A sensitive fluorescence monitor for the detection of activated Ras: totalchemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface
Cfw. Becker et al., A sensitive fluorescence monitor for the detection of activated Ras: totalchemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface, CHEM BIOL, 8(3), 2001, pp. 243-252
Background: The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic si
gnaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead t
o a continuous stimulation of downstream effecters and ultimately to cell p
roliferation. Ras mutants which increase the steady-slate 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 un
protected peptide segments by native chemical ligation. Two fluorescent ami
no acids with structures based on the nitrobenz-2-oxa-1,3-diazole and couma
ryl chromophores were incorporated at a site which is close to the RBD/Ras.
GTP binding surface. Additionally, a C-terminal lag consisting of His(6) wa
s introduced. The K-d values for binding of the site-specifically modified
proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization
of C-terminal His(6) tag-modified fluorescent RBD onto Ni-NTA-coated surfa
ces 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 ac
tivity) can also be detected in this assay system. Ras.GDP does not bind to
the immobilized RBD, thus allowing discrimination between inactive and act
ivated Ras.
Conclusions: The site-specific incorporation of a fluorescent group at a st
rategic position in a Ras effector protein allows the detection of activate
d Ras with high sensitivity. This example illustrates the fact that the che
mical synthesis of proteins or protein domains makes it possible to incorpo
rate any kind of natural or unnatural amino acid at the position of choice,
thereby enabling the Facile preparation of specific biosensors, enhanced d
etection systems for drug screening, or the synthesis of activated proteins
, e.g. phosphorylated proteins involved in signaling pathways, as defined m
olecular species. (C) 2001 Elsevier Science Ltd. All rights reserved.