Studies on the structure, function, and stability of bovine alpha-lactalbumin using site-directed mutagenesis

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Keith Brew - Committee Chair


$\alpha$-Lactalbumin is the regulatory component of the lactose synthase enzyme complex. It acts to modulate the substrate specificity of galactosyltransferase, the catalytic component, by promoting the binding of glucose and other monosaccharides while competitively inhibiting the binding of extended substrates. In addition to being of interest for its unusual role in the lactose synthase reaction, $\alpha$-lactalbumin is studied because of (i) its close evolutionary relationship with the type-c lysozymes with which it shares no overlap in function despite having a close similarity in 3D structure, (ii) its dependence on calcium ion for proper folding, and (iii) its ability to form a molten globule state under mildly denaturing conditions.A bacterial expression system has been developed from which a variant of bovine $\alpha$-lactalbumin (mLA) containing a N-terminal methionine and a valine substituted for methionine 90 is expressed in high yield. After purification and treatment to generate native fold, the protein recovered using this system is indistinguishable in physical and kinetic properties from bovine milk $\alpha$-lactalbumin. Using this system, 28 variants of mLA containing single site substitutions were expressed. Three different effects due to substitution of conserved residues were observed depending on the location of the change: (i) the modification had no significant effect on the kinetic behavior of the protein, (ii) the mutated protein was deficient in binding to galactosyltransferase, but when bound was as effective as bovine LA in promoting glucose binding, (iii) the modified protein was less able to promote glucose binding and was impaired in galactosyltransferase binding to variable extents. The results indicate that a region adjacent to the $\alpha$-lactalbumin cleft, aromatic cluster I, is very important in LA action. A section of this region that is distal from the cleft appears to act in galactosyltransferase binding, whereas a region close to the cleft influences glucose binding. Another aromatic cluster, the hydrophobic box, appears to be important for folding but not for activity.A non-native, stable tertiary interaction is formed between residues Tyr 103, Trp 104, and His 107 in the acid induced molten globule state of $\alpha$-lactalbumin. Mutations were introduced at these sites in order to study the contributions of this cluster to the relative stabilities of the native, molten globule, and unfolded states by guanidine denaturation melts. It was found that introduction of a tyrosine or a tryptophan at position 107 stabilizes the molten globule relative to the denatured state without affecting the free energy difference between the molten globule and native states. In contrast, the introduction of a proline at position 103 resulted in a protein in which the molten globule was greatly destabilized, but in which the overall stability was similar to that of mLA. (Abstract shortened by UMI.)


Biology, Molecular; Chemistry, Biochemistry

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