Abstract
Our experimental approach is based on the atomic force microscope (AFM) imaging of epitope-tagged subunits within membrane protein complexes purified in small amounts and decorated by anti-tag antibodies. Furthermore, we can produce simultaneous decoration of protein complexes using Fab fragments and IgG antibodies, which, combined with chemical modification of the substrate, allows us to determine the protein orientation across the cell membrane. Here, we describe a detailed protocol for membrane protein purification, AFM data collection, analysis, and interpretation of results. The protocol also covers basic AFM instrument settings and best practices for both observation of membrane protein complexes by AFM and automatic detection of the structures by an in-house algorithm. Once a sufficient number of membrane protein complexes have been visualized by AFM, data acquisition and processing can be completed in approximately 10 min using a scanning surface of 1 μm2.
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
Lacapere, J. J., Pebay-Peyroula, E., Neumann, J. M., and Etchebest, C. (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32, 259–270.
Muller, D. J. (2008) AFM: a nanotool in membrane biology Biochemistry 47, 7986–7998.
Shahin, V., and Barrera, N. P. (2008) Providing unique insight into cell biology via atomic force microscopy Int Rev Cytol 265, 227–252.
Barrera, N. P., and Edwardson, J. M. (2008) The subunit arrangement and assembly of ionotropic receptors Trends Neurosci 31, 569–576.
Barrera, N. P., Henderson, R. M., and Edwardson, J. M. (2008) Determination of the architecture of ionotropic receptors using AFM imaging Pflugers Arch 456, 199–209.
Barrera, N. P., Betts, J., You, H., Henderson, R. M., Martin, I. L., Dunn, S. M., and Edwardson, J. M. (2008) Atomic force microscopy reveals the stoichiometry and subunit arrangement of the alpha4beta3delta GABA(A) receptor Mol Pharmacol 73, 960–967.
Barrera, N. P., Henderson, R. M., Murrell-Lagnado, R. D., and Edwardson, J. M. (2007) The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels Biophys J 93, 505–512.
Barrera, N. P., Herbert, P., Henderson, R. M., Martin, I. L., and Edwardson, J. M. (2005) Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors Proc Natl Acad Sci USA 102, 12595–12600.
Barrera, N. P., Ormond, S. J., Henderson, R. M., Murrell-Lagnado, R. D., and Edwardson, J. M. (2005) Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize J Biol Chem 280, 10759–10765.
Barrera, N. P., Shaifta, Y., McFadzean, I., Ward, J. P., Henderson, R. M., and Edwardson, J. M. (2007) AFM imaging reveals the tetrameric structure of the TRPC1 channel Biochem Biophys Res Commun 358, 1086–1090.
Carnally, S. M., Dev, H. S., Stewart, A. P., Barrera, N. P., Van Bemmelen, M. X., Schild, L., Henderson, R. M., and Edwardson, J. M. (2008) Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging Biochem Biophys Res Commun 372, 752–755.
Ormond, S. J., Barrera, N. P., Qureshi, O. S., Henderson, R. M., Edwardson, J. M., and Murrell-Lagnado, R. D. (2006) An uncharged region within the N terminus of the P2X6 receptor inhibits its assembly and exit from the endoplasmic reticulum Mol Pharmacol 69, 1692–1700.
Schneider, S. W., Lärmer, J., Henderson, R. M., and Oberleithner, H. (1998) Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy Pflugers Arch 435, 362–367.
Barrera, N. P., Ge, H., Henderson, R. M., Fitzgerald, W. J., and Edwardson, J. M. (2008) Automated analysis of the architecture of receptors, imaged by atomic force microscopy Micron 39, 101–110.
Fotiadis, D., and Engel, A. (2004) High-resolution imaging of bacteriorhodopsin by atomic force microscopy Methods Mol Biol 242, 291–303.
Acknowledgments
We thank Dr. Robert Henderson (Department of Pharmacology, University of Cambridge) and Drs. Haifang Ge and William Fitzgerald (Department of Engineering, University of Cambridge) for their support in AFM imaging and image processing.
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
Carnally, S.M., Edwardson, J.M., Barrera, N.P. (2011). Imaging the Spatial Orientation of Subunits Within Membrane Receptors by Atomic Force Microscopy. In: Braga, P., Ricci, D. (eds) Atomic Force Microscopy in Biomedical Research. Methods in Molecular Biology, vol 736. Humana Press. https://doi.org/10.1007/978-1-61779-105-5_4
Download citation
DOI: https://doi.org/10.1007/978-1-61779-105-5_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-104-8
Online ISBN: 978-1-61779-105-5
eBook Packages: Springer Protocols