Site-specific Protein Modification Research Articles

Asymmetric Synthesis of Functionalized α-Amino Acid Derivatives via the γ-Pyrone Carbaldimine-Based Organocatalytic Mannich Reaction.

A powerful synthetic strategy for the asymmetric synthesis of enantiomerically enriched γ-functionalized α-amino acid derivatives based on the highly stereoselective proline-catalyzed Mannich-type reaction of pre-prepared or in situ-generated γ-pyrone-derived aldimines with carbonyl compounds and subsequent transformations of multifunctional reaction products has been developed. A significant positive nonlinear effect was detected for the key organocatalytic reaction. The developed strategy was applied for facile gram-scale preparation of (S)-γ-oxonorvaline, used for site-specific modification of proteins, and both enantiomers of amycolatolide A recently isolated from the lichen-derived actinomycete Amycolatopsis sp. YIM 130923.

A one-process production of completely biotinylated proteins in a T7 expression system.

Streptavidin is a tetrameric protein with high specificity and affinity for biotin. The interaction between avidin and biotin has become a valuable tool in nanotechnology. In recent years, the site-specific biotin modification of proteins using biotin ligases, such as BirA, has attracted attention. This study established an in vivo method for achieving the complete biotinylation of target proteins using a single plasmid co-expressing BirA and its target proteins. Specifically, a biotin-modified protein was produced in Escherichia coli strain BL21(DE3) using a single plasmid containing genes encoding both BirA and a protein fused to BirA's substrate sequence, Avitag. This approach simplifies the production of biotinylated proteins in E. coli and allows the creation of various biotinylated protein types through gene replacement. Furthermore, the biotin modification rate of the obtained target protein could be evaluated using Native-PAGE without performing complicated isolation operations of biotinylated proteins. In Native-PAGE, biotin-modified proteins and unmodified proteins were confirmed as clearly different bands, and it was possible to easily derive the modification rate from the respective band intensities.

Site-Specific Anchoring a Luminescent Tag in a Protein with Non-Emissive Iridium(III) Complex.

Site-specific modification of proteins with synthetic fluorescent tag effectively improves the resolution of imaging, and such a labeling method with negligible three-dimensional structural perturbations and minimal impact on the biological functions of proteins is of high interest to dissect the high-resolution activities of biomolecules in complex systems. To this end, several non-emissive iridium(III) complexes [Ir(C-N)2 (H2 O)2 ]+ OTF- (C-N denotes various cyclometalated ligands) were designed and synthesized. These complexes were tested for attaching a protein by coordinating to H/X (HisMet, HisHis, and HisCys) that are separated by i and i+4 in α-helix. Replacement of the two labile water ligands in the iridium(III) complex by a protein HisHis pair increases the luminescent intensity up to over 100 folds. This labeling approach has been demonstrated in a highly specific and efficient manner in a number of proteins, and it is also feasible for labeling target proteins in cell lysates.

Tertiary amide bond formation by an engineered asparaginyl ligase.

Transpeptidases are powerful tools for site-specific protein modification, enabling the production of tailored biologics to investigate protein function and aiding the development of next-generation therapeutics and diagnostics. Although protein labelling at the N- or C-terminus is readily accomplished using a range of established transpeptidases, these reactions are generally limited to forming products that are linked by a standard (secondary) amide bond. Here we show that, unlike other widely used transpeptidases, an engineered asparaginyl ligase is able to efficiently synthesise tertiary amide bonds by accepting diverse secondary amine nucleophiles. These reactions proceed efficiently under mild conditions (near-neutral pH) and allow the optimal recognition elements for asparaginyl ligases (P1 Asn and P2'' Leu) to be preserved. Certain products, particularly proline-containing products, were found to be protected from recognition by the enzyme, allowing for straightforward sequential labelling of proteins. Additionally, incorporation of 4-azidoproline enables one-pot dual labelling directly at the ligation junction. These capabilities further expand the chemical diversity of asparaginyl ligase-catalysed reactions and provide an alternative approach for straightforward, successive modification of protein substrates.

Affinity-Directed Site-Specific Protein Labeling and Its Application to Antibody-Drug Conjugates.

Chemically modified proteins have diverse applications; however, conventional chemo-selective methods often yield heterogeneously labeled products. To address this limitation, site-specific protein labeling holds significant potential, driving extensive research in this area. Nevertheless, site-specific modification of native proteins remains challenging owing to the complexity of their functional groups. Therefore, a method for site-selective labeling of intact proteins is aimed to design. In this study, a novel approach to traceless affinity-directed intact protein labeling is established, which leverages small binding proteins and genetic code expansion technology. By applying this method, a site-specific antibody labeling with a drug, which leads to the production of highly effective antibody-drug conjugates specifically targeting breast cancer cell lines is achieved. This approach enables traceless conjugation of intact target proteins, which is a critical advantage in pharmaceutical applications. Furthermore, small helical binding proteins can be easily engineered for various target proteins, thereby expanding their potential applications in diverse fields. This innovative approach represents a significant advancement in site-specific modification of native proteins, including antibodies. It also bears immense potential for facilitating the development of therapeutic agents for various diseases.

Enzymatic Fluoromethylation as a Tool for ATP-Independent Ligation.

S-adenosylmethionine-dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme-catalyzed F-methylation of carboxylate substrates produces F-methyl esters that readily react with N- or S-nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site-specific protein modification and native chemical ligation.

Enzymatic Fluoromethylation as a Tool for ATP-Independent Ligation.

S-adenosylmethionine-dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme-catalyzed F-methylation of carboxylate substrates produces F-methyl esters that readily react with N- or S-nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site-specific protein modification and native chemical ligation.

Highly efficient site-specific protein modification using tyrosinase from Streptomyces avermitilis: Structural insight

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.

Metal-Mediated Ligand Affinity Chemistry (MLAC).

Metal-mediated ligand affinity chemistry (MLAC) enables site-specific protein modification and represents a powerful bioorthogonal strategy. Conventional bioorthogonal methods often involve two steps: (i) incorporation of the bioorthogonal handle (e.g., non-canonical amino acid, enzyme domain, peptide sequences) and (ii) the binding of functional molecules such as drugs, affinity tags, and fluorophores. This two-step protocol often involves genetic manipulation, which makes it impossible to chemically modify endogenous proteins in living systems. Thus, we propose the development of a transition metal-based chemical strategy that is ligand-directed to the endogenous protein of interest in a single step, which we refer to as metal-mediated ligand affinity chemistry (MLAC).

Activity-based profiling of cullin–RING E3 networks by conformation-specific probes

The cullin–RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs.

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization.

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation.

Site-specific modification of proteins has wide applications in probing and perturbing biological systems. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities. Indeed, various bioorthogonal reactions have been developed, including a recently reported reaction between 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM). Here, we describe the procedure that combines genetic code expansion and TAMM condensation for site-specific modification of cellular membrane proteins. The 1,2-aminothiol functionality is introduced through a genetically incorporated noncanonical amino acid to a model membrane protein on mammalian cells. Treatment of the cells with a fluorophore-TAMM conjugate leads to fluorescent labeling of the target protein. This method can be applied to modify different membrane proteins on live mammalian cells.

Synthesis of Peptides and Proteins with Site-Specific Glutamate Arginylation.

Solid-phase peptide synthesis and protein semi-synthesis are powerful methods for site-specific modification of peptides and proteins. We describe protocols using these techniques for the syntheses of peptides and proteins bearing glutamate arginylation (EArg) at specific sites. These methods overcome challenges posed by enzymatic arginylation methods and allow for a comprehensive study of the effects of EArg on protein folding and interactions. Potential applications include biophysical analyses, cell-based microscopic studies, and profiling of EArg levels and interactomes in human tissue samples.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

Protein C-Terminal Tyrosine Conjugation via Recyclable Immobilized BmTYR.

Protein modification plays an essential role in biological and pharmaceutical research. Due to the ordinary selectivity and inevitable damage to proteins of chemical synthetic methods, increased efforts were focused on biocatalysts which exhibited high regioselectivity and mild reaction conditions. However, separation of the biocatalysts and modified proteins remained a problem, especially when scaling up. Here, we developed a simple method for site-specific protein modification with a recyclable biocatalyst. The immobilizing tyrosinase (BmTYR) on magnetic beads can oxidize C-terminal tyrosine residues of the target protein to o-quinone, followed by the spontaneous addition of different nucleophiles (e.g., aniline derivatives), resulting in a C-terminal modified protein. Compared to the homogeneous biocatalytic system reported before, this heterogeneous system leads to an easier separation. Furthermore, the solid-phase biocatalyst can be regenerated during separation, providing reusability and lower costs.

A Peptide-Based Ligand-Directed Chemistry Enables Protein Functionalization.

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide-target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.

Optimizing eRF1 to Enable the Genetic Encoding of Three Distinct Noncanonical Amino Acids in Mammalian Cells.

Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E.coli-derived tyrosyl (EcTyr)/tRNAUUA , E. coli-derived leucyl (EcLeu)/tRNACUA , and Methanosarcina mazei pyrrolysyl (MmPyl)/tRNAUCA pairs) are screened for ncAA incorporation. Furthermore, introduced combinations of eukaryotic release factor 1 (eRF1) mutants (E55R, E55D, N129D, and Y125F) significantly improve the encoding efficiency of the three premature stop codons' sites from 0.78% to 11.6%. Thus, site-specific incorporation of three distinct ncAAs into a single protein is achieved in this study. This work markedly expands the potential for multiple site-specific protein modifications within mammalian cells, thereby facilitating new in vivo applications.

Site-Specific Protein Modification with Reducing Carbohydrates.

Protein glycosylation plays critical roles in many biological processes. However, the fundamental study and application of glycobiology are hindered by the heterogeneousness of oligosaccharides in natural glycoproteins and the difficulty in constructing glycoproteins of human design. Herein, we describe a semisynthetic method to site-specifically modify proteins with reducing carbohydrates. The method involves the genetic incorporation of a side-chain-esterified aspartate, which was subsequently quantitatively converted into alanine-β-hydrazide (Aβz), and chemoselective conjugation of Aβz with a range of readily available reducing carbohydrates. The resulting Aβz-linked GlcNAc is a close mimic of native N-GlcNAc and could be installed on various proteins, including IL-17A and RNase A. Notably, Aβz-linked GlcNAc on proteins reacted with biantennary oligosaccharide oxazoline derivatives through endoglycosidase-catalyzed transglycosylation reactions to enable the assembly of homogeneous glycans on proteins.

Efficient Enzymatic Incorporation of Dehydroalanine Based on SAMDI-Assisted Identification of Optimized Tags for OspF/SpvC.

Site-specific modification of proteins has important applications in biological research and drug development. Reactive tags such as azide, alkyne, and tetrazine have been used extensively to achieve the abovementioned goal. However, bulky side-chain "ligation scars" are often left after the labeling and may hinder the biological application of such engineered protein products. Conjugation chemistry via dehydroalanine (Dha) may provide an opportunity for "traceless" ligation because the activated alkene moiety on Dha can then serve as an electrophile to react with radicalophile, thiol/amine nucleophile, and reactive phosphine probe to introduce a minimal linker in the protein post-translational modifications. In this report, we present a mild and highly efficient enzymatic approach to incorporate Dha with phosphothreonine/serine lyases, OspF and SpvC. These lyases originally catalyze an irreversible elimination reaction that converts a doubly phosphorylated substrate with phosphothreonine (pT) or phosphoserine (pS) to dehydrobutyrine (Dhb) or Dha. To generate a simple monophosphorylated tag for these lyases, we conducted a systematic approach to profile the substrate specificity of OspF and SpvC using peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. The optimized tag, [F/Y/W]-pT/pS-[F/Y/W] (where [F/Y/W] indicates an aromatic residue), results in a ∼10-fold enhancement of the overall peptide labeling efficiency via Dha chemistry and enables the first demonstration of protein labeling as well as live cell labeling with a minimal ligation linker via enzyme-mediated incorporation of Dha.