Research Paper A sensitive £uorescence monitor for the detection of activated Ras: total chemical synthesis of site-speci¢cally labeled Ras binding domain of c-Raf1 immobilized on a surface Christian F.W. Becker a , Christie L. Hunter b , Ralf P. Seidel a , Stephen B.H. Kent b , Roger S. Goody a , Martin Engelhard a ; * a Max-Planck-Institut fu «r molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany b Gryphon Sciences, 250 East Grand Ave., South San Francisco, CA 94080, USA Received 7 November 2000; revisions requested 13 December 2000; revisions received 4 January 2001; accepted 26 January 2001 First published online 13 February 2001 Abstract Background : The RasWGDP^RasWGTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated RasWGTP lead to a continuous stimulation of down- stream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of RasWGTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to RasWGTP but not to RasWGDP. Results: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromo- phores were incorporated at a site which is close to the RBD/ RasWGTP binding surface. Additionally, a C-terminal tag consist- ing of His 6 was introduced. The K d values for binding of the site- specifically modified proteins to RasWGTP are comparable to that of wild-type RBD. Immobilization of C-terminal His 6 tag- modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of RasWGTP in the 100 nM range. Likewise, RasWGTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. RasWGDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. Conclusions : The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species. ß 2001 Elsevier Science Ltd. All rights reserved. Keywords : Chemical protein synthesis ; Fluorescent biosensor ; Native chemical ligation ; Ras binding domain of c-Raf1 ; Site-speci¢c labeling 1. Introduction In recent years, chemical synthesis by means of the na- tive chemical ligation of unprotected peptides [1] has be- come a viable method for the preparation of a wide vari- ety of biologically active proteins [2]. These include cytokines, enzymes (e.g. human secretory phospholipase A2 [3]), and e¡ector molecules (e.g. the Ras binding do- main (RBD) fragment of c-Raf1 [4]). Even integral mem- brane proteins, a class of proteins which will become of great signi¢cance in research in the near future, have been prepared by total chemical synthesis [5]. Furthermore, the expressed protein ligation technique [6] has enabled the production of polypeptide thioesters by recombinant means for use in native chemical ligation. The combina- tion of chemically synthesized peptides with recombinant polypeptides greatly increases the versatility and appli- 1074-5521 / 01 / $ ^ see front matter ß 2001 Elsevier Science Ltd. All rights reserved. PII:S1074-5521(01)00003-5 Abbreviations : DAP, diaminopropionic acid ; DMCA, 6,7-dimethoxy- coumaryl-alanine; FRET, £uorescence resonance energy transfer ; GppNHp, guanosine-5P-(b,g-imido)-triphosphate ; HFIPA, hexa£uoro- isopropanol ; His, 6UHis-tag ; mant, methylanthraniloyl ; NBD, nitro- benz-2-oxa-1,3-diazole ; RBD, Ras binding domain of c-Raf1 * Correspondence: Martin Engelhard; E-mail : martin.engelhard@mpi-dortmund.mpg.de Chemistry & Biology 8 (2001) 243^252 www.elsevier.com/locate/chembiol