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 substitution(s) in the B helix, were engineered to examine the relation between the stability of the molten globule state and the hydrophobicity of these 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 protein shows a remarkably more stable molten globule than the other proteins. The recombinant proteins were expressed Escherichia coli 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 acidic 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 stabilized by the amino acid substitutions which raise the hydrophobicity of the residues, suggesting that the hydrophobic core in a globular protein plays an important role in the stability of the molten globule state. The change in stabilization free energy of the molten globule state caused by each amino acid substitution has been evaluated, and molecular mechanisms of stabilization of the molten globule state are discussed.
Uchiyama
H
H
Department of Chemistry and Biotechnology, The University of Tokyo, Hongo, Bunkyo-ku, Japan.
Perez-Prat
E M
EM
Watanabe
K
K
Kumagai
I
I
Kuwajima
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006041:Goats; D006168:Guinea Pigs; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D014508:Urea; D007768:Lactalbumin