SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
CHARACTERIZATION OF THE PHOTOCONVERSION REACTION OF THE FLUORESCENT PROTEIN KAEDE ON SINGLE MOLECULE LEVEL
Petra S. Dittrich 1, Stephan P. Schaefer 2 and Petra Schwille 2*
1 ISAS (Institute for Analytical Sciences), Dortmund
2 TU Dresden
* To whom correspondence should be addressed. E-mail: pschwil{at}gwdg.de.
Submitted on February 24, 2005
Revised on March 29, 2005
Accepted on 14 July 2005
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Abstract |
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Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein ("Kaede") was introduced (Ando et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:12651-12656), that exhibits an irreversible color-shift from green to red fluorescence after photoactivation with l=350-410 nm and thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single molecule spectroscopy. By fluorescence correlation spectroscopy (FCS), fast flickering dynamics of the chromophore group were revealed. While these dynamics on a sub-ms time scale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering time scale of the photoconverted red chromophore was constant over a large pH range but varying with intensity of the 488 nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on single molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (l=405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at l=488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 41.238.6 s-1 for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.
Key Words:
GFP, continuous flow, fluorescence correlation spectroscopy, microfluidics, photoactivation, photophysics
