Design, Total Chemical Synthesis, and Binding Properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-Binding Domain of c-Raf-1
Abstract
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 (N1-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.
- Publication:
-
Proceedings of the National Academy of Science
- Pub Date:
- July 1999
- DOI:
- 10.1073/pnas.96.14.7865
- Bibcode:
- 1999PNAS...96.7865S