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Item Backbone dynamics of bovine [beta]-lactoglobulin by ¹⁵N NMR spectroscopy : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry, Institute of Fundamental Sciences and Institute of Molecular Biosciences, Massey University, New Zealand(Massey University, 2011) Baker, KristyBovine β-lactoglobulin (β-Lg) is a small 162 residue protein of unknown function from the whey component of milk, constituting ~50 % by dry mass. The protein is of great interest to the dairy industry due, in part, to its role in the fouling of dairy plants during heat treatment, and the significant operational costs this incurs. The structure of this protein is an eight stranded β-barrel with one long and two short flanking helices. It is dimeric at neutral pH but dissociates at pH < 3. In New Zealand herds there are three genetic variants, with variants A and B of bovine β-Lg predominating, while the C variant occurs at low levels in Jersey cows. However, despite the structural similarities of the three variants, milks containing one of A, B or C behaves differently when subjected to thermal processing. A greater understanding of factors that differentiate these protein variants is therefore important. In this study, 15N nuclear magnetic (NMR) spectroscopy methods have been used to study the backbone dynamics of β-Lg A and B, at one temperature, and the hitherto unstudied C variant, at three temperatures. For follow-up functional studies a mutant protein, a covalently linked Ala34Cys dimer, was produced.Item The binding of small volatile molecules by bovine [beta]-lactoglobulin : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University(Massey University, 2008) Hsu, Yu-TingBovine 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.