Photophysics and optical switching in green fluorescent protein mutants

Photophysics and optical switching in green fluorescent protein mutants

^†Center for the Study of Excited States of Molecules, Huygens and Gorlaeus Laboratories, University of Leiden, P.O. Box 9504, 2300 RA Leiden, the Netherlands; and ^‡Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, D-37077 Göttingen, Germany

Edited by Marye Anne Fox, North Carolina State University, Raleigh, NC, and approved January 3, 2000 (received for review August 26, 1999)

Abstract

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable “one-color” systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in “color” on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.

Footnotes

↵ § To whom reprint requests should be addressed. E-mail: silvia{at}molphys.leidenuniv.nl.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.050365997.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.050365997
Abbreviations:

GFP,

green fluorescent protein;

wt,

wild-type