A sensitive photoabsorption technique for studies of gas-phase biomolecules has been used at the ELISA electrostatic heavy-ion storage ring. We show that the anion form of the chromophore of the green fluorescent protein in vacuo has an absorption maximum at 479 nm, which coincides with one of the two absorption peaks of the protein. Its absorption characteristics are therefore ascribed to intrinsic chemical properties of the chromophore. Evidently, the special beta-can structure of the protein provides shielding of the chromophore from the surroundings without significantly changing the electronic structure of the chromophore through interactions with amino acid side chains.

The absorption spectrum of the all-trans retinal chromophore in the protonated Schiff-base form, that is, the biologically relevant form, has been measured in vacuo, and a maximum is found at 610 nm. The absorption of retinal proteins has hitherto been compared to that of protonated retinal in methanol, where the absorption maximum is at 440 nm. In contrast, the new gas-phase absorption data constitute a well-defined reference for spectral tuning in rhodopsins in an environment devoid of charges and dipoles. They replace the misleading comparison with absorption properties in solvents and lay the basis for reconsidering the molecular mechanisms of color tuning in the large family of retinal proteins. Indeed, our measurement directly shows that protein environments in rhodopsins are blue- rather than red shifting the absorption. The absorption of a retinal model chromophore with a neutral Schiff base is also studied. The data explain the significant blue shift that occurs when metharhodopsin I becomes deprotonated as well as the purple-to-blue transition of bacteriorhodopsin upon acidification.

Photoabsorption spectra of deprotonated trans p-coumaric acid and two of its methyl substituted derivatives have been studied in gas phase both experimentally and theoretically. We have focused on the spectroscopic effect of the location of the two possible deprotonation sites on the trans p-coumaric acid which originate to either a phenoxide or a carboxylate. Surprisingly, the three chromophores were found to have the same absorption maximum at 430 nm, in spite of having different deprotonation positions. However, the absorption of the chromophore in polar solution is substantially different for the distinct deprotonation locations. We also report on the time scales and pathways of relaxation after photoexcitation for the three photoactive yellow protein chromophore derivatives. As a result of these experiments, we could detect the phenoxide isomer within the deprotonated trans p-coumaric acid in gas phase; however, the occurrence of the carboxylate is uncertain. Several computational methods were used simultaneously to provide insights and assistance in the interpretation of our experimental results. The calculated excitation energies S(0)-S(1) are in good agreement with experiment for those systems having a negative charge on a phenoxide moiety. Although our augmented multiconfigurational quasidegenerate perturbation theory calculations agree with experiment in the description of the absorption spectrum of anions with a carboxylate functional group, there are some puzzling disagreements between experiment and some calculational methods in the description of these systems.

The absorption spectra of two photoactive yellow protein model chromophores have been measured in vacuum using an electrostatic ion storage ring. The absorption spectrum of the isolated chromophore is an important reference for deducing the influence of the protein environment on the electronic energy levels of the chromophore and separating the intrinsic properties of the chromophore from properties induced by the protein environment. In vacuum the deprotonated trans-thiophenyl-p-coumarate model chromophore has an absorption maximum at 460 nm, whereas the photoactive yellow protein absorbs maximally at 446 nm. The protein environment thus only slightly blue-shifts the absorption. In contrast, the absorption of the model chromophore in aqueous solution is significantly blue-shifted (lambda(max) = 395 nm). A deprotonated trans-p-coumaric acid has also been studied to elucidate the effect of thioester formation and phenol deprotonation. The sum of these two changes on the chromophore induces a red shift both in vacuum and in aqueous solution.

No abstract

No abstract

No abstract

No abstract