Probing the Ground State Structure of the Green Fluorescent Protein Chromophore Using Raman Spectroscopy

Abstract

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

“…S1). Raman bands at 1,603 and 1,565 cm −1 are markers for the neutral chromophore (29). Upon photoexcitation, GEM-GECO1 without/with Ca 2+ displays key changes starting at time zero: the 1,247/1,250 cm −1 GS C-O stretching mode blueshifts to 1,265 cm −1 ; the 1,155/1,157 cm −1 mode redshifts to 1,138/1,147 cm −1 ; and the 1,174 cm −1 mode blueshifts to 1,180 cm −1 ( Table S1).…”

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

“…The off-resonance Raman instrument and analysis methods have been described previously. 22 The two instruments employed for ultrafast spectroscopy are based in the Ultrafast Spectroscopy Laboratory of the UK Central Laser Facility, and have been described in detail elsewhere. 19,24 The TRIR spectrometer measures IR difference spectra as a function of time after excitation with a sub-200 fs, 400 nm pump pulse.…”

Local complexity of amino acid interactions in a protein core

“…1a) (15). Tuning of the chromophore absorbance color depends on the net free energy interactions with the surrounding protein (22,23), and Raman spectroscopic studies show that mutations that alter the absorbance spectrum are directly related to perturbations of the ground state structure of the molecule (24). Thus the GFP absorbance spectrum provides a high-resolution assay for reporting the energetic value of perturbations at sites surrounding the chromophore, and the effects are likely to arise from the crystallographically observable structure.…”

“…The structures were solved to a resolution of at least 1.6 Å and show a . The energetic effect of each mutation is measured as change in chromophore absorbance maximum, a property that derives from changes to the ground state structure of GFP (24). Mutation of some sites has no significant energetic effect despite direct interaction with the chromophore (H148C), whereas the largest effect is seen for Q183, which only indirectly contacts the chromophore.…”