Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival.

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.

Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Ultrafast Biomimetic Oxidative Folding of Cysteine-rich Peptides and Microproteins in Organic Solvents.

Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous-buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio-inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand-fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine-rich microproteins instantaneously without observable misfolded or dead-end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200-fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine-rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.

Substrate-binding glycine residues are major determinants for hydrolase and ligase activity of plant legumains.

Peptide asparaginyl ligases (PALs) are useful tools for precision modifications of proteins and live-cell surfaces by ligating peptides after Asn/Asp (Asx). They share high sequence and structural similarity to plant legumains that are generally known as asparaginyl endopeptidases (AEPs), thus making it challenging to identify PALs from AEPs. In this study, we investigate 875 plant species from algae to seed plants with available sequence data in public databases to identify new PALs. We conducted evolutionary trace analysis on 1500 plant legumains, including eight known PALs, to identify key residues that could differentiate ligases and proteases, followed by recombinant expression and functional validation of 16 novel legumains. Previously, we showed that the substrate-binding sequences flanking the catalytic site can strongly influence the enzymatic direction of a legumain and which we named as ligase-activity determinants (LADs). Here, we show that two conserved substrate-binding Gly residues of LADs are critical, but negative determinants for ligase activity. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies, accounting for c. 1% and 88%, respectively.

Arabidopsis NPF2.13 functions as a critical transporter of bacterial natural compound tunicamycin in plant-microbe interaction.

Metabolites including antibiotics, enzymes, and volatiles produced by plant-associated bacteria are key factors in plant-microbiota interaction that regulates various plant biological processes. There should be crucial mediators responsible for their entry into host plants. However, less is known about the identities of these plant transporters. We report that the Arabidopsis Nitrate Transporter1 (NRT1)/NPF protein NPF2.13 functions in plant uptake of tunicamycin (TM), a natural antibiotic produced by several Streptomyces spp., which inhibits protein N-glycosylation. Loss of NPF2.13 function resulted in enhanced TM tolerance, whereas NPF2.13 overexpression led to TM hypersensitivity. Transport assays confirmed that NPF2.13 is a H+ /TM symporter and the transport is not affected by other substrates like nitrate. NPF2.13 exclusively showed TM transport activity among tested NPFs. Tunicamycin uptake from TM-producing Streptomyces upregulated the expression of nitrate-related genes including NPF2.13. Moreover, nitrate allocation to younger leaves was promoted by TM in host plants. Tunicamycin could also benefit plant defense against the pathogen. Notably, the TM effects were significantly repressed in npf2.13 mutant. Overall, this study identifies NPF2.13 protein as an important TM transporter in plant-microbe interaction and provides insights into multiple facets of NPF proteins in modulating plant nutrition and defense by transporting exterior bacterial metabolites.

Multi-omics analyses of serum metabolome, gut microbiome and brain function reveal dysregulated microbiota-gut-brain axis in bipolar depression.

The intricate processes of microbiota-gut-brain communication in modulating human cognition and emotion, especially in the context of mood disorders, have remained elusive. Here we performed faecal metagenomic, serum metabolomics and neuroimaging studies on a cohort of 109 unmedicated patients with depressed bipolar disorder (BD) patients and 40 healthy controls (HCs) to characterise the microbial-gut-brain axis in BD. Across over 12,000 measured metabolic features, we observed a large discrepancy (73.54%) in the serum metabolome between BD patients and HCs, spotting differentially abundant microbial-derived neuroactive metabolites including multiple B-vitamins, kynurenic acid, gamma-aminobutyric acid and short-chain fatty acids. These metabolites could be linked to the abundance of gut microbiota presented with corresponding biosynthetic potentials, including Akkermansia muciniphila, Citrobacter spp. (Citrobacter freundii and Citrobacter werkmanii), Phascolarctobacterium spp., Yersinia spp. (Yersinia frederiksenii and Yersinia aleksiciae), Enterobacter spp. (Enterobacter cloacae and Enterobacter kobei) and Flavobacterium spp. Based on functional neuroimaging, BD-related neuroactive microbes and metabolites were discovered as potential markers associated with BD-typical features of functional connectivity of brain networks, hinting at aberrant cognitive function, emotion regulation, and interoception. Our study combines gut microbiota and neuroactive metabolites with brain functional connectivity, thereby revealing potential signalling pathways from the microbiota to the gut and the brain, which may have a role in the pathophysiology of BD.

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme.

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.

Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates.

Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx-Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively. The hydrazinolysis reaction exhibits very high efficiency and can be completed in minutes at a low enzyme-to-substrate ratio. We further show the utility of the so-formed asparaginyl hydrazide in native chemical ligation and hydrazone conjugation. Using an EGFR-targeting affibody as a model protein, we have showcased our methodology in the preparation of a number of protein ligation or conjugation products, which are decorated with various functional moieties. The ZEGFR affibody-doxorubicin conjugate shows high selective binding and cytotoxicity toward the EGFR-positive A431 cells. Our results demonstrate the advantages of AEP-mediated protein hydrazinolysis as a simple and straightforward strategy for the precision manufacturing of protein bioconjugates.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.

Structural determinants for peptide-bond formation by asparaginyl ligases.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

pH-Controlled Protein Orthogonal Ligation Using Asparaginyl Peptide Ligases.

Peptide asparaginyl ligases (PALs) catalyze transpeptidation at the Asn residue of a short Asn-Xaa1-Xaa2 tripeptide motif. Due to their high catalytic activity toward the P1-Asn substrates at around neutral pH, PALs have been used extensively for peptide ligation at asparaginyl junctions. PALs also bind to aspartyl substrates, but only when the γCOOH of P1-Asp remains in its neutral, protonated form, which usually requires an acidic pH. However, this limits the availability of the amine nucleophile and, consequently, the ligation efficiency at aspartyl junctions. Because of this perceived inefficiency, the use of PALs for Asp-specific ligation remains largely unexplored. We found that PAL enzymes, such as VyPAL2, display appreciable catalytic activities toward P1-Asp substrates at pH 4-5, which are at least 2 orders of magnitude higher than that of sortase A, making them practically useful for both intra- and intermolecular ligations. This also allows sequential ligations, first at Asp and then at Asn junctions, because the newly formed aspartyl peptide bond is resistant to the ligase at the pH used for asparaginyl ligation in the second step. Using this pH-controlled orthogonal ligation method, we dually labeled truncated sfGFP with a cancer-targeting peptide and a doxorubicin derivative at the respective N- and C-terminal ends in the N-to-C direction. In addition, a fluorescein tag and doxorubicin derivative were tagged to an EGFR-targeting affibody in the C-to-N direction. This study shows that the pH-dependent catalytic activity of PAL enzymes can be exploited to prepare multifunction protein biologics for pharmacological applications.

The plant secondary compound swainsonine reshapes gut microbiota in plateau pikas (Ochotona curzoniae).

Plants produce various plant secondary compounds (PSCs) to deter the foraging of herbivorous mammals. However, little is known about whether PSCs can reshape gut microbiota and promote gut homeostasis of hosts. Using 16S rDNA sequencing to investigate the effects of PSCs on the gut microbiota of small herbivorous mammals, we studied plateau pikas (Ochotona curzoniae) fed diets containing swainsonine (SW) extracted from Oxytropis ochrocephala. Our results showed that both long- and short-term treatment of a single artificial diet in the laboratory significantly reduced alpha diversity and significantly affected beta diversity, core bacteria abundance, and bacterial functions in pikas. After SW was added to the artificial diet, the alpha diversity significantly increased in the long-term treatment, and core bacteria (e.g., Akkermansiaceae) with altered relative abundances in the two treatments showed no significant difference compared with pikas in the wild. The complexity of the co-occurrence network structure was reduced in the artificial diet, but it increased after SW was added in both treatments. Further, the abundances of bacteria related to altered alanine, aspartate, and glutamate metabolism in the artificial diet were restored in response to SW. SW further decreased the concentration of short-chain fatty acids (SCFAs) in both treatments. Our results suggest that PSCs play a key role in regulating gut microbiota community and intestinal homeostasis, thereby maintaining host health. KEY POINTS: • Swainsonine improves the intestinal bacterial diversity of plateau pikas. • Swainsonine promotes the recovery of core bacterial abundances in the gut of plateau pikas. • Swainsonine promotes the restoration of intestinal bacterial functions of plateau pikas.

Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins.

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Nγ -Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases.

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely Nγ -hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. The most potent cyclic peptide (Ki =2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization owing to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments.

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

Investigating Glyoxylate-Mediated Transamination Using Dipeptide Arrays and Proteomic Peptide Mixtures.

Glyoxylate-mediated transamination (GT) is a classic, potentially general, and N-terminus-specific protein modification method useful for the preparation of bioconjugates. However, there is a lack of information on whether and how readily a particular N-terminal amino acid (in the context of a peptide chain) can be converted to the 2-oxoacyl moiety under GT conditions. Here, we conducted a systematic investigation of GT using membrane-bound dipeptide arrays that include all the 400 possible dipeptide combinations of the 20 genetically encoded amino acids. This colorimetric method offers a convenient way to assess the GT reaction tendency of N-terminal residues by the naked eye. It also provides interesting information about the effect of the second residues on GT, which has not been reported previously. In addition, we also designed a proteomics approach to study GT in solution using tryptic peptide mixtures, which not only confirmed many of our findings in peptide array assays but also revealed potential side reaction products. Taken together, our studies will make the future use of GT for protein modification in a much more predictable way.

Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid.

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.