A mutational change in the signal sequence of ribose-binding protein (RBP) of E. coli blocks the export of the protein to the periplasm. Intragenic suppressors for this mutation that have single amino acid substitution in the mature portion of RBP at 27th and 50th have been selected previously. In order to assess the role of these amino acids in protein folding and translocation, site-specific mutagenesis approach was employed to substitute various amino acids. We obtained 9 amino acid changes for 27 position, and 11 for 50 position. Chemotactic experiments showed that 5 out of 9 changes at 27 position and 11 of 11 changes at 50 position restored taxis to ribose. Pulse chase labelling experiment showed that these proteins, less stable than wild-type RBP, were exported to the periplasm. Among these suppressor proteins, the less stable ones were translocated more efficiently than stable proteins. This implies that in wild-type RBP, Ala at 27 position might interacts with neighboring amino acid residues specifically during the folding process, whereas Val at 50th not.
It has been proposed that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export. We measured the intrinsic tyrosine fluorescence of some of the mutant ribose-binding proteins to analyse the equilibrium unfolding transition and unfolding/refolding kinetics. All the analysed mutant proteins showed decreased stability and slowed down refolding rate compared to that of wild-type by 2 to 26 folds. This result is consistent with the kinetic partitioning model. If folding rate slows down, the translocation-competent conformation would be maintained for longer time, therby increase the chance for interaction with a secretory protein factor.
