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Title: Structural and functional studies on bovine β-lactoglobulin
Author: McAlpine, Alan Scott
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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The existing medium resolution structure of the predominant bovine milk whey protein, β-lactoglobulin (B1g) showed that the fold of the monomer consisted of a barrel of 9 β-strands and one 3-turn α-helix. Its similarity to retinol binding protein (RBP), two bilin binding proteins and its close sequence homology to other transport proteins, has resulted in its inclusion in a superfamily, the lipocalycins. The β-barrel forms an hydrophobic pocket which is thought responsible for its ability to bind a series of small hydrophobic molecules. The function of the protein is uncertain. The possibility of engineering the protein's hydrophobic pocket so that it can carry small hydrophobic drug molecules through the stomach, where their presence may be detrimental is investigated in the following manner. Digestion experiments indicated that the protein has remarkable resistance to bovine pepsin and, to a lesser extent, trypsin. Its resistance to human pepsins is such that it would allow its passage through the stomach without any degradation. The presence of a ligand bound to the protein was shown to enhance the resistance to the protease trypsin. The solution of two small molecule structures is described and serves as an introduction to the technique of X-ray crystallography. Refinement of the existing model, lattice Y at pH 7.8 (space group B2212, a = 55.7 AA, b = 67.2 AA, c = 81.7 AA encounters some problems and these are discussed. A new medium resolution data set was collected and allows a more accurate model to be obtained. Refinement with the least squares package TNT gives an R-factor of 20.1&37 at 3.0 AA. The rescaling of existing high resolution data is described which will be merged with the medium resolution data set in the future. Crystals of the protein were grown from ammonium sulphate at pH 3.0 (space group P63; a = b&61 68.49 AA; c&61 143.17 AA) and data have been collected. A low pH structure will help investigate the protein's remarkable stability under these conditions. It will also help in the investigation of residues within the hydrophobic pocket which may be genetically engineered. Data have also been collected on crystals grown at the protein's isoelectric point, pH 5.2 (space group P21; a&61 72.2 AA; b&61 67.9 AA; c&61 36.2 AA; βchar61 92.0o), and examined by molecular replacement which orientates the lattice Y structure in the cell of the unknown.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available