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Proceedings Paper

Anti-Brownian ELectrokinetic (ABEL) trapping of single β2-adrenergic receptors in the absence and presence of agonist
Author(s): Samuel Bockenhauer; Alexandre Fuerstenberg; Xiao Jie Yao; Brian K. Kobilka; W. E. Moerner
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Paper 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.

Paper Details

Date Published: 13 February 2012
PDF: 16 pages
Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 822805 (13 February 2012); doi: 10.1117/12.910018
Show Author Affiliations
Samuel Bockenhauer, Stanford Univ. (United States)
Alexandre Fuerstenberg, Stanford Univ. (United States)
Univ. of Geneva (Switzerland)
Xiao Jie Yao, Stanford Univ. Medical School (United States)
Brian K. Kobilka, Stanford Univ. Medical School (United States)
W. E. Moerner, Stanford Univ. (United States)


Published in SPIE Proceedings Vol. 8228:
Single Molecule Spectroscopy and Superresolution Imaging V
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling; Ingo Gregor, Editor(s)

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