Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy

Rns proto-oncogenes play a central role in cell proliferation by the regula tion of signal transduction pathways from receptors of the outer cell membr ane to the nucleus via the activation of transcription factors. Wild-type R as cycles between the activated GTP-bound and the inactivated GDP-bound sta te, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-l protein kinase. Mutations of,ras resul ting in the loss of the intrinsic GTPase activity result in autonomous prol iferation. Mutated Ras is found in a variety of human tumors. Therefore, mo nitoring of GTP-loaded conformation of Ras related proteins could be utilis ed in cancer diagnosis. To develop a fluorescence based bioassay we have co upled the gene for the N-terminal Ras binding domain (RBD) of Raf-l protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently in vestigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of f luorescently labeled molecules. Molecular interactions increase the molecul ar weight and influence the diffusion time of RBDGFP. FCS diffusion value o f the recombinant protein was in coincidence with the molecular weight of t he construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transi tion times indicated the interaction of RBDGFP with its corresponding antib ody. Suboptimal binding of the fusion protein to activated Ras, however, re sulted in a modest influence on the diffusion value. Taken together our rat ional design and construct shows the way for a ready characterisation of no vel GFP-connected fusion proteins employing FCS.