Design, total chemical synthesis, and binding properties of a [Leu-91-N1-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 (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.
Abteilung Physikalische Biochemie, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
J R
JR
Sydor
C
C
Herrmann
S B
SB
Kent
R S
RS
Goody
M
M
Engelhard
D000595:Amino Acid Sequence; D001665:Binding Sites; D015195:Drug Design; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019908:Proto-Oncogene Proteins c-raf / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D010446:Peptide Fragments; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins