Abstract
Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10–11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter, we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptor function.
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
Whorton, M. R., Bokoch, M. P., Rasmussen, S. G., Huang, B., Zare, R. N., Kobilka, B., and Sunahara, R. K. (2007) A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci U S A 104, 7682–7. Copyright 2007 National Academy of Sciences USA.
Bayburt, T. H., and Sligar, S. G. (2003) Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12, 2476–81.
Baas, B. J., Denisov, I. G., and Sligar, S. G. (2004) Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment. Arch Biochem Biophys 430, 218–28.
Leitz, A. J., Bayburt, T. H., Barnakov, A. N., Springer, B. A., and Sligar, S. G. (2006) Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology. Biotechniques 40, 601–602, 604, 606.
Amin, D. N., and Hazelbauer, G. L. (2010) The chemoreceptor dimer is the unit of conformational coupling and transmembrane signaling. J Bacteriol 192, 1193–200.
Raschle, T., Hiller, S., Yu, T. Y., Rice, A. J., Walz, T., and Wagner, G. (2009) Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs. J Am Chem Soc 131, 17777–9.
Mi, L. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., and Springer, T. A. (2008) Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs. Biochemistry 47, 10314–23.
Banerjee, S., Huber, T., and Sakmar, T. P. (2008) Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles. J Mol Biol 377, 1067–81.
Tsukamoto, H., Sinha, A., Dewitt, M., and Farrens, D. L. (2010) Monomeric rhodopsin is the minimal functional unit required for arrestin binding. J Mol Biol 399, 501–11.
Whorton, M. R., Jastrzebska, B., Park, P. S., Fotiadis, D., Engel, A., Palczewski, K., and Sunahara, R. K. (2008) Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer. J Biol Chem 283, 4387–94.
Kuszak, A. J., Pitchiaya, S., Anand, J. P., Mosberg, H. I., Walter, N. G., and Sunahara, R. K. (2009) Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. J Biol Chem 284, 26732–41.
Rogers, D. P., Roberts, L. M., Lebowitz, J., Datta, G., Anantharamaiah, G. M., Engler, J. A., and Brouillette, C. G. (1998) The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions. Biochemistry 37, 11714–25.
Rogers, D. P., Roberts, L. M., Lebowitz, J., Engler, J. A., and Brouillette, C. G. (1998) Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure. Biochemistry 37, 945–55.
Segrest, J. P. (1977) Amphipathic helixes and plasma lipoproteins: thermodynamic and geometric considerations. Chem Phys Lipids 18, 7–22.
Nolte, R. T., and Atkinson, D. (1992) Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism. Biophys J 63, 1221–39.
Gorshkova, I. N., Liu, T., Kan, H. Y., Chroni, A., Zannis, V. I., and Atkinson, D. (2006) Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation. Biochemistry 45, 1242–54.
Bhat, S., Sorci-Thomas, M. G., Alexander, E. T., Samuel, M. P., and Thomas, M. J. (2005) Intermolecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemical cross-linking and mass spectrometry. J Biol Chem 280, 33015–25.
Thomas, M. J., Bhat, S., and Sorci-Thomas, M. G. (2006) The use of chemical cross-linking and mass spectrometry to elucidate the tertiary conformation of lipid-bound apolipoprotein A-I. Curr Opin Lipidol 17, 214–20.
Li, H., Lyles, D. S., Thomas, M. J., Pan, W., and Sorci-Thomas, M. G. (2000) Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer. J Biol Chem 275, 37048–54.
Panagotopulos, S. E., Horace, E. M., Maiorano, J. N., and Davidson, W. S. (2001) Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins. J Biol Chem 276, 42965–70.
Koppaka, V., Silvestro, L., Engler, J. A., Brouillette, C. G., and Axelsen, P. H. (1999) The structure of human lipoprotein A-I. Evidence for the “belt” model. J Biol Chem 274, 14541–4.
Gan, K. N., Smolen, A., Eckerson, H. W., and La Du, B. N. (1991) Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab Dispos 19, 100–6.
Rogers, D. P., Brouillette, C. G., Engler, J. A., Tendian, S. W., Roberts, L., Mishra, V. K., Anantharamaiah, G. M., Lund-Katz, S., Phillips, M. C., and Ray, M. J. (1997) Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation. Biochemistry 36, 288–300.
Attie, A. D., Kastelein, J. P., and Hayden, M. R. (2001) Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis. J Lipid Res 42, 1717–26.
Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., and Sligar, S. G. (2004) Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126, 3477–87.
Bayburt, T. H., Leitz, A. J., Xie, G., Oprian, D. D., and Sligar, S. G. (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282, 14875–81.
Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001) Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30, 544–546, 548, 550.
Lefkowitz, R. J., and Shenoy, S. K. (2005) Transduction of receptor signals by beta-arrestins. Science 308, 512–7.
Kobilka, B. K. (1995) Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 231, 269–71.
Swaminath, G., Deupi, X., Lee, T. W., Zhu, W., Thian, F. S., Kobilka, T. S., and Kobilka, B. (2005) Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists. J Biol Chem 280, 22165–71.
Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., and Duong, F. (2007) Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J 26, 1995–2004.
Boldog, T., Grimme, S., Li, M., Sligar, S. G., and Hazelbauer, G. L. (2006) Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A 103, 11509–14.
Devanathan, S., Yao, Z., Salamon, Z., Kobilka, B., and Tollin, G. (2004) Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor. Biochemistry 43, 3280–8.
Civjan, N. R., Bayburt, T. H., Schuler, M. A., and Sligar, S. G. (2003) Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. Biotechniques 35, 556–60; 562–3.
Forte, T., Norum, K. R., Glomset, J. A., and Nichols, A. V. (1971) Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: structure of low and high density lipoproteins as revealed by elctron microscopy. J Clin Invest 50, 1141–8.
Lima, E. S., and Maranhao, R. C. (2004) Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin Chem 50, 1086–8.
Segrest, J. P., Jones, M. K., Klon, A. E., Sheldahl, C. J., Hellinger, M., De Loof, H., and Harvey, S. C. (1999) A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein. J Biol Chem 274, 31755–8.
Acknowledgments
This work is supported through funding of the National Institutes of Health (GM-068603 and GM-083118), the University of Michigan Biological Sciences Scholars Program and the Cellular and Molecular Biology Training Grant and the University of Michigan Rackham Merit Program.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Vélez-Ruiz, G.A., Sunahara, R.K. (2011). Reconstitution of G Protein-Coupled Receptors into a Model Bilayer System: Reconstituted High-Density Lipoprotein Particles. In: Luttrell, L., Ferguson, S. (eds) Signal Transduction Protocols. Methods in Molecular Biology, vol 756. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-160-4_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-160-4_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-159-8
Online ISBN: 978-1-61779-160-4
eBook Packages: Springer Protocols