PROTOCOL
High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy
Aaron J. Wolfe,
Kyle J. Parella,
Liviu Movileanu,
Aaron J. Wolfe
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorKyle J. Parella
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorCorresponding Author
Liviu Movileanu
Department of Physics, Syracuse University, Syracuse, New York
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York
Corresponding author: [email protected]Search for more papers by this authorAaron J. Wolfe,
Kyle J. Parella,
Liviu Movileanu,
Aaron J. Wolfe
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorKyle J. Parella
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorCorresponding Author
Liviu Movileanu
Department of Physics, Syracuse University, Syracuse, New York
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York
Corresponding author: [email protected]Search for more papers by this authorFirst published: 30 August 2019
Abstract
This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd, the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd, the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.
Literature Cited
- Current Protocols. (1998). Commonly used reagents. Current Protocols in Protein Science, 12, A.2E.1–A.2E.5. doi: 10.1002/0471140864.psa02es12.
- Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., & Welte, W. (1998). Siderophore-mediated iron transport: Crystal structure of FhuA with bound lipopolysaccharide. Science, 282(5397), 2215–2220. doi: 10.1126/science.282.5397.2215.
- Gallagher, S. R. (2012). One-dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
- Gradinaru, C. C., Marushchak, D. O., Samim, M., & Krull, U. J. (2010). Fluorescence anisotropy: From single molecules to live cells. Analyst, 135(3), 452–459. doi: 10.1039/b920242k.
- Grosse, W., Psakis, G., Mertins, B., Reiss, P., Windisch, D., Brademann, F., … Essen, L. O. (2014). Structure-based engineering of a minimal porin reveals loop-independent channel closure. Biochemistry, 53(29), 4826–4838. doi: 10.1021/bi500660q.
- Hagel, L. (1998). Gel-filtration chromatography. Current Protocols in Proteins Science, 14, 8.3.1–8.3.30. doi: 10.1002/0471140864.ps0803s14.
- Hermanson, G. T. (2013). Bioconjugate techniques ( 3rd ed.). Cambridge, MA: Academic Press.
- Jameson, D. M., & Ross, J. A. (2010). Fluorescence polarization/anisotropy in diagnostics and imaging. Chemical Reviews, 110(5), 2685–2708. doi: 10.1021/cr900267p.
- Kimple, M. E., Brill, A. L., & Pasker, R. L. (2018). Overview of affinity tags for protein purification. Current Protocols in Protein Science, 73, 9.9.1–9.9.23. 10.1002/0471140864.ps0909s73.
- Kleinschmidt, J. H., & Popot, J. L. (2014). Folding and stability of integral membrane proteins in amphipols. Archives of Biochemistry and Biophysics, 564, 327–343. doi: 10.1016/j.abb.2014.10.013.
- Kwok, K. C., & Cheung, N. H. (2010). Measuring binding kinetics of ligands with tethered receptors by fluorescence polarization and total internal reflection fluorescence. Analytical Chemistry, 82(9), 3819–3825. doi: 10.1021/ac1002245.
- Lakowicz, J. R. (2006). Principles of fluorescence microscopy ( 2nd ed.). New York, NY: Springer.
- le Maire, M., Champeil, P., & Moller, J. V. (2000). Interaction of membrane proteins and lipids with solubilizing detergents. Biochimica et Biophysica Acta, 1508(1–2), 86–111. doi: 10.1016/s0304-4157(00)00010-1.
- D. R. E. Lide (ed.) (2008). CRC handbook of chemistry and physics—A ready reference book of chemical and physical data ( 88th ed.). Boca Raton, FL: CRC Press–Taylor and Francis Group.
- Moerke, N. J. (2009). Fluorescence polarization (FP) assays for monitoring peptide-protein or nucleic acid-protein binding. Current Protocols in Chemical Biology, 1(1), 1–15. doi: 10.1002/9780470559277.ch090102.
- Mohammad, M. M., Howard, K. R., & Movileanu, L. (2011). Redesign of a plugged beta-barrel membrane protein. Journal of Biological Chemistry, 286(10), 8000–8013. doi: 10.1074/jbc.M110.197723.
- Mohammad, M. M., Iyer, R., Howard, K. R., McPike, M. P., Borer, P. N., & Movileanu, L. (2012). Engineering a rigid protein tunnel for biomolecular detection. Journal of the American Chemical Society, 134(22), 9521–9531. doi: 10.1021/ja3043646.
- Movileanu, L., Cheley, S., Howorka, S., Braha, O., & Bayley, H. (2001). Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules. Journal of General Physiology, 117(3), 239–251. doi: 10.1085/jgp.117.3.239.
- Nazari, M., Kurdi, M., & Heerklotz, H. (2012). Classifying surfactants with respect to their effect on lipid membrane order. Biophysical Journal, 102(3), 498–506. doi: 10.1016/j.bpj.2011.12.029.
- Petty, K. J. (1996). Metal-chelate affinity chromatography. Current Protocols in Protein Science, 4, 9.4.1–9.4.16. 10.1002/0471140864.ps0904s04.
- Prazeres, T. J. V., Fedorov, A., Barbosa, S. P., Martinho, J. M. G., & Berberan-Santos, M. N. (2008). Accurate determination of the limiting anisotropy of rhodamine 101. Implications for its use as a fluorescence polarization standard. Journal of Physical Chemistry A, 112(23), 5034–5039. doi: 10.1021/jp710625j.
- Prinz, H. (2010). Hill coefficients, dose-response curves and allosteric mechanisms. Journal of Chemical Biology, 3(1), 37–44. doi: 10.1007/s12154-009-0029-3.
- Prive, G. G. (2007). Detergents for the stabilization and crystallization of membrane proteins. Methods, 41(4), 388–397. doi: 10.1016/j.ymeth.2007.01.007.
- Prive, G. G. (2009). Lipopeptide detergents for membrane protein studies. Current Opinion in Structural Biology, 19(4), 379–385. doi: 10.1016/j.sbi.2009.07.008.
- Roos, M., Ott, M., Hofmann, M., Link, S., Rossler, E., Balbach, J., … Saalwachter, K. (2016). Coupling and decoupling of rotational and translational diffusion of proteins under crowding conditions. Journal of the American Chemical Society, 138(32), 10365–10372. doi: 10.1021/jacs.6b06615.
- Rossi, A. M., & Taylor, C. W. (2011). Analysis of protein-ligand interactions by fluorescence polarization. Nature Protocols, 6(3), 365–387. doi: 10.1038/nprot.2011.305.
- Splinter, R. H., & Hooper, B.A. (2007). An introduction to biomedical optics. New York, NY: Taylor & Francis.
- Stoddart, L. A., White, C. W., Nguyen, K., Hill, S. J., & Pfleger, K. D. (2016). Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding. British Journal of Pharmacology, 173(20), 3028–3037. doi: 10.1111/bph.13316.
- Subbarao, G. V., & van den Berg, B. (2006). Crystal structure of the monomeric porin OmpG. Journal of Molecular Biology, 360(4), 750–759. doi: 10.1016/j.jmb.2006.05.045.
- Swonger, K. N., & Robinson, A. S. (2018). Using fluorescence anisotropy for ligand binding kinetics of membrane proteins. Current Protocols in Protein Science, 93(1), e63. doi: 10.1002/cpps.63.
- Textor, M., & Keller, S. (2015). Automated analysis of calorimetric demicellization titrations. Analytical Biochemistry, 485, 119–121. doi: 10.1016/j.ab.2015.06.009.
- Thakur, A. K., Larimi, M. G., Gooden, K., & Movileanu, L. (2017). Aberrantly large single-channel conductance of polyhistidine arm-containing protein nanopores. Biochemistry, 56(36), 4895–4905. doi: 10.1021/acs.biochem.7b00577.
- Thakur, A. K., & Movileanu, L. (2019). Real-time measurement of protein-protein interactions at single-molecule resolution using a biological nanopore. Nature Biotechnology, 37(1), 96–101. doi: 10.1038/nbt.4316.
- Titus, J. A., Haugland, R., Sharrow, S. O., & Segal, D. M. (1982). Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. Journal of Immunological Methods, 50(2), 193–204. doi: 10.1016/0022-1759(82)90225-3.
- Turman, D. L., Nathanson, J. T., Stockbridge, R. B., Street, T. O., & Miller, C. (2015). Two-sided block of a dual-topology F- channel. Proceedings of the National Academy of Sciences of the United States of America, 112(18), 5697–5701. doi: 10.1073/pnas.1505301112.
- Wolfe, A. J., Gugel, J. F., Chen, M., & Movileanu, L. (2018a). Detergent desorption of membrane proteins exhibits two kinetic phases. Journal of Physical Chemistry Letters, 9(8), 1913–1919. doi: 10.1021/acs.jpclett.8b00549.
- Wolfe, A. J., Gugel, J. F., Chen, M., & Movileanu, L. (2018b). Kinetics of membrane protein-detergent interactions depend on protein electrostatics. Journal of Physical Chemistry B, 122(41), 9471–9481. doi: 10.1021/acs.jpcb.8b07889.
- Wolfe, A. J., Hsueh, Y. C., Blanden, A. R., Mohammad, M. M., Pham, B., Thakur, A. K., … Movileanu, L. (2017). Interrogating detergent desolvation of nanopore-forming proteins by fluorescence polarization spectroscopy. Analytical Chemistry, 89(15), 8013–8020. doi: 10.1021/acs.analchem.7b01339.
- Wolfe, A. J., Mohammad, M. M., Thakur, A. K., & Movileanu, L. (2016). Global redesign of a native beta-barrel scaffold. Biochimica et Biophysica Acta, 1858(1), 19–29. doi: 10.1016/j.bbamem.2015.10.006.
- Wolfe, A. J., Si, W., Zhang, Z., Blanden, A. R., Hsueh, Y. C., Gugel, J. F., … Movileanu, L. (2017). Quantification of membrane protein-detergent complex interactions. Journal of Physical Chemistry B, 121(44), 10228–10241. doi: 10.1021/acs.jpcb.7b08045.
- Yildiz, O., Vinothkumar, K. R., Goswami, P., & Kuhlbrandt, W. (2006). Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation. The EMBO Journal, 25(15), 3702–3713. doi: 10.1038/sj.emboj.7601237.
- Yusko, E. C., Bruhn, B. R., Eggenberger, O. M., Houghtaling, J., Rollings, R. C., Walsh, N. C., … Mayer, M. (2017). Real-time shape approximation and fingerprinting of single proteins using a nanopore. Nature Nanotechnology, 12(4), 360–367. doi: 10.1038/nnano.2016.267.
- Zhang, H., Wu, Q., & Berezin, M. Y. (2015). Fluorescence anisotropy (polarization): From drug screening to precision medicine. Expert Opinion on Drug Discovery, 10(11), 1145–1161. doi: 10.1517/17460441.2015.1075001.
- Zumstein, L. (1995). Dialysis. Current Protocols in Protein Science, 00, A.3B.1–A.3B.4. doi: 10.1002/0471140864.psa03bs00|.
