Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha -lactalbumin has been studied by stopped-flo w circular dichroism and molecular dynamics simulations. The effects of fou r single mutations and a double mutation on the stability of the protein un der a native condition were studied. The mutations were introduced into res idues located at a hydrophobic core in the alpha -domain of the molecule. H ere we show that an amino acid substitution (T29I) increases the native-sta te stability of goat alpha -lactalbumin against the guanidine hydrochloride -induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild -type and mutant goat alpha -lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was n ot constructed in the transition state of the folding reaction. To characte rize the local structural change around the Thr29 side chain to an atomic l evel of resolution, we performed high-temperature (at 400 K and 600 K) mole cular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 porti on of the molecule experienced structural disruption accompanied with the l oss of inter-residue contacts and with the water molecule penetration in th e 400-K simulation as well as in four of the six 600-K simulations. Disrupt ion of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous r eport. Proteins 2001;42:49-65. (C) 2000 Wiley-Liss, Inc.