Balance between ultrafast parallel reactions in the Green Fluorescent Protein has a structural origin

¹ Imperial College London
² Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory
³ Northeastern University

^* To whom correspondence should be addressed. E-mail: j.vanthor{at}imperial.ac.uk.

Submitted on January 22, 2008
Revised on February 28, 2008
Accepted on 4 April 2008

	Abstract

The fluorescence photocycle of the Green Fluorescent Protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein sidechain conformation of Glutamate 222 and reactivity is established, particularly of the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin and we report on the vibrational properties of responsive aminoacids on an ultrafast time-scale. Blue excitation of GFP drives two parallel excited state deuteron transfer reactions (ESPT) with 10 ps and 75 ps time-constants to the buried carboxylic acid sidechain of Glutamate 222 via a hydrogen bonding network. Assignment of 1456 cm^-1 and 1441 cm^-1 modes to _sym and 1564 cm^-1 and 1570 cm^-1 features to _asym of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant, and consistent with photoselection measurements. In contrast to the wild type, measurements of E222D can be described with only one difference spectrum, with the _sym mode at 1435 cm^-1 and the _asym mode at 1567 cm^-1, also correlating a large _asym-sym with slow ESPT kinetics. DFT calculations and published model compound and theoretical studies relate differences in _asym-sym to the strength and number of hydrogen-bonding interactions that is detected via equilibrium geometry and COO^- stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall ESPT process, consistent with recent theoretical predictions. Photoselection measurements show agreement for localised C=O vibrations of chromophore, Q69 and E222 with DFT and ab initio calculations placed in the X-ray geometry and provide their vibrational response in the intermediates in the photocycle

Key Words: DFT, Excited State Proton Transfer, GFP, Green Fluorescent Protein, ultrafast infrared spectroscopy