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Biophys. J. BioFAST: First Published September 15, 2006. doi:10.1529/biophysj.106.087411
© 2006 by the Biophysical Society.

A more recent version of this article appeared on December 1, 2006.
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PROTEINS

CHROMOPHORE-PROTEIN INTERACTIONS IN THE ANTHOZOAN GREEN FLUORESCENT PROTEIN asFP499

Karin Nienhaus 1, Fabiana Renzi 2, Beatrice Vallone 2, Joerg Wiedenmann 1 and Gerd Ulrich Nienhaus 1*

1 University of Ulm
2 University of Rome

* To whom correspondence should be addressed. E-mail: uli{at}uiuc.edu.

Submitted on April 18, 2006
Revised on June 1, 2006
Accepted on 30 August 2006


  Abstract
Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The X-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 Å, shows the typical {beta}-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln63-Tyr64-Gly65, is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The X-ray structure provides interesting clues as to how the spectroscopic properties are fine-tuned by the chromophore environment.

Key Words: X-ray structure analysis, absorption spectroscopy, excited state proton transfer, fluorescence spectroscopy, protonation




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Copyright © 2006 by the Biophysical Society.