Understanding blue-to-red conversion in monomeric fluorescent timers and hydrolytic degradation of their chromophores

Abstract

Fast-FT is a fluorescent timer (FT) engineered from DsRed-like fluorescent protein mCherry. Crystal structures of Fast-FT (chromophore Met66-Tyr67-Gly68) and its precursor with blocked blue-to-red conversion Blue102 (chromophore Leu66-Tyr67-Gly68) have been determined at the resolution of 1.15 A and 1.81 A, respectively. Structural data suggest that blue-to-red conversion, taking place in Fast-FT and in related FTs, is associated with the oxidation of Calpha2-Cbeta2 bond of Tyr67. Site directed mutagenesis revealed a crucial role of Arg70 and Tyr83 in the delayed oxidation of Calpha2-Cbeta2 bond, introducing the timing factor in maturation of the timer. Substitutions Ser217Ala and Ser217Cys in Fast-FT substantially slow down formation of an intermediate blue chromophore but do not affect much blue-to-red conversion, whereas mutations Arg70Lys or Trp83Leu, having little effect on the blue chromophore formation rate, markedly accelerates formation of the red chromophore. The chromophore of FTs adopts a cis-conformation stabilized by a hydrogen bond between its phenolate oxygen and the side chain hydroxyl of Ser146. In Blue102, a bulky side chain of Ile146 precludes the chromophore from adopting a "cis-like" conformation, blocking its blue-to-red conversion. Both Fast-FT and Blue102 structures revealed hydrolytic degradation of the chromophores. In Fast-FT, chromophore-forming Met66 residue is eliminated from the polypeptide chain, whereas Leu66 in Blue102 is cleaved out from the chromophore, decarboxylated and remains attached to the preceding Phe65. Hydrolysis of the chromophore competes with chromophore maturation and is driven by the same residues that participate in chromophore maturation.

Figure 1

Chemical structure of Fast-FT chromophore.

Figure 2

Amino acid sequence alignment of mCherry, Fast-FT, Blue124, Blue102, and Aequorea victoria GFP (in gray). Differences between the residues are indicated in red. Chromophore-forming residues are underlined. Catalytic Arg95 and Glu215 are highlighted in blue. Numbering above (in black) and below (in gray) correspond to mCherry and to avGFP, respectively.

Figure 3

Stereo representation of the residues that differ between (a) Fast-FT and mCherry and (b) Blue102 and mCherry. Fast-FT and Blue102 chromophores are shown in pink and blue, respectively; residues of Fast-FT and Blue102 that differ from those in mCherry are shown in green.

Figure 4

Stereo representation of the chromophore environment in Fast-FT and Blue102. (a) A view of the Fast-FT chromophore from the side of its phenolic ring. A degraded chromophore moiety and the preceding Phe65 (occupancies 0.77) are shown in green. An intact red-emitting chromophore and an alternative conformation of Phe65 (occupancies 0.23) are shown in pink. Hydrogen bonds and important close contacts are shown in red and blue dashed lines, respectively. (b) A similar view of the Blue102 chromophore. A degraded non-fluorescent chromophore moiety, decarboxylated Leu66, Phe65, and the important residues of the chromophore environment are shown in green. Hydrogen bonds and important close contacts are shown in red and blue dashed lines, respectively.

Figure 5

(a) Stereo representation of the intact and degraded Fast-FT chromophore moieties in their electron density. The density was calculated in the presence of degraded chromophore moiety and the preceding Phe65 in major conformation, and in the absence of intact chromophore and the preceding Phe65 in minor conformation. Blue-- 2Fo-Fc electron density contoured at 2.0 σ level corresponding to the degraded chromophore and Phe65 in the major conformation (model shown in green). Green-- difference Fo-Fc electron density contoured at 2.0 σ level corresponding to the intact chromophore and Phe65 in the minor conformation (model shown in pink). (b) Stereo representation of a hydrolyzed Blue102 chromophore moiety, decarboxylated Leu66, and preceding Phe65 in a 2Fo-Fc electron density contoured at 1.5 σ level.

Figure 6

Absorbance spectra of the mutants recorded at maturation points corresponding to the maximum intensities of their blue forms (blue curves) and red forms (red curves), and normalized to the intensity of a 280 nm band.

Scheme 1

Posttranslational events taking place in fluorescent timers and their precursors. Intact polypeptide (A) undergoes backbone cyclization followed by dehydration of its 5-membered ring producing a keto intermediate (B). Deprotonation of compound (B) leads to a non-fluorescent enolate form of chromophore precursor (C). Oxidation of N1-Cα1 bond to acylimine produces a blue-emitting chromophore moiety in which an active π-electron system is delocalized over imidazolone and acylimine groups of the chromophore and carbonyl of Phe65 (D). (Compound D is the final product in Blue102 and Blue124 FPs.) Subsequent oxidation of Cα2-Cβ2 bond occurring via unknown intermediate engages phenolic ring in conjugation resulting in red chromophore (E). Alternatively, a chromophore precursor may undergo hydrolytic degradation which starts from the cleavage of cyclized keto (B) or enolate (C) intermediates to give products (F) and (G), respectively. Compound G is the final product of the hydrolysis pathway in Blue102 and Blue124, and is observed in Blue102 structure. Oxidation of Cα2-Cβ2 bond results in compound (H) that is the final product of hydrolytic degradation in Fast-FT. The first chromophore-forming residue (Met66 in Fast-FT and Leu66 in Blue102) (I) that, after hydrolysis, becomes cleaved out from the chromophore, undergoes further chemical transformations. In Blue102, Leu66 undergoes decarboxylation but remains attached to Phe65 (K), whereas in Fast-FT, Met66 is completely removed from the protein. Two scenarios are possible, Met66 with either first undergoing decarboxylation (K) and than hydrolysis (L), or getting cleaved directly, skipping the decarboxylation step (J).