HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 39, 33652-33659, September 30, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/39/33652    most recent M505473200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Thor, J. J. Articles by Sage, J. T. Search for Related Content PubMed PubMed Citation Articles by van Thor, J. J. Articles by Sage, J. T. Social Bookmarking                      What's this?

Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein^*

Jasper J. van Thor, Georgi Y. Georgiev1, Michael Towrie¶, and J. Timothy Sage

From the Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom, The Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115, and the ^¶Central Laser Facility, Council for the Central Laboratory of the Research Councils, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, United Kingdom

Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO₂. Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 Å of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 Å resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm^-1 (+) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence.

Received for publication, May 18, 2005 , and in revised form, June 29, 2005.

The atomic coordinates and structure factors (codes 1W7S, 1W7SSF, 1W7T, 1W7TSF, 1W7U, and 1W7USF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work is supported by grants from The Royal Society (University Research Fellowship to J. J. v. T.) and by National Institutes of Health and National Science Foundation Grants GM-52002 and PHY-0240955 (to J. T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 44-(0)1865-285352; Fax:44-(0)1865-275182; E-mail: jasper{at}biop.ox.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J.-P. Colletier, D. Bourgeois, B. Sanson, D. Fournier, J. L. Sussman, I. Silman, and M. Weik Shoot-and-Trap: Use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography PNAS, August 19, 2008; 105(33): 11742 - 11747. [Abstract] [Full Text] [PDF]