Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy.

Abstract:

:Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Schenk A,Ivanchenko S,Röcker C,Wiedenmann J,Nienhaus GU

doi

10.1016/S0006-3495(04)74114-4

subject

Has Abstract

pub_date

2004-01-01 00:00:00

pages

384-94

issue

1 Pt 1

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(04)74114-4

journal_volume

86

pub_type

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