title: Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches.
abstract: The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Horii
authors.forename: M
authors.initials: M
authors.lastname: Saito
authors.forename: T
authors.initials: T
authors.lastname: Yoda
authors.forename: K
authors.initials: K
authors.lastname: Tsumoto
authors.forename: M
authors.initials: M
authors.lastname: Matsushima
authors.forename: K
authors.initials: K
authors.lastname: Kuwajima
authors.forename: I
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002942:Circular Dichroism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006041:Goats; D006860:Hydrogen Bonding; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry* / Q000235:genetics
chemical_list: D020033:Protein Isoforms; D013912:Threonine; D007768:Lactalbumin