EFFECTS OF AMINO-ACID SUBSTITUTIONS IN THE HYDROPHOBIC CORE OF ALPHA-LACTALBUMIN ON THE STABILITY OF THE MOLTEN GLOBULE STATE

Five mutant alpha-lactalbumins, with one or two amino acid substitutio n(s) in the B helix, were engineered to examine the relation between t he stability of the molten globule state and the hydrophobicity of the se amino acids. The mutation sites (Thr29, Ala30 and Thr33) have been chosen on the basis of comparison of the amino acid sequences of goat, bovine and gunea pig alpha-lactalbumin, in which the guinea pig prote in shows a remarkably more stable molten globule than the other protei ns, The recombinant proteins were expressed Escherichia call and then purified and refolded efficiently to produce the active proteins. The stability of the molten globule state of these engineered proteins has been investigated by urea-induced unfolding transition under an acidi c condition (pH 2.0), where the molten globule state is stable in the absence of urea, The results show that the molten globule state is sta bilized by the amino acid substitutions which raise the hydrophobicity of the residues, suggesting that the hydrophobic core in a globular p rotein plays an important role in the stability of the molten globule state, The change in stabilization free energy of the molten globule s tate caused by each amino acid substitution has been evaluated, and mo lecular mechanisms of stabilization of the molten globule state are di scussed.