We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1  cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35-Wat-Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.

Department of Anatomy and Structural Center, Albert Einstein College of Medicine vladislav.verkhusha@einstein.yu.edu Macromolecular Crystallography Laborat Biomedical Research Inc., Basic Research Pr Shemyakin-Ovchinnikov Institute of Bioo Sciences, Moscow 117997, Russian Federati Department of Molecular and Cellular Bi California 95616, USA Department of Biochemistry and Develo University of Helsinki, Helsinki 00029, Finla † Electronic supplementary information ( crystallographic data collection and additional 6 gures and 2 tables. See DOI ‡ These authors contributed equally. Cite this: Chem. Sci., 2017, 8, 4546

The crystal structure of the dimeric green fluorescent protein EGFP-K162Q with C-terminal deletion MDELYK (EGFPv) has been determined in space group P6 at resolution 1.34 A. The obtained structure has been compared with that of the monomeric form of EGFP (green biomarker with enhanced photophysical properties) determined in other crystal space group P2(1)2(1)2(1) at resolution 1.50 and 1.35 A [1, 2]. Two subunits in the EGFPv structure are packed at 75 degrees with the contact surface approximately 800 A2. The dimeric structure is stabilized by six hydrogen bonds and the central hydrophobic core built of six residues. The RMSD value for Calpha atoms of 3-230 residues in the superimposed P61 and P2(1)2(1)2(1) structures is 0.55 A. The distinguishing feature of EGFPv- P6(1) structure, compared with that of EGFP-P2(1)2(1)2(1), is the noticeable difference in orientation of the Glu222 side chain and also new conformation of the loop fragment 155-159 with deviations among the Calpha atoms of superimposed structures reaching for Lys156 - 4.6 A and Lys158 - 5.5 A

The 13,000 member short-chain oxidoreductase (SCOR) family of enzymes includes at least 300 biochemically characterized enzymes in prokaryotes and eukaryotes. They catalyze oxidation, reduction, epimerization and synthase reactions. Over 70% (∼7,900) of the putative family members belong to a subfamily that contains the signature sequence TGxxxGIG in the (2(3 turn of the Rossmann fold. The crystal structures of 50 unique SCORs, have now been reported. Although there is not one residue fully conserved, 40 fingerprint residues are conserved at 70% identity or greater. We are determining the precise roles of each of the fingerprint residues in controlling protein folding, cofactor binding, catalysis, and function. Cofactor selectivity is controlled by two adjacent residues in the β2α2 turn of the Rossmann fold, seven residues may be critical to catalysis, C-H..O hydrogen bonds may play a significant role in catalysis (Fig. 1a) and a 310 kink and patterns of aromatic amino acid substitution on helix 5 may control dimer formation. The φ,ψ values of seven of the 11 Gly residues in the fingerprint fall in a region of the Ramachandran plot where the other 19 amino acids are rarely observed. Gly residues in these positions are indispensable for the maintenance of the Rossmann fold. The substrates for the family include steroids, sugars, prostaglandins, alcohols, acids, aromatics, dyes, and xenobiotics and none of the 40 fingerprint residues are substrate specific. Substrate binding specificity is determined by amino acids in three flexible loops. In crystal complexes, substrates and inhibitors make hydrogen or van der Waals contacts with amino acids in sequence specific locations on the three loops. A co-conserved set of amino acids (T,D,N,G,Q,F,M,T, and L) in nine positions on the three loops are found in 177 members of the SCOR family. Many of these 177 proteins have been biochemically characterized as β-keto [acyl carrier protein] reductases (Fig. 1b). Different combinations of amino acids in the same nine sequence positions on the three flexible loops identified 11 subgroups of SCORs that have different substrates. Supported by NIH Grant No. DK26546.

The three-dimensional structure of the red fluorescent protein (RFP) zRFP574 from the button polyp Zoanthus sp. (two dimers per asymmetric unit, 231 x 4 amino acids) has been determined at 2.4 A resolution in space group C222(1). The crystal structure, refined to a crystallographic R factor of 0.203 (R(free) = 0.249), adopts the beta-barrel fold composed of 11 strands similar to that of the yellow fluorescent protein zYFP538. The zRFP574 chromophore, originating from the protein sequence Asp66-Tyr67-Gly68, has a two-ring structure typical of GFP-like proteins. The bond geometry of residue 66 shows the strong tendency of the corresponding C(alpha) atom to sp(2) hybridization as a consequence of N-acylimine bond formation. The zRFP574 chromophore contains the 65-66 cis-peptide bond characteristic of red fluorescent proteins. The chromophore phenolic ring adopts a cis conformation coplanar with the imidazolinone ring. The crystallographic study has revealed an unexpected chemical feature of the internal chromophore. A decarboxylated side chain of the chromophore-forming residue Asp66 has been observed in the structure. This additional post-translational modification is likely to play a key role in the bathochromic shift of the zRFP574 spectrum.