Abstract
Biophysical measurements of multidrug transporters in vitro can often be of limited relevance to the natural in vivo behavior. In particular, the properties of transporters when removed from their native bilayer and solubilized in detergents or lipids can differ significantly from their in vivo properties, reducing the value of in vitro measurements for the design of antagonists to the transporters. This problem can be addressed by studying the transport protein in liposomes in which the properties of the liposome bilayer are altered through systematic changes in lipid composition. Isothermal titration calorimetry can be used to determine the properties of the lipid-reconstituted protein in bilayers of different lipid compositions as well as to quantify the percentage recovery of functional protein in different lipids. Both these measurements lead to an accurate analysis of substrate binding activity. The approach is illustrated here for the small multidrug transport protein, EmrE from Escherichia coli. The percentage of functional EmrE successfully reconstituted into liposome depends on lipid composition. Differences in ligand binding and subtle differences in the secondary structure also occur in different lipid compositions.
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
Rotem D, Schuldiner S (2004) EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J Biol Chem 279(47):48787-48793
Morein S, Andersson A, Rilfors L, Lindblom G (1999) Wild type Escherichia coli cells regulate the membrane lipid composition in a “window” between gel and non-lamellar structures. J Biol Chem 271:6801-6809
Johnson AE, van Waes MA (1999) The translocon: a dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 15:799-842
Cornell R, Arnold R (1996) Modulation of the activities of enzymes of membrane lipid metabolism by non-bilayer-forming lipids. Chem Phys Lipids 81(2):215-227
Perozo E, Kloda A, Cortes DM, Martinac B (2002) Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nat Struct Biol 9(9):696-703
Moe P, Blount P (2005) Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups. Biochemistry 44(36):12239-12244
Perozo E, Cortes DM, Sompornpisut P, Kloda A, Martinac B (2002) Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418(6901):942-948
van den Brink-van der Laan E, Chupin V, Killian JA, de Kruijff B (2004) Stability of KcsA tetramer depends on membrane lateral pressure. Biochemistry 43(14):4240-4250
van den Brink-van der Laan E, Chupin V, Killian JA, de Kruijff B (2004) Small alcohols destabilize the KcsA tetramer via their effect on the membrane lateral pressure. Biochemistry 43(20):5937-5942
Butler PJ, Ubarretxena-Belandia I, Warne T, Tate CG (2004) The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state. J Mol Biol 340(4):797-808
Wallace BA (2000) Synchrotron radiation circular-dichroism spectroscopy as a tool for investigating protein structures. J Synchrotron Radiat 7(Pt 5):289-295
Tate CG, Kunji ER, Lebendiker M, Schuldiner S (2001) The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution. Embo J 20(1-2):77-81
Muth TR, Schuldiner SA (2000) membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE. Embo J 19(2):234-240
MicroCal (1998) ITC Data Analysis in Origin. MicroCal
Acknowledgments
This work was supported by the BBSRC (B19845), Leverhulme Trust (F/00182/AW) and The Royal Society. PJB holds a Royal Society Wolfson Research Merit Award. We thank Kalypso Charalambous, Paul Curnow, Mark Lorch, and other members of PJB’s research group for discussions of experimental work and Kath Moreton for excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Miller, D., Booth, P.J. (2010). The Use of Isothermal Titration Calorimetry to Study Multidrug Transport Proteins in Liposomes. In: Weissig, V. (eds) Liposomes. Methods in Molecular Biology™, vol 606. Humana Press. https://doi.org/10.1007/978-1-60761-447-0_17
Download citation
DOI: https://doi.org/10.1007/978-1-60761-447-0_17
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-446-3
Online ISBN: 978-1-60761-447-0
eBook Packages: Springer Protocols