A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins

Abstract

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 E_c 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.

Keywords: chromophore; crystal structure; fluorescent protein; gene reporter; lancelet; mutagenesis; protein engineering.

FIGURE 1

Improving the solubility of lanFP10A. (a) Amino acid sequence of proteins generated during improvement of solubility of lanFP10A; the chromophore‐forming triad is highlighted in yellow. bfVFP was added to the sequence alignment. (b) Phenotype of E. coli cells expressing the proteins whose sequence is shown above. (c) SDS–PAGE of cells expressing the proteins shown above revealed by Western blot using anti‐His antibody coupled to alkaline phosphatase. M: protein marker, 1 and 2: lanFP10A, 3 and 4 lanFP10A2, 5 and 6: lanFP10A3, 7 and 8: lanFP10A2‐F70Y. Samples 1, 3, 5, and 7: soluble fractions; samples 2, 4, 6, and 8: total extracts

FIGURE 2

bfVFP compared with two other blue fluorescent proteins: Sirius and mTagBFP2. (a) Visualization of the pure proteins at 1 mg/ml under irradiation at different lights. (b) UV–Vis absorbance of the three BFPs. (c) Excitation and emission spectra of the three BFPs

FIGURE 3

Crystal structure of lanFP10A2 (PDB ID: 7FSA) showing the amino acid changes acquired during the recombination PCR process to improve the solubility of lanFP10A (PDB ID: 6M9X)

FIGURE 4

Structural analysis of bfVFP crystal. (a) Chemical structure of the chromophore found in bfVFP: oxidized tyrosine 68 in cis conformation. (b) Contrary to lanFP10A2 (shown in green), the chromophore‐forming triad in bfVFP (shown in purple) forms no H‐bonds with either Arg97 or Glu219. (c) The position of the bfVFP chromophore is stabilized by two H‐bonds between the p‐hydroxyphenyl ring and the NH group of His148 (2.8 Å) and the C═O of Thr150 (2.5 Å)

FIGURE 5

Absorbance spectra of lanFP10A2, bfVFP, and its mutants

SCHEME 1

Alternative pathways for maturation of the GYA tripeptide in bfVFP and its mutants