Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1
AUTOR(ES)
Becker, Christian F. W.
FONTE
The National Academy of Sciences
RESUMO
Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein–protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras–RBD interaction as well as from nucleotide replacement reactions in Ras. An unnatural amino acid bearing a nitrobenzofurazan side chain was introduced into position 91 of the RBD, providing unique fluorescence properties. The association transient of nitrobenzofurazan labeled with Ras⋅guanosine 5′-β,γ-imidotriphosphate showed a slow phase that had not been detected in earlier work by using other signals.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=154300Documentos Relacionados
- Design, total chemical synthesis, and binding properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-binding domain of c-Raf-1
- RAS signalling is abnormal in a c-raf1 MEK1 double mutant.
- Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product.
- Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae.
- Mammalian assay for site-specific DNA damage processing using the human H-ras proto-oncogene.