Protein Silencing with Self-Peptides.

Designing functional molecules which can recognize and modify the activity of a specific protein is a frequently encountered challenge in biology and pharmaceutical chemistry, and requires major effort for each specific protein target. Here we demonstrate that "self-peptides", parts of folded proteins which by their nature are recognizable by the rest of the protein, provide a general route to developing such molecules. Such a synthetic peptide with a chemically prestabilized conformation can incorporate into the target protein during its folding, and can potentially displace its native counterpart to cause functional deficits. This strategy is especially promising for proteins with β-barrel topology, as the seam of the barrel provides a vulnerable target. We demonstrate this strategy by using green fluorescent protein (EGFP) as a model, as its fluorescence is a direct reporter of its conformation and function. Refolding EGFP in the presence of 35 μM of a disulfide-stabilized 20-residue self-peptide (SP1, which resembles a seam, strands 3 and 11, of GFP) quenches the fluorescence by 97%. A peptide with the same composition but a different sequence is only 40% as effective, demonstrating that silencing is relatively specific. Fluorescence correlation spectroscopy and time-resolved fluorescence lifetime measurements show that SP1 causes complete long-term fluorescence silencing of the EGFP molecules it incorporates into. This result can in principle have a biological application if the self-peptide incorporates into a protein during its synthesis, before the nascent protein folds. We show that SP1 can indeed silence nascent sfGFP (closely related to EGFP) during its ribosomal synthesis in an in vitro translation system. Therefore, self-peptides present a potentially general strategy for developing protein-specific silencers for physiological applications.