Several peptides and a protein with an inter- or intramolecular cation-π interaction between tryptophan (Trp) and an amine cation are shown to absorb and fluoresce in the visible region of the spectrum. Titration of indole with sodium hydroxide or ammonium hydroxide yields an increasing visible fluorescence as well. Visible absorption and multi-peaked fluorescence excitation spectra correlate with experimental absorption spectra and the vibrational modes of calculated absorption spectra for the neutral Trp radical. The radical character of the cation-indole interaction is predicted to stem from the electrostatic dislocation of indole highest occupied molecular orbital (HOMO) charge density towards the cation with a subsequent electronic transition from the HOMO-2 to the HOMO. Because this is a vertical transition, fluorescence is possible. Hydrogen bonding at the indole amine most likely stabilizes the radical-like state. These results provide new spectroscopic tools for the investigation of cation-π interactions in numerous biological systems, among them, proteins and their myriad ligands, and show that one, or at most, two, point mutations with natural amino acids are all that is required to impart visible fluorescence to proteins.
Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.
Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK1, pK2, and pKR) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the 1La transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes.
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