Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2.

Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the novel severe acute respiratory syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and angiotensin-converting enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 µM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 µM. The current study demonstrates that two kinetically controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7.

Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nε-(7-azidoheptanoyl)-l-lysine (AzHeK) by amber codon in Escherichia coli for recombinant expression of a number of AzHeK-containing histone H3 proteins. We assembled in vitro acyl-nucleosomes from these recombinant acyl-H3 histones. All these acyl-nucleosomes contained an azide functionality that allowed quick click labeling with a strained alkyne dye for in-gel fluorescence analysis. Using these acyl-nucleosomes as substrates and click labeling as a detection method, we systematically investigated chromatin deacylation activities of SIRT7, a class III NAD+-dependent histone deacylase with roles in aging and cancer biology. Besides confirming the previously reported histone H3K18 deacylation activity, our results revealed that SIRT7 has an astonishingly high activity to catalyze deacylation of H3K36 and is also catalytically active to deacylate H3K37. We further demonstrated that this H3K36 deacylation activity is nucleosome dependent and can be significantly enhanced when appending the acyl-nucleosome substrate with a short double-stranded DNA that mimics the bridging DNA between nucleosomes in native chromatin. By overexpressing SIRT7 in human cells, we verified that SIRT7 natively removes acetylation from histone H3K36. Moreover, SIRT7-deficient cells exhibited H3K36 hyperacetylation in whole cell extracts, at rDNA sequences in nucleoli, and at select SIRT7 target loci, demonstrating the physiologic importance of SIRT7 in determining endogenous H3K36 acetylation levels. H3K36 acetylation has been detected at active gene promoters, but little is understood about its regulation and functions. Our findings establish H3K36 as a physiologic substrate of SIRT7 and implicate this modification in potential SIRT7 pathways in heterochromatin silencing and genomic stability.

Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids.

By evolving the N-terminal domain of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNAPyl , a mutant clone displaying improved amber-suppression efficiency for the genetic incorporation of Nϵ -(tert-butoxycarbonyl)-l-lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ -acetyl-l-lysine and Nϵ -(4-azidobenzoxycarbonyl)-l-δ,ϵ-dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N-terminal domain of PylRS that was separated from its catalytic domain for charging tRNAPyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system-based noncanonical amino-acid mutagenesis.

A Versatile Approach for Site-Specific Lysine Acylation in Proteins.

Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

A Genetically Encoded Allysine for the Synthesis of Proteins with Site-Specific Lysine Dimethylation.

Using the amber suppression approach, Nϵ -(4-azidobenzoxycarbonyl)-δ,ϵ-dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site-specific lysine dimethylation. Using this approach, dimethyl-histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.

Phospha-Michael Addition as a New Click Reaction for Protein Functionalization.

A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site-specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha-Michael addition with an alkyl phosphine. Our kinetic study indicated a second-order rate constant of 0.07 m(-1) s(-1) for the reaction between tris(2-carboxyethyl)phosphine and acrylamide at pH 7.4. To demonstrate its application in protein functionalization, we used a dansyl-phosphine conjugate to successfully label proteins that were site-specifically installed with N(ɛ) -acryloyl-l-lysine and employed a biotin-phosphine conjugate to selectively probe human proteins that were metabolically labeled with N-acryloyl-galactosamine.

A click chemistry approach to site-specific immobilization of a small laccase enables efficient direct electron transfer in a biocathode.

Controlled orientation of a small laccase on a multi-walled carbon nanotube electrode was achieved via copper-free click chemistry mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer.

Genetically encoded unstrained olefins for live cell labeling with tetrazine dyes.

A number of non-canonical amino acids (NCAAs) with unstrained olefins are genetically encoded using mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pairs. These NCAAs readily undergo inverse electron-demand Diels-Alder cycloadditions with tetrazine dyes, leading to selective labeling of proteins bearing these NCAAs in live cells.

Two rapid catalyst-free click reactions for in vivo protein labeling of genetically encoded strained alkene/alkyne functionalities.

Detailed kinetic analyses of inverse electron-demand Diels­Alder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these "click" reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.

The genetic incorporation of thirteen novel non-canonical amino acids.

Thirteen novel non-canonical amino acids were synthesized and tested for suppression of an amber codon using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair. Suppression was observed with varied efficiencies. One non-canonical amino acid in particular contains an azide that can be applied for site-selective protein labeling.

E1-catalyzed ubiquitin C-terminal amidation for the facile synthesis of deubiquitinase substrates.

Will Ub my partner? The ubiquitin (Ub)-activating enzyme (E1) was used to catalyze an amidation reaction to functionalize the C terminus of Ub with unique functional groups, such as thiol, azide, alkyne, and alkene groups, with high efficiency and yield (>90 %). These groups were then applied for the facile synthesis of fluorophore-conjugated ubiquitin and specifically conjugated diubiquitin substrates for deubiquitinases.

Reversal of the hofmeister series: specific ion effects on peptides.

Ion-specific effects on salting-in and salting-out of proteins, protein denaturation, as well as enzymatic activity are typically rationalized in terms of the Hofmeister series. Here, we demonstrate by means of NMR spectroscopy and molecular dynamics simulations that the traditional explanation of the Hofmeister ordering of ions in terms of their bulk hydration properties is inadequate. Using triglycine as a model system, we show that the Hofmeister series for anions changes from a direct to a reversed series upon uncapping the N-terminus. Weakly hydrated anions, such as iodide and thiocyanate, interact with the peptide bond, while strongly hydrated anions like sulfate are repelled from it. In contrast, reversed order in interactions of anions is observed at the positively charged, uncapped N-terminus, and by analogy, this should also be the case at side chains of positively charged amino acids. These results demonstrate that the specific chemical and physical properties of peptides and proteins play a fundamental role in ion-specific effects. The present study thus provides a molecular rationalization of Hofmeister ordering for the anions. It also provides a route for tuning these interactions by titration or mutation of basic amino acid residues on the protein surface.

Effects of End Group Termination on Salting-Out Constants for Triglycine.

Salting out constants for triglycine were calculated for a series of Hofmeister salts using molecular dynamics simulations. Three variants of the peptide were considered with both termini capped, just the N-terminus capped, and without capping. The simulations were supported by NMR and FTIR measurements. The data provide strong evidence that earlier experimental values of salting out constants assigned to the fully capped peptide (as previously assumed) should have been assigned to the half-capped peptide instead. Therefore, these values cannot be used to directly establish Hofmeister ordering of ions at the peptide backbone, since they are strongly influenced by interactions of the ions with the negatively charged C-terminus.