The single-molecule approach to membrane protein stoichiometry

Michael G. Nichols, Richard Hallworth

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages189-199
Number of pages11
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1427
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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