This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.104.044412v1 88/3/1932    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jung, G. Articles by Zumbusch, A. Search for Related Content PubMed PubMed Citation Articles by Jung, G. Articles by Zumbusch, A.

The Photophysics of Green Fluorescent Protein: Influence of the Key Amino Acids at Positions 65, 203, and 222

Gregor Jung ^*, Jens Wiehler  and Andreas Zumbusch ^*

^* Department Chemie and Center for Nanoscience, LMU Munich, Munich, Germany; and Genzentrum der LMU Munich, Munich, Germany

Correspondence: Address reprint requests to Andreas Zumbusch, Dept. Chemie and Center for Nanoscience, LMU Munich, Butenandtstr. 11, D-81377 Munich, Germany. Tel.: 49-0-89-2180-77544; Fax: 49-0-89-2180-77545; E-mail: andreas.zumbusch{at}cup.uni-muenchen.de.

The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.

This article has been cited by other articles:

				J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs Dark States in Monomeric Red Fluorescent Proteins Studied by Fluorescence Correlation and Single Molecule Spectroscopy Biophys. J., May 15, 2008; 94(10): 4103 - 4113. [Abstract] [Full Text] [PDF]

				D. Arosio, G. Garau, F. Ricci, L. Marchetti, R. Bizzarri, R. Nifosi, and F. Beltram Spectroscopic and Structural Study of Proton and Halide Ion Cooperative Binding to GFP Biophys. J., July 1, 2007; 93(1): 232 - 244. [Abstract] [Full Text] [PDF]

				K. Nienhaus, F. Renzi, B. Vallone, J. Wiedenmann, and G. U. Nienhaus Chromophore-Protein Interactions in the Anthozoan Green Fluorescent Protein asFP499 Biophys. J., December 1, 2006; 91(11): 4210 - 4220. [Abstract] [Full Text] [PDF]

				R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram Development of a Novel GFP-based Ratiometric Excitation and Emission pH Indicator for Intracellular Studies Biophys. J., May 1, 2006; 90(9): 3300 - 3314. [Abstract] [Full Text] [PDF]

				V. V. Demidov, N. V. Dokholyan, C. Witte-Hoffmann, P. Chalasani, H.-W. Yiu, F. Ding, Y. Yu, C. R. Cantor, and N. E. Broude Fast complementation of split fluorescent protein triggered by DNA hybridization PNAS, February 14, 2006; 103(7): 2052 - 2056. [Abstract] [Full Text] [PDF]

				J. A. Sniegowski, J. W. Lappe, H. N. Patel, H. A. Huffman, and R. M. Wachter Base Catalysis of Chromophore Formation in Arg96 and Glu222 Variants of Green Fluorescent Protein J. Biol. Chem., July 15, 2005; 280(28): 26248 - 26255. [Abstract] [Full Text] [PDF]