Protein Science Sheba protein
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online before print October 26, 2007, 10.1110/ps.073112007
Protein Science (2007), 16:2703-2710. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow All Versions of this Article:
ps.073112007v1
16/12/2703    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shu, X.
Right arrow Articles by Remington, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shu, X.
Right arrow Articles by Remington, S. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V

Xiaokun Shu1, Pavel Leiderman2, Rinat Gepshtein2, Nicholas R. Smith1, Karen Kallio1, Dan Huppert2, and S. James Remington1

1 Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403-1229, USA
2 Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel

(RECEIVED July 9, 2007; FINAL REVISION September 6, 2007; ACCEPTED September 6, 2007)

Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at ~395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place ~30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

Keywords: active sites; structure/function studies; crystallography; fluorescence; kinetics


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




HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2007 by The Protein Society.