Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state |
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Authors: | Uchiyama Hidefumi; Perez-Prat Eva M; Watanabe Kimitsuna; Kumagai Izumi; Kuwajima Kunihiro |
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Affiliation: | Department of Chemistry and Biotechnology, Faculty of Engineering
2Department of Physics, School of Science The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan |
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Abstract: | Five mutant lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by ureainduced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed. |
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Keywords: | -lactalbumin/" target="_blank">gif" ALT="{alpha}" BORDER="0">-lactalbumin/ amino acid substitutions/ hydrophobic core/ molten globule/ refolding |
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