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A Guide to Tracking Single Membrane Proteins and Their Interactions in Supported Lipid Bilayers

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Lipid-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2003))

Abstract

The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single proteins in planar supported biomimetic membranes. This chapter describes, in detail, the construction of a simple single molecule microscope, which includes: (1) a parts list, (2) temperature control, (3) an alignment procedure, (4) a calibration procedure, and (5) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported bilayers on hydrophilically treated borosilicate and quartz. These include (1) POPC bilayers, (2) POPC/PEG-PE cushioned bilayers, (3) POPC/PEG-PE cushioned bilayers on BSA passivated substrates, and (4) a cushioned biomimetic membrane of the endoplasmic reticulum (ER). A procedure for the detergent mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. A procedure for the detergent-free incorporation of cytochrome P450 reductase (CPR) and cytochrome P450 enzymes (P450) into an ER biomimetic is also described. The final experimental section of this chapter details different procedures for data analysis including (1) quantitative analysis of mean squared displacements from individually tracked proteins, (2) gamma distribution analysis of diffusion coefficients from a small ensemble of individually tracked proteins, (3) average mean squared displacement analysis, (4) Gaussian analysis of step-size distributions, (5) Arrhenius analysis of temperature dependent data, (6) the determination of equilibrium constants from a step-size distribution, and (7) a perspective associated with the interpretation of single particle tracking data.

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Notes

  1. 1.

    Needed if no optical bench is available.

  2. 2.

    All ½″ broadband dielectric mirror (BB05-E02, Thorlabs) are mounted in a kinematic mirror mount (KM05T, Thorlabs) at 7½″ above the base of the microscope using ½″ diameter by 4″ length optical posts (TR4, Thorlabs) in the appropriate post holder(UPH4, Thorlabs) and sit on a translation stage (PT1, Thorlabs).

  3. 3.

    The final power of the laser beam should be adjusted so get the best compromise between signal-to-noise ratio and the longevity of the fluorescence probe. 1 mW is a good place to start but should be optimized for each series of experiments.

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Author information

Authors and Affiliations

  1. Department of Chemistry, Washington State University, Pullman, WA, USA

    Evan L. Taylor & James A. Brozik

  2. Analytical Development, Juno Therapeutics, Seattle, WA, USA

    Kumud Raj Poudel

Authors
  1. Evan L. Taylor
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  2. Kumud Raj Poudel
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  3. James A. Brozik
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Corresponding author

Correspondence to James A. Brozik .

Editor information

Editors and Affiliations

  1. Department of Biophysics, Institute of Biology, FB10 and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Kassel, Germany

    Jörg H. Kleinschmidt

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Taylor, E.L., Poudel, K.R., Brozik, J.A. (2019). A Guide to Tracking Single Membrane Proteins and Their Interactions in Supported Lipid Bilayers. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 2003. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9512-7_17

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  • DOI: https://doi.org/10.1007/978-1-4939-9512-7_17

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  • Publisher Name: Humana, New York, NY

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  • Online ISBN: 978-1-4939-9512-7

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