Iben B. Nielsen

Experimental studies of the photophysics of gas-phase fluorescent protein chromophores

To better understand the photophysics of photoactive proteins, the absorption bands of several gas-phase biomolecules have been studied recently at the electrostatic heavy-ion storage ring ELISA by a photo-fragmentation technique. In the present paper we discuss the involved photophysics and photochemistry for protonated and deprotonated model chromophores of the Green Fluorescent Protein (GFP) and the Red Fluorescent Protein (RFP). We show specifically that the delayed dissociation after photoabsorption can be understood in terms of a thermally activated process of the Arrhenius type. The rate of dissociation as a function of time after laser excitation is modeled in a calculation which is based on calculated heat capacities of the chromophores. Absorption of only one photon is enough to dissociate the deprotonated GFP chromophore on a time scale of milliseconds whereas absorption of two to three photons occurs for other chromophore ions. The difference is attributed to different activation energies, pre-exponential factors and locations of the absorption bands. We obtain activation energies for the dissociation that are of the order of 1–3 eV. Collision-induced dissociation experiments were performed to help identifying the fragmentation channels. Loss of methyl is found to be the dominant fragmentation channel for the deprotonated GFP chromophore and is also likely to be important for the protonated GFP chromophore at high temperatures. Other channels are open for the RFP chromophores. For the deprotonated RFP chromophore there is evidence that dissociation occurs through a non-trivial dissociation with substantial rearrangement.

Gas-phase absorption properties of DsRed model chromophores

The absorption spectra of two compounds, RFP(1) and RFP(2), designed to model the chromophore of the Red Fluorescent Protein DsRed have been recorded in the gas phase with a heavy-ion storage ring technique. Both anions and cations were investigated. The electronic delocalization is greater in RFP(2) than in RFP(1) due to an additional CC double bond in conjugation with the π system, and the absorption bands of RFP(2) are red-shifted compared to those of RFP(1). Band maxima of the RFP(2) and RFP(1) anions are 549 nm and 521 nm, respectively, and of the cations 448 nm and 441 nm, respectively. These values are in good agreement with calculated HOMO–LUMO gaps at the B3LYP/6-311++G(2d,p)//PM3 level of theory: 559 nm and 496 nm for the RFP(2) and RFP(1) anions and 452 nm and 436 nm for the corresponding cations. The protein absorbs maximally at 558 nm and it is assumed that the chromophore is anionic. Hence, the electronic structure of the RFP(2) anion is close to that of the in vivo chromophore in its protein environment. A comparison is made between the two model chromophores and their well-known Green Fluorescent Protein homologue chromophore, as well as between different media (gas phase, solution phase). For the anionic gas-phase spectra, vibrational structures are clearly resolved for both compounds (hν0 = 382 ± 10 cm−1 for RFP(1) and 518 ± 10 cm−1 for RFP(2)) and are assigned to harmonic vibrational progressions due to collective motion of the entire chromophores. Based on calculations on model chromophores closer to the wild-type DsRed chromophore, we suggest that the protein environment forces the chromophore to adopt a planar geometry.

Gas-phase absorption properties of a green fluorescent protein-mutant chromophore: The W7 clone

The gas-phase absorption properties of a blue GFP-mutant chromophore have been investigated in an electrostatic heavy-ion storage ring combined with an electrospray ion source. From the production of neutral photofragments, the gas-phase absorption profiles of both protonated and deprotonated forms have been obtained and compared with their homologues in the liquid phase and in the protein. Maximum absorption for the anion is found around 456 nm in solution and in the gas phase. It matches one absorption maximum in the W7 protein, which suggests that this is due to an anionic form of the chromophore—similar to the case of the GFP. For the W7 chromophore cation, the gas-phase absorption band exhibits a doublet feature in the gas-phase with maxima at 454 and 477 nm. Solvation effects are more pronounced for the cation than for the anion and the observed shifts in the absorption maxima may be explained by charge delocalization.

Ground state proton transfer in phenol-(NH3)(n) (n ≤ 11) clusters studied by mid-IR spectroscopy in 3-10 μm range.

The infrared (IR) spectra of size-selected phenol-ammonia clusters, PhOH-(NH(3))(n) (n ≤ 11) in the 3-10 μm wavelength region were measured to investigate the critical number of solvent molecules necessary to promote the ground state proton transfer (GSPT) reaction. While the N-H stretching vibrations did not provide clear information, characteristic changes that are assigned to the GSPT reaction were observed in the skeletal vibrational region. The production of phenolate anion (PhO(-)), which is a product of the GSPT reaction, was established from the appearance of characteristic bands assignable to C-C stretching and C-H bending vibrations of PhO(-) and from the corresponding disappearance of C-O-H bending vibration of PhOH at n = 9. The mid-IR spectroscopy directly proves the structural change induced by the deprotonation from the O-H bond and thus establishes the GSPT reaction as complete at n = 9. No such absorptions were observed for n ≤ 5 in line with a previous report. For n = 6-8, both the proton transferred and the nontransferred signatures were observed in the spectra, showing coexistence of both species for the first time.

Novel retinylidene iminium salts for defining opsin shifts: synthesis and intrinsic chromophoric properties

Retinal Schiff bases serve as chromophores in many photoactive proteins that carry out functions such as signalling and light-induced ion translocation. The retinal Schiff base can be found as neutral or protonated, as all-trans, 11-cis or 13-cis isomers and can adopt different conformations in the protein binding pocket. Here we present the synthesis and characterisation of isomeric retinylidene iminium salts as mimics blocked towards isomerisation at the C11 position and conformationally restrained. The intrinsic chromophoric properties are elucidated by gas phase absorption studies. These studies reveal a small blue-shift in the S0-->S1 absorption for the 11-locked derivative as compared to the unlocked one. The gas phase absorption spectra of all the cationic mimics so far investigated show almost no absorption in the blue region. This observation stresses the importance of protein interactions for colour tuning, which allows the human eye to perceive blue light.