Modulation of intracellular protein activity at level of protein folding by beta-turn engineering

Control of the intracellular protein activity is very important in various biological studies and biotechnology. This has generally been achieved at the transcription and translation levels. Although control of the intracellular activity at the protein folding level is conceptually possible, but there have been few studies. The present study examined this possibility by modulating the in vivo protein folding rate of green fluorescence protein (GFP) through beta-turn engineering. A type II’ two residue beta-turn in GFP was targeted to generate two sets of mutants. First, a switch-off mutant was designed to stop the protein activity completely. The modulation mutants were then constructed to change the rates of GFP folding. The design of mutants was based on the rationale that residues i+1 and i+2 of a beta-turn have defined residue preferences, and their perturbation affects the rate of protein folding. The in vivo fluorescence activity of the designed GFP variants was switched off and modulated as expected. The change in the in vivo folding patterns of the mutants was confirmed by SDS-PAGE and found to be similar to the intracellular fluorescence activities of the mutants. The in vitro refolding kinetics performed with purified variants showed correlations with the in vivo folding patterns. These results showed that the beta-turns in a protein can be a target for modulating the in vivo protein folding pattern and activity.