title: Structural parameters controlling the fluorescence properties of phytochromes.
abstract: Phytochromes constitute a class of photoreceptors that can be photoconverted between two stable states. The tetrapyrrole chromophore absorbs in the red spectral region and displays fluorescence maxima above 700 nm, albeit with low quantum yields. Because this wavelength region is particularly advantageous for fluorescence-based deep tissue imaging, there is a strong interest to engineer phytochrome variants with increased fluorescence yields. Such targeted design efforts would substantially benefit from a deeper understanding of those structural parameters that control the photophysical properties of the protein-bound chromophore. Here we have employed resonance Raman (RR) spectroscopy and molecular dynamics simulations for elucidating the chromophore structural changes in a fluorescence-optimized mutant (iRFP) derived from the PAS-GAF domain of the bacteriophytochrome RpBphP2 from Rhodopseudomas palustris . Both methods consistently reveal the structural consequences of the amino acid substitutions in the vicinity of the biliverdin chromophore that may account for lowering the propability of nonradiative excited state decays. First, compared to the wild-type protein, the tilt angle of the terminal ring D with respect to ring C is increased in iRFP, accompanied by the loss of hydrogen bond interactions of the ring D carbonyl function and the reduction of the number of water molecules in that part of the chromophore pocket. Second, the overall flexibility of the chromophore is significantly reduced, particularly in the region of rings D and A, thereby reducing the conformational heterogeneity of the methine bridge between rings A and B and the ring A carbonyl group, as concluded from the RR spectra of the wild-type proteins.
authors: Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors: Francisco
authors: F
authors: Velazquez Escobar
authors: Thomas
authors: T
authors: Hildebrandt
authors: Tillmann
authors: T
authors: Utesch
authors: Franz Josef
authors: FJ
authors: Schmitt
authors: Ina
authors: I
authors: Seuffert
authors: Norbert
authors: N
authors: Michael
authors: Claudia
authors: C
authors: Schulz
authors: Maria Andrea
authors: MA
authors: Mroginski
authors: Thomas
authors: T
authors: Friedrich
authors: Peter
authors: P
authors: Hildebrandt
mesh_terms: D019943:Amino Acid Substitution; D056004:Molecular Dynamics Simulation; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D012241:Rhodopseudomonas / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
chemical_list: D010834:Phytochrome