ABSTRACT
BACKGROUND: The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. RESULTS: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. CONCLUSIONS: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.
