Crystal Structure and Raman Studies of dsFP483, a Cyan Fluorescent Protein from Discosoma striata

Abstract

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins, we have determined the 2.1 Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan color class of Anthozoa fluorescent proteins, and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437-400 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the proteinintrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stablility. The refined crystal structure of the biological tetramer (spacegroup C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical to that in green fluorescent protein (avGFP). To test the roles of specific residues in color modulation, the optical properties of the H163Q and K70M variants were investigated. Although absorbance bands remain broad, peak excitation maxima are red-shifted to 455 and 460 nm, emitting cyan and green light respectively. To probe chromophore ground state features, Raman spectra were collected using 752 nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral avGFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan color is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.

Structural Basis of Enhanced Photoconversion Yield in Green Fluorescent Protein-like Protein Dendra2

“…The reduced extent of charge delocalization upon excitation should result in blue-shifted absorption and emission spectra of amFP486. This explanation may be challenged by recent results from another cyan FP, dsFP483 (78), which has strongly blue-shifted spectra but a (neutral) threonine instead of a histidine residue at the corresponding position.…”

One-Photon and Two-Photon Excitation of Fluorescent Proteins

“…Understanding the origin of the blueshifted spectral properties of this group has proven rather challenging. The broad absorption/excitation band hints at structural heterogeneity of either the chromophore or the surroundings [86]. By contrast, the narrower emission spectrum signals a unique emitting state.…”

“…It is present in Dronpa, a green-emitting FP, and it is missing in cyan dsFP483, in which His at av203 is replaced by a Thr. However, in dsFP483, a different charged residue, a lysine (K70), is in close contact with the chromophore-bridging carbon and points toward the phenolate [86], suggesting that such proximity of a charged residue is indeed necessary.…”

“…7 Relevant residues and water molecules in the chromophore environment of three cyan FPs dsFP483, amFP486, mTFP0.7 and the green FP Dronpa. The coordinates were taken from the Protein Data Bank, with codes 3CGL [86], 2A46 [87], 2OTB [88], and 2IOV [99]. The Arginine residue at position av96 is omitted for clarity.…”

Raman scattering investigation of selenomethionine replacement in protein SOUL crystals

“…[12] Many other studies have shown that the combined spectroscopic/crystallographic approach is a powerful new tool to investigate these systems. [13] In this work, we use Raman microscopy to investigate the vibrational properties of crystals obtained from wild-type and from SeMet-labelled protein SOUL. We have identified several methionine (Met) and SeMet Raman peaks as useful markers in crystal spectra and managed to unambiguously assign them by comparing the experimental Raman spectra of pure amino acids, recorded in solid-state phase and in aqueous solution with the Raman intensities computed using quantum chemical calculations.…”