Kimiko Saeki

Acid Denaturation and Refolding of Green Fluorescent Protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.

Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody

The position of the N terminus of myosin light chain 1 (LC1) and myosin light chain 2 (LC2) of rabbit skeletal muscle was mapped on the myosin head with a monoclonal antibody (SI304), which recognized the amino acid sequence N-trimethylalanyl-prolyl-lysyl-lysyl at the N terminus of LC1 and LC2. The complex of the antibody and myosin was observed by electron microscopy. By selective cleavage of the N terminus of LC1 or LC2 with papain or chymotrypsin, the position of the N terminus of LC1 and LC2 was determined separately. The N terminus of LC2 is located at the head-rod junction. The N terminus of LC1 is 11 nm (+/- 3 nm, standard deviation) from the head-rod junction. This position is near the actin-binding site of the myosin head.

Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants

Equilibria and kinetics of folding/unfolding of α‐lactalbumin and its two N‐terminal variants were studied by circular dichroism spectroscopy. The two variants were wild‐type recombinant and Glu1‐deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α‐lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped‐flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst‐phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding‐limb curvature was interpreted in terms of accumulation of the burst‐phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding‐limb curvature. We thus assumed a sequential four‐state mechanism, in which the folding from the burst‐phase intermediate takes place via two transition states separated by a high‐energy intermediate. We estimated changes in the free energies of the burst‐phase intermediate and two transition states, caused by the N‐terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca2+‐binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo‐wild‐type α‐lactalbumin in future studies. Proteins 2012;