A biosynthetic route to photoclick chemistry on proteins

Abstract

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.

Figure 1

Coomassie blue stained SDS-PAGE gel showing expression of the TAG4 mutant myoglobin in the presence and absence of 1 mM p-Tpa (1).

Figure 2

ESI-TOF mass spectrometry analysis of p-Tpa-encoded mutant myoglobin. The charge ladder (top) and the deconvoluted intact mass (bottom) were shown. Calculated intact mass = 18482 Da, found 18481.0 Da. The small peak with the intact mass of 18349.0 corresponds to the p-Tpa-encoded mutant myoglobin with the first Met deleted.

Figure 3

Time courses of the pyrazoline cycloadduct formation for the photoinduced cycloaddition reactions between 2-tolyltetrazole and dimethyl fumarate at three different concentrations: ■ = 1 mM 2-tolyltetrazole and 20 mM dimethyl fumarate; = 200 μM 2-tolyltetrazole and 20 mM dimethyl fumarate; green triangle; = 200 μM 2-tolyltetrazole and 4 mM dimethyl fumarate.

Figure 4

Selective fluorescent labeling of the p-Tpa-encoded myoglobin via photoclick chemistry: (a) Reaction scheme showing that a mixture of p-Tpa-encoded mutant myoglobin (or wild-type, 27 μM) and FITC-fumarate 5 (200 μM) in PBS buffer supplemented with 3 mM TCEP was exposed to 5-min photoirradiation of a handheld 254-nm UV lamp followed by additional 1.5-hr incubation at room temperature. (b) Coomassie blue stain (left) and inverted fluorescence image (right) after resolving the reaction mixtures on SDS-PAGE gel. (c) Fluorescent labeling of p-Tpa-encoded mutant myoglobin (MT) with FITC-fumarate 5 requires photoactivation: A solution of p-Tpa-encoded mutant myoglobin (27 μM) and FITC-fumarate 5 (200 μM) in PBS buffer supplemented with 3 mM TCEP was either simply incubated or subjected to photoirradiation by a handheld 254-nm UV lamp for 5 min followed by 1.5-hr incubation at room temperature. The inverted fluorescence image (right) were shown along with the Coomassie blue stain (left).

Figure 5

Crystal structure of p-TpaRS-1 bound to p-Tpa: (a) A ribbon model showing the overall fold and the bound p-Tpa ligand in CPK model; (b) A close-up view of the p-Tpa binding pocket (rotated 180° along horizontal axis relative to a) with the six mutated residues rendered in tube models. The complex structure has been deposited into the Protein Data Bank with access code of 3N2Y.

Scheme 1

Synthesis of p-(2-tetrazole)phenylalanine (1)^{a a}Conditions: a) NBS, BPO, CCl₄, reflux, 61%; b) LiHMDS/THF, −78°C; c) 3, THF, −78°C to r.t.; d) 2 N HCl, MeOH; e) (Boc)₂O, TEA, THF; 69% for steps b–e; f) NaOH, EtOH/H₂O; g) TFA/DCM; 95% for steps f–g.