New chimera proteins for fluorescence correlation spectroscopy.

A new class of chimera proteins has been developed. They are ideally suited for detection by fluorescence correlation spectroscopy (FCS), a new technology to analyze molecular interactions. The molecular structure of these chimera proteins consists of four domains: a N-terminal (His)6-tag for affinity chromatography followed by an eight amino acid epitope for immunodetection, a polypeptide affinity domain (ADF) for target specific interaction and a C-terminal Green Fluorescent Protein (GFPuv) for reporting of interaction with the target by FCS. We designed, prepared and characterized a prototype of ADF-GFP proteins capable of specific interaction with DNA fragments bearing nuclear factor (NF)-kappaB sites. ADF NF-kappaB p50 and a non-DNA-binding deletion mutant (p35) combined with GFPuv were inserted in a procaryotic vector and expressed in E. coli. Following affinity purification the fluoroproteins p50-GFPuv and p35-GFPuv were employed in specific protein-protein and protein-DNA interaction studies. FCS analysis as well as EMSA showed that p50-GFPuv revealed a fully functional ADF. We present a model for the preparation of GFP fusion proteins capable of specific interaction with proteins, lipids or nucleic acids. The rational design allows any polypeptide fragment to be incorporated into the chimeric protein. So a new series of bio-molecules with different binding specificities and assays can be developed.

Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study.

Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm). hRepA starts to aggregate under acidic conditions (

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.

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

Ras proto-oncogenes play a central role in cell proliferation by the regulation of signal transduction pathways from receptors of the outer cell membrane to the nucleus via the activation of transcription factors. Wild-type Ras cycles between the activated GTP-bound and the inactivated GDP-bound state, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-1 protein kinase. Mutations of ras resulting in the loss of the intrinsic GTPase activity result in autonomous proliferation. Mutated Ras is found in a variety of human tumors. Therefore, monitoring of GTP-loaded conformation of Ras related proteins could be utilised in cancer diagnosis. To develop a fluorescence based bioassay we have coupled the gene for the N-terminal Ras binding domain (RBD) of Raf-1 protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently investigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of fluorescently labeled molecules. Molecular interactions increase the molecular weight and influence the diffusion time of RBDGFP. FCS diffusion value of the recombinant protein was in coincidence with the molecular weight of the construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transition times indicated the interaction of RBDGFP with its corresponding antibody. Suboptimal binding of the fusion protein to activated Ras, how ever, resulted in a modest influence on the diffusion value. Taken together our rational design and construct shows the way for a ready characterisation of novel GFP-connected fusion proteins employing FCS.