Abstract
Fluorescence spectroscopic techniques have been widely applied to the study of soluble proteins over the past 20 yr, and they are being increasingly applied to membrane proteins, including membrane transporters. Some advantages of fluorescence approaches include high sensitivity (so that relatively small amounts of protein are required), commonly available and affordable instrumentation, and the ability to explore several different (but complementary) aspects of membrane protein structure and function. For comprehensive coverage of the principles and practice of modern fluorescence approaches for studying proteins, the reader is referred to the excellent book authored by Lakowicz (1).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, Kluwer Academic, New York.
Schneider, E. and Hunke, S. (1998) ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol. Rev. 22, 1–20.
Holland, I. B. and Blight, M. A. (1999) ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. J. Mol. Biol. 293, 381–399.
Sharom, F. J. (1997) The P-glycoprotein efflux pump: how does it transport drugs? J. Membr. Biol. 160, 161–175.
Ambudkar, S. V., Dey, S., Hrycyna, C. A., et al. (1999) Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 39, 361–398.
Ford, J. M. (1996) Experimental reversal of P-glycoprotein-mediated multidrug resistance by pharmacological chemosensitisers. Eur. J. Cancer 32A, 991–1001.
Higgins, C. F. and Gottesman, M. M. (1992) Is the multidrug transporter a flippase? Trends Biochem. Sci. 17, 18–21.
Sharom, F. J., Yu, X., Chu, J. W. K., et al. (1995) Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Biochem. J. 308, 381–390.
Urbatsch, I. L., al-Shawi, M. K., and Senior, A. E. (1994) Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. Biochemistry 33, 7069–7076.
Doige, C. A. and Sharom, F. J. (1992) Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells. Biochim. Biophys. Acta 1109, 161–171.
Sharom, F. J., Yu, X., DiDiodato, G., et al. (1996) Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport. Biochem. J. 320, 421–428.
Lu, P., Liu, R., and Sharom, F. J. (2001) Drug transport by reconstituted P-glycoprotein in proteoliposomes—effect of substrates and modulators, and dependence on bilayer phase state. Eur. J. Biochem. 268, 1687–1697.
Sharom, F. J., Liu, R., and Romsicki, Y. (1998) Spectroscopic and biophysical approaches for studying the structure and function of the P-glycoprotein multidrug transporter. Biochem. Cell Biol. 76, 695–708.
Sharom, F. J., Liu, R., Romsicki, Y., et al. (1999) Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies. Biochim. Biophys. Acta 1461, 327–345.
Sharom, F. J., Liu, R., Qu, Q., et al. (2001) Exploring the structure and function of the P-glycoprotein multidrug transporter using fluorescence spectroscopic tools. Semin. Cell Dev. Biol. 12, 257–266.
Liu, R., Siemiarczuk, A., and Sharom, F. J. (2000) Intrinsic fluorescence of the P-glycoprotein multidrug transporter: sensitivity of tryptophan residues to binding of drugs and nucleotides. Biochemistry 39, 14,927–14,938.
Wu, J. and Kaback, H. R. (1994) Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site. 2. Site-directed fluorescence studies. Biochemistry 33, 12,166–12,171.
Kwaw, I., Zen, K. C., Hu, Y. L., et al. (2001) Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding. Biochemistry 40, 10,491–10,499.
Liu, R. and Sharom, F. J. (1996) Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. Biochemistry 35, 11,865–11,873.
Sharom, F. J., Yu, X., and Doige, C. A. (1993) Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J. Biol. Chem. 268, 24,197–24,202.
Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., et al. (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J. Biol. Chem. 271, 31,543–31,548.
Doige, C. A. and Sharom, F. J. (1991) Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Protein Express Purif. 2, 256–265.
Parker, C. A. (1968) Photoluminescence of Solutions, Elsevier, Amsterdam.
Doppenschmitt, S., Spahn-Langguth, H., Regardh, C. G., et al. (1998) Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter. Pharm. Res. 15, 1001–1006.
Peterson, G. L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346–356.
Doige, C. A., Yu, X., and Sharom, F. J. (1992) ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Biochim. Biophys. Acta 1109, 149–160.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Sharom, F.J., Russell, P.L., Qu, Q., Lu, P. (2003). Fluorescence Techniques for Studying Membrane Transport Proteins. In: Yan, Q. (eds) Membrane Transporters. Methods in Molecular Biology, vol 227. Humana Press. https://doi.org/10.1385/1-59259-387-9:109
Download citation
DOI: https://doi.org/10.1385/1-59259-387-9:109
Publisher Name: Humana Press
Print ISBN: 978-1-58829-104-2
Online ISBN: 978-1-59259-387-3
eBook Packages: Springer Protocols