Femtosecond-to-nanosecond dynamics of flavin mononucleotide monitored by stimulated Raman spectroscopy and simulations

Abstract

The ultrafast excited-state dynamics of flavin mononucleotide (FMN) was monitored upon light irradiation by a hybrid experimental/computational approach.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

“…S8-S10, ESI †). As is known from calculations on the chromophore itself, 35 and YtvA, 13 T 1 tends to be more stable by B10 kcal mol À1 than the equivalent S 1 state. In the protein cluster environment this also holds true, except for RsLOV where the stability of the triplets falls to 6 kcal mol À1 .…”

“…It was reported by our group, via Raman calculations in free FMN, that vibrations of the ribityl-phosphate moiety can be expected up to 1450 cm À1 , usually coupled to isoalloxazine modes. 35 However, the ribityl chain retains an unfolded conformation in the protein environment, unlike in solution. A possible interaction that might register in the spectra is between ribityl hydroxy groups and the side chain of Q454 and Q59 in AsLOV2 and RsLOV, respectively which is missing from the calculations.…”

“…27 To monitor the above light-triggered changes involved in LOV signal transduction, time-resolved vibrational spectroscopy, and in particular transient infrared spectroscopy, has proven to be an invaluable instrument in the researchers' toolbox. 7,[28][29][30][31][32][33][34] A challenge is posed by the need to separate spectral contributions originating from the chromophore and the protein environment, a task that can be approached by a combination of (i) analysis of the spectra of the isolated chromophore, 35,36 (ii) isotopic labelling of key residues such as the glutamine (achieved currently only for a BLUF domain), 37 and the chromophore, 33,38 (iii) mutation of key residues 32,39,40 or (iv) by inserting non-canonical amino acid probes in the protein sequence. 41 More recently, femtosecond-stimulated Raman experiments indicate that FMN modes can be selectively enhanced under appropriate resonance conditions.…”

“…41 More recently, femtosecond-stimulated Raman experiments indicate that FMN modes can be selectively enhanced under appropriate resonance conditions. 35,42,43 Computational efforts have been instrumental in the elucidation of large scale changes of LOV domains via molecular dynamics simulations. 6,7,[44][45][46][47][48][49] However, from a theoretical spectroscopy point of view, few vibrational studies have tried to tackle protein-chromophore interactions, [50][51][52][53] with most limiting their scope to the chromophore.…”

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

“…In the present experiment it was centered at 750 nm (4 µJ 100 µm spot size), a wavelength which was selected to be resonant with the excited state transient absorption of both singlet and triplet states of FMN; as described by Andrikopoulos et al there is a broad transient absorption at ca 800 nm for S1, which evolves into a more triplet-triplet absorption with a much larger transition dipole moment and a peak at 712 nm. 23 Pulses were overlapped and focused to the sample position and the stimulated Raman signal was collected in the phase matched directions and dispersed in a SPEX 500M spectrometer with CCD detector. Optical choppers were used to modulate the actinic and Raman pump pulses resulting in four sets of pulse sequences (a) Actinic Pump-Probe+Raman, (b) Raman+Probe, (c) Actinic Pump+Probe, and (d) probe only such that the excited state FSRS signals can be extracted from the transient absorption using:…”