Genetic study on protein folding of ribose-binding protein

Abstract

Although details of the protein folding in vitro were investigated with the recent technical advances, little is known about the folding of protein inside the cell that occurs in a quite different physicochemical environment because of the lack of tools to monitor the folding in vivo. In an attempt to compare protein folding in vitro with the folding in vivo, the protein translocation system of Escherichia coli was employed. According to the kinetic partitioning model of the protein translocation, mutations that affect the in vivo folding of ribose-binding protein (RBP) were isolated by selecting intragenic suppressors for the export-defective signal sequence. Along with 3 suppressor mutations in the signal sequence, eleven different amino acid substitutions were found in the mature region. In the three-dimensional structure of RBP, ten mutational changes were localized in the hydrophobic region of the N-domain in this two-domain protein together with the previously reported two suppressors [Kim et al. (1992) J. Bacteriol. 174, 5219-5227]. The stability of the purified mutant proteins were decreased by 2.1 to 5.1 kcal/mol as determined by the equilibrium unfolding study with the tyrosine fluorescence in the six mutational changes investigated. Kinetic study for refolding and unfolding induced by guanidine hydrochloride revealed that refolding of mutant proteins was retarded by 4.4 to 63-fold compared to that of wild type while unfolding were little affected. Together with the concentration of the randomly selected folding mutations in the N-domain, this kinetic result suggests that folding events in the N-domain are critical in the rate-limiting step of the refolding of RBP in vitro. By analyzing the unique translocation kinetics of mutant proteins according to the theoretical model that describes translocation kinetics in terms of the rates of export and folding in vivo, the rate coefficients for the folding in vivo were separated and compared with the folding...