to BioTechniques free email alert service to receive content updates.
Heterogeneous transition metal–based fluorescence polarization (HTFP) assay for probing protein interactions
 
Alexander Riechers1, Jennifer Schmidt2, Burkhard König1, and Anja Katrin Bosserhoff2
Full Text (PDF)

Multimerization studies

Although aggregation can lead to artifacts in other binding experiments, we hypothesized that our assay, with its long lifetime of the luminescent label, should be beneficial for investigating proteins prone to multimerization. The addition of an excess of unlabeled MIA protein to MIA-Ru(bpy)3 does not change FP (Figure 4A), indicating that the size of the multimers does not change and that there are no aggregates consisting of ≥10 molecules. We estimate this from the lifetime of the label and the molecular weight of the protein by the Perrin equation. To demonstrate the existence of smaller aggregates, we coated wells with a MIA-biotin conjugate. Indeed, a large increase in FP was detected, indicating the presence of direct MIA-MIA interactions. The formation of multimeric structures of MIA protein was also confirmed by Western blot analysis as shown in Figure 4B. These aggregates appear to be extraordinarily stable since they can even be observed after treatment with denaturing and reducing Laemmli buffer at 70°C.



Discussion

Several methods have been developed for the investigation of protein interactions. While surface plasmon resonance (SPR) (25) can also be used for small-molecule interactions with the help of antibodies, it is still costly because proprietary chips are generally required. Far-Western blotting is time consuming and not suitable for high-throughput applications (26). Furthermore, this method relies on the refolding of the protein to its native conformation on the membrane, which may not always be successful. Immuno-precipitation and pull-down experiments are also far more time-consuming than fluorescence-based investigations (27). Binding experiments using the 1-anilino-8-naphthalene sulfonate (ANS) probe (28), while compatible with multiwell plate–based assays, suffer from short excitation and emission wavelengths. Automated isothermal calorimetry measurements offer the advantage of label-free detection, but still require relatively large amounts of reagents.

Methods capable of handling high-throughput screening include various types of microarrays using enzymes, isotopes, or fluorescent labels. However, these techniques require special safety precautions, antibodies, and washing steps that may lead to cross-contamination and other artifacts.

FP detection is both high-throughput–capable and self-referenced, meaning that no washing steps are required. This is clearly an advantage over the traditional enzyme-linked immunosorbent assay (ELISA) platform. However, traditional homogeneous FP assays are limited by the molecular weight of the interaction partner to be investigated, due to the short lifetime of the required organic fluorophores. We extend this range by immobilizing a known interaction partner of the protein of interest, which is itself labeled with a long-lifetime luminescent transition metal chelate. The maximum acceptable molecular weight of the interaction partner obviously depends on the decay time of the label on the target. Given the decay time of Ru(bpy)3, we estimate from the Perrin equation (29) that interactions with binding partners of up to 500 kDa should still be observable; however, that limit could be raised by using a transition metal with a longer decay time.

The results show that our HTFP assay allows the investigation of protein–small molecule and protein–protein interactions. In contrast to traditional homogeneous FP assays, interactions with both high-and low–molecular weight compounds can be investigated. As presented for the interaction of MIA protein with AR54, this FP assay should also be amenable for the screening of libraries of potential drug candidates. Additionally, our HTFP assay is suitable for the investigation of protein aggregation and compounds cleaving these aggregates. This tolerance of the HTFP assay for aggregation makes it unique and should allow for the investigation of proteins that show aggregation-related artifacts in other assays. However, one limitation of our assay is that it will be difficult to estimate aggregate sizes from the polarization values due to the long decay time.

Since our assay format is applicable to a variety of situations, it is conceivable that it might also be used for other analytical or diagnostic applications. For instance, the HTFP assay would be expected to enable the investigation of protein complexes, such as cell signaling molecules, transport proteins, or transcription factors. It could also be used for the identification of an initiator or regulator of polymerization reactions (i.e., for actin or tubulin subunits within the dynamic processes of the cytoskeleton). This assay may also serve to identify activators or co-activators for enzymatic reactions as well as for the design of immunoassays in the field of serology and diagnostics. Since the HTFP assay is based on a luminescent transition metal complex label, it benefits from all the associated advantages over organic fluorophores. While the inherent photostability is obviously convenient, the large Stokes shift increases the signal-to-noise ratio and allows a broader selection of suitable emission filters for the spectrometer. Furthermore, complex biological matrices in the samples are also tolerable because the autofluorescence of biological material has a very short lifetime and can thus be eliminated. Finally, the long lifetime of transition metal complex labels opens the possibility of time-gated measurements. This may be employed for multi-label experiments with different transition metal complexes of different lifetimes, allowing these labels to be resolved regardless of spectral overlap.

Acknowledgments

We thank the Center of Excellence Fluorescent Bioanalytics (KFB) for providing access to the providing access to the Polarstar microplate reader and Jörg Plümpe (Active MotifChromeon) for the generous gift of the Ru(bpy)3-isothiocyanate dye. This work was supported by the University of Regensburg and a grant from the DFG (Deutsche Forschungsgemeinschaft).

The authors declare no competing interests.

Correspondence
Address correspondence to Anja Bosserhoff, University of Regensburg, Institute of Pathology, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. email: [email protected]

References
1.) Dürkop, A., F. Lehmann, and O.S. Wolfbeis. 2002. Polarization immunoassays using reactive rutheniummetal-ligand complexes as luminescent labels. Anal. Bioanal. Chem. 372:688-694.

2.) Guo, X.Q., F.N. Castellano, L. Li, and J.R. Lakowicz. 1998. Use of a long-lifetime Re(I) complex in fluorescence polarization immunoassays of high molecular weight analytes. Anal. Chem. 70:632-637.

3.) Szmacinski, H., F.N. Castellano, E. Terpetschnig, J.D. Dattelbaum, J.R. Lakowicz, and G.J. Meyer. 1998. Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics. Biochim. Biophys. Acta 1383:151-159.

4.) Guo, X.Q., F.N. Castellano, L. Li, and J.R. Lakowicz. 1998. Use of a long-lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular-weight analytes. Anal. Chem. 70:632-637.

5.) Terpetschnig, E., J.D. Dattelbaum, H. Szmacinski, and J.R. Lakowicz. 1997. Synthesis and spectral characterization of a thiol-reactive long-lifetime Ru(II) complex. Anal. Biochem. 251:241-245.

6.) Terpetschnig, E., H. Szmacinski, and J.R. Lakowicz. 1996. Fluorescence polarization immunoassay of a high-molecular-weight antigen using a long wavelength-absorbing and laser diode-excitable metal-ligand complex. Anal. Biochem. 240:54-59.

7.) Szmacinski, H., E. Terpetschnig, and J.R. Lakowicz. 1996. Synthesis and evaluation of Ru-complexes as anisotropy probes for protein-hydrodynamics and immunoassays of high-molecular-weight antigens. Biophys. Chem. 62:109-120.

8.) Terpetschnig, E., H. Szmacinski, H. Malak, and J.R. Lakowicz. 1995. Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics. Biophys. J. 68:342-350.

9.) Terpetschnig, E., H. Szmacinski, and J.R. Lakowicz. 1995. Fluorescence polarization immunoassay of a high-molecular-weight antigen based on a long-lifetime Ru-ligand complex. Anal. Biochem. 227:140-147.

10.) Nasir, M., and M.E. Jolley. 1999. Fluorescence polarization: an analytical tool for immunoassay and drug discovery. Comb. Chem. High Throughput Screen. 2:177-190.

11.) Hun, X., and Z. Zhang. 2007. Fluoroimmunoassay for tumor necrosis factor in human serum using Ru(bpy)3Cl2-doped fluorescent silica nanoparticles as labels. Talanta 73:366-371.

12.) Sanchez-Martinez, M.L., M.P. Aguilar-Caballos, and A. Gomez-Hens. 2007. Long-wavelength fluorescence polarization immunoassay: determination of amikacin on solid surface and gliadins in solution. Anal. Chem. 79:7424-7430.

13.) Sanchez-Martinez, M.L., M.P. Aguilar-Caballos, S.A. Eremin, and A. Gomez-Hens. 2007. Long-wavelength fluorescence polarization immuno-assay for surfactant determination. Talanta 72:243-248.

14.) Bosserhoff, A.K., M. Kaufmann, B. Kaluza, I. Bartke, H. Zirngibl, R. Hein, W. Stolz, and R. Buettner. 1997. Melanoma-inhibiting activity, a novel serum marker for progression of malignant melanoma. Cancer Res. 57:3149-3153.

15.) Bosserhoff, A.K., and R. Buettner. 2003. Establishing the protein MIA (melanoma inhibitory activity) as a marker for chondrocyte differentiation. Biomaterials 24:3229-3234.

16.) Dreau, D., A.K. Bosserhoff, R.L. White, R. Buettner, and W.D. Holder. 1999. Melanoma-inhibitory activity protein concentrations in blood of melanoma patients treated with immunotherapy. Oncol. Res. 11:55-61.

17.) Stahlecker, J., A. Gauger, A. Bosserhoff, R. Buttner, J. Ring, and R. Hein. 2000. MIA as a reliable tumor marker in the serum of patients with malignant melanoma. Anticancer Res. 20:5041-5044.

18.) Bosserhoff, A.K., and R. Buettner. 2002. Expression, function and clinical relevance of MIA (melanoma inhibitory activity). Histol. Histopathol. 17:289-300.

19.) Bauer, R., M. Humphries, R. Fassler, A. Winklmeier, S.E. Craig, and A.K. Bosserhoff. 2006. Regulation of integrin activity by MIA. J. Biol. Chem. 281:11669-11677.

20.) Bosserhoff, A.K., R. Stoll, J.P. Sleeman, F. Bataille, R. Buettner, and T.A. Holak. 2003. Active detachment involves inhibition of cell-matrix contacts of malignant melanoma cells by secretion of melanoma inhibitory activity. Lab. Invest. 83:1583-1594.

21.) Stoll, R., S. Lodermeyer, and A.K. Bosserhoff. 2006. Detailed analysis of MIA protein by mutagenesis. Biol. Chem. 387:1601-1606.

22.) Stoll, R., C. Renner, M. Zweckstetter, M. Bruggert, D. Ambrosius, S. Palme, R.A. Engh, M. Golob. 2001. The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold. EMBO J. 20:340-349.

23.) Schmidt, J., and A.K. Bosserhoff. 2006. Processing of MIA protein during melanoma cell migration. Int. J. Cancer. 125:1587-1594.

24.) Ismail, K.Z., and S.G. Weber. 1991. Tris(2,2′-bipyridine)ruthenium (II) as a peroxide-producing replacement for enzymes as chemical labels. Biosens. Bioelectron. 6:699-705.

25.) Myszka, D.G. 2004. Analysis of small-molecule interactions using Biacore S51 technology. Anal. Biochem. 329:316-323.

26.) Wu, Y., Q. Li, and X.Z. Chen. 2007. Detecting protein-protein interactions by far western blotting. Nat. Protocols 2:3278-3284.

27.) Arany, I., A. Faisal, Y. Nagamine, and R.L. Safirstein. 2008. p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells. J. Biol. Chem. 283:6110-6117.

28.) Gasymov, O.K., and B.J. Glasgow. 2007. ANS fluorescence: Potential to augment the identification of the external binding sites of proteins. Biochim. Biophys. Acta 1774:403-411.

29.) Lakowicz, J.R. 2006. Principles of fluorescence spectroscopy, 3rd editio. Springer Science+Business Media, New York.

  1    2    3    4