Anti-Brownian ELectrokinetic (ABEL) trapping of single β 2- adrenergic receptors in the absence and presence of agonist

Samuel Bockenhauer, Alexandre Fürstenberg, Xiao Jie Yao, Brian K. Kobilka, W. E. Moerner

Research output: Chapter in Book/Report/Conference proceedingConference PaperOtherpeer-review

4 Citations (Scopus)

Abstract

The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics, as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist. A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting single-molecule distributions.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging V
Volume8228
DOIs
Publication statusPublished - 2012
Externally publishedYes
EventSingle Molecule Spectroscopy and Superresolution Imaging V - San Francisco, CA, United States of America
Duration: 21 Jan 201222 Jan 2012

Conference

ConferenceSingle Molecule Spectroscopy and Superresolution Imaging V
CountryUnited States of America
CitySan Francisco, CA
Period21/01/1222/01/12

Keywords

  • b2AR
  • environment-sensitive dyes
  • fluctuations
  • fluorescence
  • GPCR
  • nonparametric statistics
  • radiative rate
  • single-molecule
  • stretched exponential

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