Design, total chemical synthesis, and binding properties of a [Leu-91-N(1)-methyl-7-azaTrp]Ras-binding domain of c-Raf-1

The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N(1)-methyl-7-azatryptophan) at position 91 of RBD, producing a protein with fluorescent properties distinct from and distinguishable from those of proteins containing the natural fluorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RBD based on transient kinetic monitoring of the binding event. Modified RBD containing the l-isomer of the unnatural amino acid or its racemic d,l mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein–protein interactions and for the introduction of an unnatural backbone structure at strategic positions.