglobulin nanoparticles by heating, then coating them with pectin; Type 2 – forming particles by heating β-lactoglobulin and pectin together. Type 2 particles had smaller diameters and had better pH and salt stability than Type 1 particles. It was proposed that Type 2 particles had a pectin-saturated surface that limited their aggregation, whereas Type 1 particles had “gaps” in the pectin surface coverage that led to greater aggregation. Finally, the possibility of controlling the size and concentration of biopolymer particles formed by heating β-lactoglobulin-pectin complexes by controlling preparation conditions was studied. Biopolymer particle size and concentration increased with increasing holding time (0 to 30 minutes), decreasing holding temperature (90 to 70 ºC), increasing protein concentration (0 to 2 wt%), increasing pH (4.5 to 5.0), and increasing salt concentration (0 to 50 mol/kg). The influence of these factors on biopolymer particle size was attributed to their impact on protein-polysaccharide interactions, protein denaturation, and protein aggregation kinetics. The knowledge gained from this study will facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes that can be used in the food and other industries, e.g., for encapsulation, texture modification, optical properties modification.

Bovine ß-lactoglobulin (ß-Lg) has been studied extensively but there is no clear identification of its biological function. Hydrophobic molecules have been observed binding into the hydrophobic calyx of ß-Lg. By comparison with other members of lipocalin family, it is probable that ß-Lg plays a role of transport of ligands, as ligands also bind into the central cavity of lipocalins. The structurally similar MUP is a pheromone-binding protein; therefore, it is possible that ß-Lg may also fulfil a similar role. This study has begun to test this hypothesis by investigating the interactions between bovine ß-Lg and several small volatile molecules (2-sec-4,5-dihydrothiazole, 3-methyl-2-butenal, 3-methyl-2-buten-1-ol and phenylacetic acid). The interactions between the volatile molecules and ß-Lg were studied by both two-dimensional NMR spectroscopy and X-ray crystallographic methods. TOCSY spectra were recorded for ß-Lg and the complex between ß-Lg and the ligands. The observed chemical shifts in the HN-Ha region are sensitive to the proximity of ligands, and hence chemical shift changes on ligand binding provide information on possible binding sites. It appears that several amino acids with hydrophobic sidechains are affected by interaction with volatile molecules at pH 2.0. The X-ray crystallographic study at pH 8.5 showed that the potential ligand, 2-sec-4,5-dihydrothiazole, may have decomposed into a linear 2-methyl-butanol. The refined structure (R=0.281, Rfree=0.354 for reflections to 2.6 Å resolution) reveals that the potential ligand may bind to the central cavity in a manner similar to the binding of 12-bromodecanoic acid to ß-Lg.

Protein-protein and protein-molecule interactions are complicated phenomena due to the tendency of proteins to change shape and function in response to their environment. Protein aggregation whether onto surfaces or in solution, can pose numerous problems in industry. Surface plasmon resonance (SPR) devices and quartz crystal microbalances (QCM) are two real-time, label free methods that can be used to detect the interactions between molecules on surfaces. These devices often employ self-assembled monolayers (SAMs) to produce specific surfaces for studying protein-protein interactions. The objective of this work was to develop methodologies utilizing SPR to better understand protein-protein and protein-molecule interactions with possible applications in the food and separation industrial sectors. A very well characterized whey protein, β-lactoglobulin (BLG), is used in numerous applications in the food industry. BLG can undergo different types of self-aggregation due changes in external environment factors such as buffer strength, pH or temperature. In this work, a hydrophilic SAM was developed and used to study the interaction and non-specific adsorption of BLG and palmitic acid (PA), a molecule which is known to bind to BLG. It was found that PA tended to reduce BLG conformational changes once on the surface, resulting in a decrease in its surface adhesion. Fluorescent excitation emission matrices (EEM’s) using a novel fluorescence probe technique were utilized to detect protein on the surface as well as conformational changes on the surface of the sensor, although the extent these changes could not be quantified. Another whey protein, α-lactoglobulin (AL), was utilized as a surrogate protein to study the adsorption of colloidal/particulate and protein matter (CPP) extracted from filtration studies of river water. A large fraction of natural organic matter (NOM), the major foulant in membrane based water filtration, is CPP and protein. Understanding the interactions between these components is essential in abating NOM membrane fouling. Several SPR methods were investigated in order to verify the interactions. A mixture of AL and CPP particles in solution prevented the non-specific adsorption of AL to the SAM surface. This change in association was then detected through SPR. Fluorescent EEM’s of the sensor surface verified that CPP and AL bound to the surface. This finding has fundamental significance in the interpretation of NOM-based membrane fouling. To better understand the mechanisms behind non-specific adsorption, a mechanistic mathematical model was developed to describe the adsorption of BLGs onto the hydrophilic SAM. The resulting model performed well in terms of predicting adsorption based on SPR data. The model incorporated the monomer-dimer equilibrium of BLG in solution, highlighting the impact of protein aggregation on non-specific adsorption mechanisms. For future studies, improvement in fluorescent FOP surface scan methodology would help identify different protein/molecules and conformations on the surface.

Biosynthetic natural products are invaluable resources that have been gleaned from the environment for generations, and they play an essential role in drug development. Natural product biosynthesis also possesses the latent ability to affect biological systems adversely. This work implements recent advances in genomic, proteomic and microbiological technologies to understand further biosynthetic molecules that may influence progression of human disease. Azinomycin A and B are antitumor metabolites isolated from the terrestrial bacterium Streptomyces sahachiroi. The azinomycins possess an unusual aziridine [1,2-a] pyrrolidine ring that reacts in concert with an epoxide moiety to produce DNA interstrand cross-links. Genomic sequencing of S. sahachiroi revealed a putative azinomycin resistance protein (AziR). Overexpression of AziR in heterologous hosts demonstrated the protein increases cell viability and decreases DNA damage response in the presence of azinomycin. Fluorescence titration indicated AziR functions as an azinomycin binding protein. An understanding of azinomycin resistance is important for future engineering and drug delivery strategies. Additionally, the S. sahachiroi draft genome obtained via 454 pyrosequencing and Illumina sequencing revealed several silent secondary metabolic pathways that may provide new natural products with biomedical application. β-lactoglobulin (BLG), the most abundant whey protein in bovine milk, has been observed to promote the self-condensation of retinal and similar α,β-unsaturated aldehydes. BLG is a possible non-genetic instigator of cycloretinal and A2E accumulation in the macula, a condition associated with age-related macular degeneration. BLG-mediated terpenal condensation has been optimized for in vitro study with the retinal mimic citral. In rabbits fed retinal and BLG or skim milk, cycloretinal formation was detected in the blood by 1H-NMR, and SDS-PAGE analysis indicated BLG was present in blood serum, suggesting the protein survives ingestion and retains catalytic activity. Mass spectrometry and site-directed mutagenesis provided mechanistic insight toward this unusual moonlighting behavior. The experiments described in this dissertation serve to further natural product biosynthesis discovery and elucidation as they relate to consequences for human health. Efforts to solve azinomycin biosynthesis via enzymatic reconstitution, characterize compounds produced by orphan gene clusters within S. sahachiroi, and obtain a clear mechanism for BLG-promoted cycloterpenal formation are immediate goals within the respective projects.

The main aim of this study is to determine genetic diversity of milk protein genes associated with milk traits, namely beta-casein, kappa-casein and betalactoglobulin, in native Turkish cattle breeds (Turkish Grey, Eastern Anatolian Red, Anatolian Black, and Southern Anatolian Red) and Turkish Holstein. Only 11% deviation from the Hardy-Weinberg equilibrium and insignificant Fis values for the populations were observed, indicating that samples are free of inbreeding. B alleles of these genes, which are positively related with cheese yield and quality, seem to be relatively high in native Turkish breeds. Therefore, the results suggest that milk of Turkish native breeds is advantageous for producing high-quality and -yield cheese. A1 allele of beta-casein, which releases a bioactive peptide called BCM-7 after successive gastrointestinal proteolytic digestions, has been claimed to have adverse health effects on humans. Another aim of this study is to develop a protocol and assess the potential detrimental effects of BCM-7 on human peripheral blood cells. Despite the fact that the results are inconclusive, the optimized experimental protocol will guide further researchers while judging the effect of BCM-7 on human health. Even though A1 beta-casein, which has a low frequency in native Turkish breeds, and hence BCM-7 have no adverse health effects on humans, this probability should be enough to keep its frequency low in native cattle breeds. Bulls must be screened for A1 allele of beta-casein as well as E allele of kappa-casein, which is absent in native breeds and known to have detrimental effects on cheese quality.

In this thesis, mechanisms for enhancing the stability of whey protein emulsions using two approaches were investigated. First, the physicochemical and emulsifying properties of whey protein isolate (WPI), and its two main proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (β-LG), were investigated in response to changes in pH and temperature pre-treatments. Solvent conditions which inhibit protein aggregation, such as pHs away from the isoelectric point, were found to form stable emulsions. In contrast, thermal treatments were found to negatively affect emulsion stability, where the most stable emulsions for WPI, ALA and β-LG were formed at room temperature (i.e. 25°C) at pH 7.0. It was also determined that emulsions formed using WPI, ALA and β-LG were stabilized by electrostatically repulsive forces which prevent flocculation and creaming. Secondly, the use of tailored protein-polysaccharide interactions involving WPI and carrageenan (CG) were explored as a means of enhancing emulsion stability. Carrageenan (CG) partakes in electrostatic attraction with WPI when acidified, leading to the formation of coupled gel networks. CG was selected for its anionic properties and for its well-characterized structure in that kappa-, iota- and lambda-type CG contain 1-, 2- and 3-sulfated groups per disaccharide repeating unit respectively. WPI-CG mixtures formed gel networks once acidified, where WPI-kappa-CG and WPI-iota-CG mixtures formed stiff networks, whereas WPI-lambda-CG formed a weak fluid network. WPI-CG complexes were found to be surface active, causing changes to the interfacial tension and interfacial rheology at pHs corresponding to where electrostatic attraction occurs upon acidification. Electrostatically coupled gel networks were formed in an emulsion, where oil droplets became entrapped within the biopolymer matrix. WPI-CG mixtures were sensitive to WPI-CG mixing ratio as stiffer gels were formed at higher CG content. Furthermore, WPI-iota-CG gels were stiffer than those made with WPI-kappa-CG gels presumably due to the higher number of sulfated groups lending greater opportunities for iota-CG to form bonds with neighboring polymers compared to kappa-CG.

Protein-protein and protein-molecule interactions are complicated phenomena due to the tendency of proteins to change shape and function in response to their environment. Protein aggregation whether onto surfaces or in solution, can pose numerous problems in industry. Surface plasmon resonance (SPR) devices and quartz crystal microbalances (QCM) are two real-time, label free methods that can be used to detect the interactions between molecules on surfaces. These devices often employ self-assembled monolayers (SAMs) to produce specific surfaces for studying protein-protein interactions. The objective of this work was to develop methodologies utilizing SPR to better understand protein-protein and protein-molecule interactions with possible applications in the food and separation industrial sectors. A very well characterized whey protein, β-lactoglobulin (BLG), is used in numerous applications in the food industry. BLG can undergo different types of self-aggregation due changes in external environment factors such as buffer strength, pH or temperature. In this work, a hydrophilic SAM was developed and used to study the interaction and non-specific adsorption of BLG and palmitic acid (PA), a molecule which is known to bind to BLG. It was found that PA tended to reduce BLG conformational changes once on the surface, resulting in a decrease in its surface adhesion. Fluorescent excitation emission matrices (EEM’s) using a novel fluorescence probe technique were utilized to detect protein on the surface as well as conformational changes on the surface of the sensor, although the extent these changes could not be quantified. Another whey protein, α-lactoglobulin (AL), was utilized as a surrogate protein to study the adsorption of colloidal/particulate and protein matter (CPP) extracted from filtration studies of river water. A large fraction of natural organic matter (NOM), the major foulant in membrane based water filtration, is CPP and protein. Understanding the interactions between these components is essential in abating NOM membrane fouling. Several SPR methods were investigated in order to verify the interactions. A mixture of AL and CPP particles in solution prevented the non-specific adsorption of AL to the SAM surface. This change in association was then detected through SPR. Fluorescent EEM’s of the sensor surface verified that CPP and AL bound to the surface. This finding has fundamental significance in the interpretation of NOM-based membrane fouling. To better understand the mechanisms behind non-specific adsorption, a mechanistic mathematical model was developed to describe the adsorption of BLGs onto the hydrophilic SAM. The resulting model performed well in terms of predicting adsorption based on SPR data. The model incorporated the monomer-dimer equilibrium of BLG in solution, highlighting the impact of protein aggregation on non-specific adsorption mechanisms. For future studies, improvement in fluorescent FOP surface scan methodology would help identify different protein/molecules and conformations on the surface.