Rotational diffusion of the α(2a) adrenergic receptor revealed by FlAsH labeling in living cells.

Abstract:

:The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α(2A) adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ∼50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557-573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113) and revealed a discrepancy of a factor between 10 and 100.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Spille JH,Zürn A,Hoffmann C,Lohse MJ,Harms GS

doi

10.1016/j.bpj.2010.08.080

subject

Has Abstract

pub_date

2011-02-16 00:00:00

pages

1139-48

issue

4

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(10)01192-6

journal_volume

100

pub_type

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