Raman Study of Chromophore States in Photochromic Fluorescent Proteins

Abstract

The photophysical mechanism underlying the photochromic behavior of green fluorescent protein (GFP) mutants is investigated by means of preresonant Raman spectroscopy and model calculations. The studied molecules are reversibly switchable fluorophores that can be repeatedly converted between fluorescent and nonfluorescent states by irradiation and are attracting a broad interest for a number of new applications. Experimental results on chemically synthesized isolated chromophores are analyzed within a density functional theory approach that allows us to link the detected vibrational modes to specific ground-state configurations before and after photoconversion. These data are compared to results obtained for the case of complete folded proteins including the same chromophores. Our results highlight the impact of chromophore cis-trans isomerization and protonation state in the photophysics of these proteins and provide useful guidelines for novel mutant design.

Light-Activated Reassembly of Split Green Fluorescent Protein

“…The spectra were obtained at 170K in a frozen aqueous solution to avoid aggregation of cis GFP: at the high concentrations required to obtain Raman spectra (1 – 3mM), and 633 nm light, which is not absorbed by either form of truncated GFP, was used to obtain the Raman spectrum. EYQ1 is a GFP with the F64L, T203Y, and E222Q mutations that has been shown previously to undergo light-activated photoisomerization11. In EYQ1, the thermally stable isomerization state of the chromophore is the cis configuration, so with this truncated GFP the Raman spectrum of the light-activated form is expected to be similar to the Raman spectrum of EYQ1 prior to light activation.…”

“…Photoisomerization from the cis to the trans configuration was initially considered because there are many reversibly photoswitchable GFPs21,22 and cis / trans isomerization of the chromophore has been shown to be their underlying mechanism in mTFP0.7, asFP595-A143S, Padron, and Dronpa by crystallography18,23,24,25, and in both EYQ1 and eqFP611 by pre-resonance Raman spectroscopy11,26. The Raman spectra shown in Figure 7 are not identical to Raman spectra of cis or trans model GFP chromophores obtained previously, but some of the changes are very similar to the changes observed upon converting trans model chromophore to cis model chromophore11.…”

A diabatic three-state representation of photoisomerization in the green fluorescent protein chromophore

“…Bridge stretching modes are prominent in the resonance Raman spectrum of HBDI anion, 58,89 and resonance and preresonance Raman spectra of GFPs. 89,90 There is spectroscopic evidence of asymmetric vibrations in the spectroscopy of other fluorogenic monomethine dyes. 91 The resonance in Fig.…”

The Single T65S Mutation Generates Brighter Cyan Fluorescent Proteins with Increased Photostability and pH Insensitivity

“…The photoconversion quantum efficiencies in both directions are very high for the CFP chromophore (Φ cis  = 20%, Φ trans  = 90%), while its different diastereoisomers have strongly overlapping absorption bands [42]. Similar cis trans chromophore photoisomerizations were shown to be involved in the photoswitching properties of nearly all RSFPs [44]–[48]. The efficiency of ECFP photoreactions, and the fact that the photoconverted CFPs retain a strong absorption in the normal chromophore band (allowing a very efficient photoactivated return to the “on” state), show that these reactions principally reflect, as for other RSFPs, a chromophore photoisomerization process.…”