Amit Sachdeva

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.

DNA-catalyzed sequence-specific hydrolysis of DNA

Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is intriguing because many natural DNA nucleases are bimetallic protein enzymes.

Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins

Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.

Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog.

Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

DNA-catalyzed serine side chain reactivity and selectivity

New deoxyribozymes are shown to catalyze reactions of serine side chains, forming nucleopeptide linkages and discriminating between serine and tyrosine or between two competing serines.

Temperature-dependent rotational relaxation in a viscous alkane: Interplay of shape factor and boundary condition on molecular rotation

Rotational relaxation of three organic solutes, coumarin 6 (C6), 2,5-dimethyl-1, 4-dioxo3,6-diphenylpyrrolo[3,4-c]pyrrole (DMDPP), and nile red (NR), that are similar in size but distinct in shape has been studied in a nonpolar solvent, squalane as a function of temperature to find out how the mechanical friction experienced by the solute molecule is influenced by its shape. It has been observed that C6 rotates slowest followed by NR and DMDPP. The results are analyzed using Stokes–Einstein–Debye (SED) hydrodynamic theory and also quasihydrodynamic theories of Gierer and Wirtz, and Dote, Kivelson, and Schwartz. Analysis of the data using the SED theory reveals that the measured reorientation times of C6 and DMDPP follow subslip behavior whereas those of NR are found to match slip predictions. While no single model could mimic the observed trend even in a qualitative manner, the reorientation times of C6 and DMDPP when normalized by their respective shape factors and boundary-condition parameters can be scaled on a common curve over the entire range of temperature studied. The probable reasons for the distinctive rotational behavior of NR as compared to C6 and DMDPP are explained in terms of its molecular shape and how this in turn influences the boundary-condition parameter are discussed.

Covalent tagging of phosphorylated peptides by phosphate-specific deoxyribozymes.

Many natural peptides and proteins are phosphorylated on tyrosine (Tyr) or serine (Ser) residues. Phosphorylated peptides are important within neurochemistry (neuropeptides), immunology (cytokines), and endocrinology (hormones). For such peptides as well as for larger proteins, side chain phosphorylation is frequently associated with modulation of biological function.[1] Methods for analysis of phosphopeptides often depend upon their initial chromatographic separation from nonphosphorylated analogues using support-bound chelators or covalent binders of phosphate groups or products derived from them.[2] Alternatively, phosphotyrosine-specific antibodies can be generated, albeit with the attendant investments in cost and time.[2g,3] Here we describe proof of principle for an entirely different approach to phosphopeptide analysis in which DNA catalysts (deoxyribozymes) covalently tag phosphorylated amino acid side chains of peptides. In this approach, it is critical to ensure high selectivity for modification of phosphorylated amino acid side chains over their nonphosphorylated analogues. Deoxyribozymes were originally identified for the catalysis of RNA cleavage,[7] and their use has expanded to encompass a range of chemical reactions.[8] Our lab has reported a variety of deoxyribozymes for different chemical reactions,[9] including the covalent modification of amino acid side chains.[5,10] In particular, we have recently shown that tripeptide substrates can be covalently modified by the attachment of an RNA strand at nonphosphorylated Tyr or Ser.[6] Here we sought to identify deoxyribozymes that covalently modify phosphorylated TyrP (YP), using in vitro selection as shown in Figure 1. The hexapeptide substrate AAAYPAA was connected to a DNA anchor oligonucleotide via either a short or long tether [see structures in Supporting Information; the short tether connects the hexapeptide directly via its α-amino group to the DNA anchor, whereas the long tether includes an intervening hexa(ethylene glycol) moiety]. In vitro selection was used to identify deoxyribozymes that attach a 5′-triphosphorylated RNA tag to TyrP, with pyrophosphate as the leaving group. Figure 1 Strategy for selective covalent modification of phosphorylated Tyr (TyrP, YP) within a peptide substrate. In vitro selection identifies deoxyribozymes that function with a TyrP-containing hexapeptide substrate, catalyzing attachment of 5′-triphosphorylated ... Two new deoxyribozymes from the selection process, 8VM1 and 8VP1 (one from each of the two selection experiments), were examined in more detail on the basis of their high catalytic activities with the TyrP- and analogous phosphoserine (SerP)-containing hexapeptides. Both deoxyribozymes were highly selective (>200:1) for each phosphorylated peptide over its nonphosphorylated analogue, with no detectable reaction at TyrOH or SerOH (<0.5%; Figure 2). 8VM1, which was identified by selection with the short tether, favored as its substrate the TyrP peptide over the SerP peptide by about 4- to 5-fold. In contrast, 8VP1, which was found via selection with the long tether, functioned equally well with TyrP or SerP peptides. 8VP1 was also found to accept a range of different amino acid identities—including hydrophobic and charged residues—flanking the TyrP that it covalently modifies (Figure 3), suggesting broad generality for different phosphopeptide sequences. Figure 2 The 8VM1 and 8VP1 deoxyribozymes covalently modify phosphotyrosine and phosphoserine. (a) PAGE image showing high selectivity for TyrP over TyrOH and for SerP over SerOH (50 mM HEPES, pH 7.5, 40 mM Mg2+, 20 mM Mn2+, 150 mM NaCl, 37 °C; single-turnover ... Figure 3 Sequence generality for the phosphopeptide substrate. Covalent modification by 8VP1 was examined with DNA-anchored hexapeptide substrate CAAYPAA and several illustrated sequence variants, for which one amino acid adjacent to YP (on either side) was changed ... To examine the applicability of deoxyribozymes for analysis of mixtures of phosphorylated and nonphosphorylated peptides, such a mixture (each peptide attached via a disulfide to the DNA anchor) was tagged with RNA by the 8VP1 deoxyribozyme. Analysis of the unpurified product mixture by MALDI mass spectrometry (after DTT cleavage of the DNA anchor) revealed selective RNA tagging of only the phosphopeptides, despite the presence of a large amount of nonphosphorylated peptide (Figure 4). Figure 4 Analysis of a peptide mixture by mass spectrometry using the DNA-catalyzed tagging approach. See diagram of this experiment in the Supporting Information. Each of a mixture of nonphosphorylated and phosphorylated peptides (100 pmol nonphosphorylated peptides; ... In summary, we have demonstrated proof of principle that DNA can catalyze highly selective covalent modification of phosphorylated Tyr or Ser residues in phosphopeptides by attaching an RNA tag at those positions. To our knowledge, this is the first report of any chemical approach for covalent, specific tagging of phosphopeptide side chains. In downstream applications, this RNA tag should be useful to report upon the amount of phosphorylated peptides present in a sample, e.g., by RT-PCR, which may help to avoid issues encountered during mass spectrometric analysis of peptide phosphorylation.[11] A wide range of peptide sequence contexts are accepted by the investigated deoxyribozymes, suggesting that this general approach may be made competitive with more traditional chromatographic separations of phosphopeptides.[2] The phosphopeptide analytical approach outlined here is distinct from methods that depend upon engineering of individual kinases to accept modified ATP substrates.[12] The present findings also expand the repertoire of DNA catalysis to include covalent modification of phosphorylated amino acid side chains. Independently, we have shown that RNA-tagging deoxyribozymes can discriminate against phosphorylated residues in favor of their nonphosphorylated analogues with promising selectivity (>20:1; data not shown). That observation along with the present work suggests the viability of ratiometric analyses in which both phosphorylated and nonphosphorylated peptides are covalently modified with different tags in the same sample. Several important issues must be addressed in future development of this approach. We will seek DNA catalysts that tag specific sequences of phosphopeptides, rather than accepting a broad range of peptide sequences. The approach also must be developed to work with free, rather than oligonucleotide-anchored, peptide substrates as well as with large phosphorylated proteins, ideally in complex mixtures such as cell lysates. Towards this goal, we have recently demonstrated the first steps towards DNA-catalyzed reactivity of free peptides;[6,13] such efforts must be merged with the present work to establish a useful analytical method.