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Andrikopoulos, P. C.

Publications and source records attributed to Andrikopoulos, P. C..

2 recordsLinked to original sources

Extensive Benchmark Study of the Resonance Raman Spectra of Lumiflavin

An extensive computational TDDFT resonance Raman study is presented here including forty-two different DFT functionals. The functionals were checked against the experimental FSRS Evolution Associated Spectrum of the equilibrated S1 state of FMN published earlier. Off-resonance spectra were computed first and yielded adequate agreement with the experimental spectrum. Fine-tuning of the correlation was achieved with the inclusion of specific scaling factors for each DFT functional, aimed to align the highest computed peak (symmetric C=O stretch) to the corresponding experimental peak in the fingerprint region. Subsequently, resonance Raman intensities were calculated with a careful choice of the resonant states. The experimental Evolution Associated Spectrum utilized for the comparisons was taken under resonance conditions, hence the inclusion of resonance enhancements in the calculations improved the agreement in most of the DFT functionals. For six particular DFT functionals, namely HCTH/407, OLYP, OPBE, O3LYP, tHCTHhyb and TPSSh, the theoretical/experimental correlation was particularly facile, and their agreement was predicted superior to the other functionals. The narrowing down to the above set was achieved by the evaluation of all DFT functionals according to five criteria ranging from the percent error of the main flavin excitations to the experimental values, to visual inspection of the spectra, and the determination of whether the inclusion of resonance in the calculation improved the agreement with experiment. Owing to the extent of the data set, valuable insights were gained to assist similar studies.

biophysics↗

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of -helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A extension of the LOV domain in controlling EL222 photocycle length. SignificanceThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C{equiv}N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.

biophysics↗