Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches

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. W e carried out protein stability measurements, X-ray crystallographic analys es, and free energy calculations based on molecular dynamics simulation. Th e equilibrium unfolding transitions induced by guanidine hydrochloride demo nstrated that the Thr29Val and Thr29Ile, mutants were, respectively, 1.9 an d 3.2 kcal/mol more stable than the wild-type protein (WT). The overall str uctures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the sidechain O-H group of Thr2 9 and the main-chain C=O group of Glu25. To analyze the stabilization mecha nism 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 t he denatured state. Futhermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unf avorably interacts electrostatically with the main-chain C=O group of Thr29 . The difference in the free energy profile of the unfolding path between W T and the Thr29Ile mutant is discussed in light of our experimental and the oretical results. (C) 2001 Wiley-Liss, Inc.