Ryan A. Mehl

Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific In Vivo Bioorthogonal Ligation with trans-Cyclooctenes

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Protein 19F NMR in Escherichia coli

Although overexpression and (15)N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, (15)N enrichment alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using (19)F-labeled proteins. Resonances from small (approximately 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the (19)F resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using (19)F NMR.

Photo-cross-linking interacting proteins with a genetically encoded benzophenone

A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions1–4. Solid-phase peptide synthesis has allowed the photo-crosslinkable amino acid p-benzoyl-L-phenylalanine (pBpa; Fig. 1a) to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes5,6. This method, however, is limited to the in vitro study of peptides and small proteins. An innovative development allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro cross-linking of proteins7–9. The method relies on an orthogonal aminoacyl tRNA synthetase–tRNACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli7 (Fig. 1b). The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid containing an amber mutant of the gene of interest. To produce the photocross-linker–containing protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, respectively, are exposed to UV light (Fig. 2).

Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer. A series of quadruplet decoding tRNAs has been developed to form an optimized orthogonal translation system. These tRNAs enable efficient, site-specific incorporation of multiple unnatural amino acids into a protein, with a substantial increase in yield over previous methods. The amino acids are then used to site-specifically label a protein with a pair of fluorophores, enabling studies of the proteins dynamics.

Oxidative coupling of peptides to a virus capsid containing unnatural amino acids.

This Communication describes the chemo- and site-selective coupling of cell type-specific targeting peptides to a virus capsid containing aminophenylalanine residues.

Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan

The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.

The biosynthesis of nicotinamide adenine dinucleotides in bacteria.

The nicotinamide adenine dinucleotides (NAD, NADH, NADP, and NADPH) are essential cofactors in all living systems and function as hydride acceptors (NAD, NADP) and hydride donors (NADH, NADPH) in biochemical redox reactions. The six-step bacterial biosynthetic pathway begins with the oxidation of aspartate to iminosuccinic acid, which is then condensed with dihydroxyacetone phosphate to give quinolinic acid. Phosphoribosylation and decarboxylation of quinolinic acid gives nicotinic acid mononucleotide. Adenylation of this mononucleotide followed by amide formation completes the biosynthesis of NAD. An additional phosphorylation gives NADP. This review focuses on the mechanistic enzymology of this pathway in bacteria.

Genetically encoding protein oxidative damage.

Posttranslational modification of tyrosine residues in proteins, to produce 3-nitrotyrosine (3-NT), is associated with over 50 disease states including transplant rejection, lung infection, central nervous system and ocular inflammation shock, cancer, and neurological disorders (for example, Alzheimers disease, Parkinsons disease, and stroke). The levels of 3-NT increase in aging tissue, and levels of 3-NT in proteins are a predictor of disease risk. Here we report the evolution and characterization of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of 3-NT into proteins at genetically encoded sites. To demonstrate the utility of our approach for studying the effect on protein function of nitration on sites defined in vivo, we prepared manganese superoxide dismutase (MnSOD) that is homogeneously nitrated at a site known to be modified in disease-related inflammatory responses, and we measured the effect of this defined modification on protein function.

Nitration of Hsp90 induces cell death

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

Conformationally Strained trans-Cyclooctene with Improved Stability and Excellent Reactivity in Tetrazine Ligation.

Computation has guided the design of conformationally-strained dioxolane-fused trans-cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative of 3,6-dipyridyl-s-tetrazine reacts with d-TCO with a second order rate k2 366,000 (+/- 15,000) M-1s-1 at 25 °C in pure water. Furthermore, d-TCO derivatives can be prepared easily, are accessed through diastereoselective synthesis, and are typically crystalline bench-stable solids that are stable in aqueous solution, blood serum, or in the presence of thiols in buffered solution. GFP with a genetically encoded tetrazine-containing amino acid was site-specifically labelled in vivo by a d-TCO derivative. The fastest bioorthogonal reaction reported to date [k2 3,300,000 (+/- 40,000) M-1s-1 in H2O at 25 °C] is described herein with a cyclopropane-fused trans-cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest trans-cyclooctene reagents, and also display enhanced stability and aqueous solubility.