Protein Modification Reactions Research Articles

Carbon dioxide enhances sulphur-selective conjugate addition reactions.

Sulphur-selective conjugate addition reactions play a central role in synthetic chemistry and chemical biology. A general tool for conjugate addition reactions should provide high selectivity in the presence of competing nucleophilic functional groups, namely nitrogen nucleophiles. We report CO2-mediated chemoselective S-Michael addition reactions where CO2 can reversibly control the reaction pHs, thus providing practical reaction conditions. The increased chemoselectivity for sulphur-alkylation products was ascribed to CO2 as a temporary and traceless protecting group for nitrogen nucleophiles, while CO2 efficiently provide higher conversion and selectivity sulphur nucleophiles on peptides and human serum albumin (HSA) with various electrophiles. This method offers simple reaction conditions for cysteine modification reactions when high chemoselectivity is required.

Challenges in the use of sortase and other peptide ligases for site-specific protein modification

Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.

DNA-assisted site-selective protein modification.

Protein modification is important for various types of biomedical research, including proteomics and therapeutics. Many methodologies for protein modification exist, but not all possess the required level of efficiency and site selectivity. This review focuses on the use of DNA to achieve the desired conversions and levels of accuracy in protein modification by using DNA (i) as a template to help concentrate dilute reactants, (ii) as a guidance system to achieve selectivity by binding specific proteins, and (iii) even as catalytic entity or construct to enhance protein modification reactions.

1H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high-mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as Congenital Disorders of Glycosylation. Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C, and 15N resonance assignments for Ost4 and Ost4V23D in dodecylphosphocholine micelles.

Protein Substrates for Reaction Discovery: Site-Selective Modification with Boronic Acid Reagents.

Chemical modification of natural proteins must navigate difficult selectivity questions in a complex polyfunctional aqueous environment, within a narrow window of acceptable conditions. Limits on solvent mixtures, pH, and temperature create challenges for most synthetic methods. While a protein's complex polyfunctional environment undoubtedly creates challenges for traditional reactions, we wondered if it also might create opportunities for pursuing new bioconjugation reactivity directly on protein substrates. This Account describes our efforts to date to discover and develop new and useful reactivity for protein modification by starting from an open-ended screen of potential transition-metal catalysts for boronic acid reactivity with a model protein substrate. By starting from a broad screen, we were hoping to take advantage of the very many potential reactive sites on even a small model protein. And perhaps more importantly, whole proteins as reaction screening substrates might exhibit uniquely reactive local environments, the results of a dense combination of functional groups that would be nearly impossible to mimic in a small-molecule context. This effort has resulted in the discovery of four new protein modification reactions with boronic acid reagents, including a remarkable modification of specific backbone N-H bonds. This histidine-directed Chan-Lam coupling, based on specific proximity of an imidazole and two amide groups, is one important example of powerful reactivity that depends on a combination of functional groups that proteins make possible. Other bioconjugation reactions uncovered include a three-component tyrosine metalation with rhodium(III), a nickel-catalyzed cysteine arylation, and an unusual ascorbate-mediated oxidative process for N-terminal modification. The remarkably broad scope of reactivity types encountered in this work is a testament to the breadth of boronic acid reactivity. It is also a demonstration of the diverse reactivities that are possible by the combined alteration of boronic acid structure and metal promoter. The discovery of specific backbone modification chemistry has been a broadly empowering reactivity. Pyroglutamate, a naturally occurring posttranslational modification, exhibits remarkably high reactivity in histidine-directed backbone modification, which allows us to treat pyroglutamate as a reactive bioorthogonal handle that is readily incorporated into proteins of interest by natural machinery. In another research direction, the development of a vinylogous photocleavage system has allowed us to view backbone modification as a photocaging modification which is released by exposure to light.

Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.

Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.

Production and biophysical characterization of a mini-membrane protein, Ost4V23D: A functionally important mutant of yeast oligosaccharyltransferase subunit Ost4p

N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc. Complete loss of N-glycosylation is lethal in all organisms. In Saccharomyces cerevisiae, OST consists of nine non-identical protein subunits. Ost4p is the smallest subunit containing 36 residues. It bridges catalytic subunit Stt3p to Ost3p/Ost6p subunit. Mutation of Valine (V) at position 23 in Ost4p to Aspartate (D) causes defects in the N-glycosylation process. To understand the structure, function and role of Ost4p in N-glycosylation, characterization of Ost4p and its functionally important mutant/s are critical. We report the mutagenesis, heterologous overexpression, purification, reconstitution in DPC micelles and biophysical characterization of Ost4V23D and compare its secondary structure and conformation to that of Ost4p. CD and NMR data suggest that mutation of Val23 to Asp impacts the secondary structure and conformation of Ost4p.

Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation.

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. Metazoan organisms express two oligosaccharyltransferase complexes that are composed of a catalytic subunit (STT3A or STT3B) assembled with a shared set of accessory subunits and one to two complex specific subunits. siRNA mediated knockdowns of STT3A and STT3B in HeLa cells have shown that the two OST complexes have partially non-overlapping roles in N-linked glycosylation. However, incomplete siRNA mediated depletion of STT3A or STT3B reduces the impact of OST complex loss, thereby complicating the interpretation of experimental results. Here, we have used the CRISPR/Cas9 gene editing technology to create viable HEK293 derived cells lines that are deficient for a single catalytic subunit (STT3A or STT3B) or two STT3B-specific accessory subunits (MagT1 and TUSC3). Analysis of protein glycosylation in the STT3A, STT3B and MagT1/TUSC3 null cell lines revealed that these cell lines are superior tools for investigating the in vivo role and substrate preferences of the STT3A and STT3B complexes.

Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions.

We evaluate the ability of hexahistidine (His6) tags on peptide and protein substrates to recruit deoxyribozymes for modifying those substrates. For two different deoxyribozymes, one that creates tyrosine-RNA nucleopeptides and another that phosphorylates tyrosine side chains, we find substantial improvements in yield, kobs, and Km for peptide substrates due to recruiting by His6/Cu(2+). However, the recruiting benefits of the histidine tag are not observed for larger protein substrates, likely because the tested deoxyribozymes either cannot access the target peptide segments or cannot function when these segments are presented in a structured protein context.

Development of oxidative coupling strategies for site-selective protein modification.

As the need to prepare ever more complex but well-defined materials has increased, a similar need for reliable synthetic strategies to access them has arisen. Accordingly, recent years have seen a steep increase in the development of reactions that can proceed under mild conditions, in aqueous environments, and with low concentrations of reactants. To enable the preparation of well-defined biomolecular materials with novel functional properties, our laboratory has a continuing interest in developing new bioconjugation reactions. A particular area of focus has been the development of oxidative reactions to perform rapid site- and chemoselective couplings of electron rich aromatic species with both unnatural and canonical amino acid residues. This Account details the evolution of oxidative coupling reactions in our laboratory, from initial concepts to highly efficient reactions, focusing on the practical aspects of performing and developing reactions of this type. We begin by discussing our rationale for choosing an oxidative coupling approach to bioconjugation, highlighting many of the benefits that such strategies provide. In addition, we discuss the general workflow we have adopted to discover protein modification reactions directly in aqueous media with biologically relevant substrates. We then review our early explorations of periodate-mediated oxidative couplings between primary anilines and p-phenylenediamine substrates, highlighting the most important lessons that were garnered from these studies. Key mechanistic insights allowed us to develop second-generation reactions between anilines and anisidine derivatives. In addition, we summarize the methods we have used for the introduction of aniline groups onto protein substrates for modification. The development of an efficient and chemoselective coupling of anisidine derivatives with tyrosine residues in the presence of ceric ammonium nitrate is next described. Here, our logic and workflow are used to highlight the challenges and opportunities associated with the optimization of site-selective chemistries that target native amino acids. We close by discussing the most recent reports from our laboratory that have capitalized on the unique reactivity of o-iminoquinone derivatives. We discuss the various oxidants and conditions that can be used to generate these reactive intermediates from appropriate precursors, as well as the product distributions that result. We also describe our work to determine the nature of iminoquinone reactivity with proteins and peptides bearing free N-terminal amino groups. Through this discussion, we hope to facilitate the use of oxidative approaches to protein bioconjugation, as well as inspire the discovery of new reactions for the site-selective modification of biomolecular targets.

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems.

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.

Activation of poly(ADP-ribose) polymerase-1 delays wound healing by regulating keratinocyte migration and production of inflammatory mediators.

Poly(ADP-ribosyl)ation (PARylation) is a protein modification reaction regulating various diverse cellular functions ranging from metabolism, DNA repair and transcription to cell death. We set out to investigate the role of PARylation in wound healing, a highly complex process involving various cellular and humoral factors. We found that topically applied poly[ADP-ribose] polymerase (PARP) inhibitors 3-aminobenzamide and PJ-34 accelerated wound closure in a mouse model of excision wounding. Moreover, wounds also closed faster in PARP-1 knockout mice as compared with wild-type littermates. Immunofluorescent staining for poly(ADP-ribose) (PAR) indicated increased PAR synthesis in scattered cells of the wound bed. Expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase and matrix metalloproteinase-9 was lower in the wounds of PARP-1 knockout mice as compared with control, and expression of IL-1β, cyclooxygenase-2, TIMP-1 and -2 also were affected. The level of nitrotyrosine (a marker of nitrating stress) was lower in the wounds of PARP-1 knockout animals as compared with controls. In vitro scratch assays revealed significantly faster migration of keratinocytes treated with 3-aminobenzamide or PJ34 as compared with control cells. These data suggest that PARylation by PARP-1 slows down the wound healing process by increasing the production of inflammatory mediators and nitrating stress and by slowing the migration of keratinocytes.

New players to the field of ADP-ribosylation make the final cut

ADP‐ribose‐based intermediates, including PARP‐generated mono‐ and poly(ADP‐ribose) post‐translational modifications, are important to a number of cellular signalling processes. The reversal of poly(ADP‐ribosyl)ation is mostly attributed to PARG, which however cannot remove the final protein‐linked mono(ADP‐ribose) residue. Three recent studies, one of them in The EMBO Journal , now report that certain macrodomains remove terminal ADP‐ribose modifications from acidic residues. ADP‐ribosylation is a post‐translational modification (PTM) in which the ADP‐ribose moiety of NAD+ is covalently transferred to a target protein (see Figure 1). This reaction is catalysed by poly(ADP‐ribose) polymerase (PARP) enzymes, some of which also extend the modification by adding additional ADP‐ribose molecules through ribose‐ribose bonds, thus generating poly(ADP‐ribose) (PAR). PARP1 and PARP2 account for the majority of PAR formation in response to cellular stress such as DNA damage. Figure 1. Pathway of reversible protein modification reactions that involve NAD+ consumption. Consumption of NAD+ through nicotinamide cleavage drives the catalysis of mono‐ and poly(ADP‐ribosyl)ation of proteins by ADP‐ribosyl transferases, as well as deacetylation of proteins by Sirtuins. Formation of these modifications is important to various cell signalling events such as DNA repair, chromatin remodelling, transcription, telomere homeostasis, and cell death. ADP‐ribose modifications are short‐lived due to the activity of hydrolase enzymes reversing the modification to yield ADP‐ribose. The recently identified macrodomains C6orf130/TARG, MacroD1, and MacroD2 now fill in previously unidentified roles of ADP‐ribose and PAR hydrolysis from acidic residues. Poly(ADP‐ribosyl)ation is a dynamic and transient modification event, since an enzyme called poly(ADP‐ribose) glycohydrolase (PARG) counters PARP activities by degrading PAR through endo‐ and exo‐glycosidic activities (see Figure 1). However, neither PARG nor the unrelated ADP‐ribosyl hydrolase 3 (ARH‐3) also implicated …

Neuronal PAD4 expression and protein citrullination: Possible role in production of autoantibodies associated with neurodegenerative disease

Peptidyl arginine deiminases (PADs) catalyze a post-translational protein modification reaction called citrullination, where arginine is converted to citrulline. This modification has been linked to the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA). More recently, several studies have suggested that Alzheimer's disease (AD), a devastating neurodegenerative disorder, may have an autoimmune component. In the present study, we have investigated the possibility that expression of PADs and protein citrullination plays a role in the production of brain-reactive autoantibodies that may contribute to Alzheimer's-related brain pathology. Here, we report the selective expression of the PAD isoforms, PAD2 and PAD4, in astrocytes and neurons, respectively, and the concomitant accumulation of citrullinated proteins within PAD4-expressing cells, including neurons of the hippocampus and cerebral cortex. Expression of PADs and citrullinated proteins is prominent in brain regions engaged in neurodegenerative changes typical for AD pathology. Furthermore, we also demonstrate that the pentatricopeptide repeat domain2 (PTCD2) protein, an antigen target of a prominent AD diagnostic autoantibody, is present in a citrullinated form in AD brains. Our results suggest that disease-associated neuronal loss results in the release of cellular contents, including citrullinated proteins, into the brain interstitium. We propose that these citrullinated proteins and their degradation fragments enter into the blood and lymphatic circulation, and some are capable of eliciting an immune response that results in the production of autoantibodies. The long-term and progressive nature of AD and other neurodegenerative diseases results in chronic exposure of the immune system to these citrullinated products and may drive the continual production of autoantibodies.

Decreased protein expression and activity of transglutaminases 1 and 2 in arteries from DOCA‐salt rats

Transglutaminases (TGs) catalyze a variety of protein modification reactions in the vasculature, and TG‐mediated protein remodeling has been implicated in hypertension. We hypothesized that there would be a higher expression of TGs in hypertensive vs normotensive vasculature due to protein cross‐linking catalyzed by the TGs. We used aorta (RA) from deoxycorticosterone acetate (DOCA)‐salt (hypertensive) and sham (normotensive) control rats. Methods included immunohistochemistry (IHC), Western blot analysis, and in situ fluorescent detection of active TGs using the K5 (TG1) and T26 (TG2) peptides. IHC showed a decreased expression of TG1 and TG2 in DOCA‐salt rat aorta (TG1: DOCA 1.22, sham 1.44; TG2: DOCA 0.61, sham 1.61 arbitrary rank units) and in situ detection showed decreased activity of TG1 and TG2 in DOCA‐salt rat aorta (TG1: DOCA 2.0, sham 2.72; TG2: DOCA 1.79, sham 2.56 arbitrary rank units). Similarly, protein expressions of TG1 and TG2 in DOCA‐salt rat aorta were diminished compared to the control (TG1: DOCA 27.8 ± 2.52, sham 51.9 ± 13.0; TG2: DOCA 83.2 ± 3.85, sham 108 ± 5.83 arbitrary densitometry units/alpha actin; p<0.05). We conclude that there is less activity and decreased levels of TG1 and TG2 in hypertensive rat aorta compared to sham. This contradicts our hypothesis and supports the idea that lack of compensatory TGs may a role in the pathology of hypertension.POIHC70687

A novel and simple method of production and biophysical characterization of a mini‐membrane protein, Ost4p: A Subunit of yeast oligosaccharyl transferase

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction. In eukaryotes, oligosaccharyl transferase (OT), a multi-subunit membrane-associated enzyme complex, catalyzes this reaction in newly synthesized proteins. In Saccharomyces cerevisiae, OT consists of nine nonidentical membrane proteins. Ost4p, the smallest subunit, bridges the catalytic subunit Stt3p with Ost3p. Mutation of transmembrane residues 18-24 in Ost4p has negative effect on OT activity, disrupts the Stt3p-Ost4p-Ost3p complex, results in temperature-sensitive phenotype, and hypoglycosylation. Heterologous expression and purification of integral membrane proteins are the bottleneck in membrane protein research. The authors report the cloning, successful overexpression and purification of recombinant Ost4p with a novel but simple method producing milligram quantities of pure protein. GB1 protein was found to be the most suitable tag for the large scale production of Ost4p. The cleavage of Ost4p conveniently leaves GB1 protein in solution eliminating further purification. The precipitated pure Ost4p is reconstituted in appropriate membrane mimetic. The recombinant protein is highly helical as indicated by the far-UV CD spectrum. The well-dispersed heteronuclear single quantum coherence spectrum indicates that this minimembrane protein is well-folded. The successful production of pure recombinant Ost4p with a novel yet simple method may have important ramification for the production of other membrane proteins.

Choosing an effective protein bioconjugation strategy

The collection of chemical techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biological understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate production, and a number of techniques can be used to label a single protein in a complex biological mixture. This review examines the way in which experimental circumstances influence one's selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.

Using Synthetically Modified Proteins to Make New Materials

The uniquely diverse structures and functions of proteins offer many exciting opportunities for creating new materials with advanced properties. Exploiting these capabilities requires a set of versatile chemical reactions that can attach nonnatural groups to specific locations on protein surfaces. Over the years, we and others have developed a series of new techniques for protein bioconjugation, with a particular emphasis on achieving high site selectivity and yield. Using these reactions, we have been able to prepare a number of new materials with functions that depend on both the natural and the synthetic components. In this Account, we discuss our progress in protein bioconjugation over the past decade, focusing on three distinct projects. We first consider our work to harness sunlight artificially by mimicking features of the photosynthetic apparatus, with its beautifully integrated system of chromophores, electron transfer groups, and catalytic centers. Central to these photosystems are light-harvesting antennae having hundreds of precisely aligned chromophores with positions that are dictated by the proteins within the arrays. Our approach to generating similar arrangements involves the self-assembly of tobacco mosaic virus coat proteins bearing synthetic chromophore groups. These systems offer efficient light collection, are easy to prepare, and can be used to build complex photocatalytic systems through the modification of multiple sites on the protein surfaces. We then discuss protein-based carriers that can deliver drugs and imaging agents to diseased tissues. The nanoscale agents we have built for this purpose are based on the hollow protein shell of bacteriophage MS2. These 27 nm capsids have 32 pores, which allow the entry of relatively large organic molecules into the protein shell without requiring disassembly. Our group has developed a series of chemical strategies that can install dyes, radiolabels, MRI contrast agents, and anticancer drugs on the inside surface of these capsids. We have also developed methods to decorate the external surfaces with binders for specific proteins on cancer cells. As a third research area, our group has developed protein-polymer hybrid materials for water remediation. To reduce the toxicity of heavy metals in living cells, Nature has evolved metallothioneins, which are sulfur-rich polypeptides that bind mercury, cadmium, and other toxic ions at sub-parts-per-billion concentrations. Unfortunately, these proteins are very difficult to incorporate into polymers, largely because typical protein modification reactions target the very cysteine, lysine, and carboxylate-containing residues that are required for their proper function. To address this challenge, we developed a new way to attach these (and many other) proteins to polymer chains by expressing them as part of an N- and C-terminal modification "cassette". The resulting materials retain their selectivity and can remove trace amounts of toxic metal ions from ocean water. Each of these examples has presented a new set of protein bioconjugation challenges that have been met through the development of new reaction methodology. Future progress in the generation of protein-based materials will require scalable synthetic techniques with improved yields and selectivities, inexpensive purification methods for bioconjugates, and theoretical and dynamical treatments for designing new materials through protein self-assembly.

Porthole to catalysis

The crystal structure of a sugar-transferring enzyme offers insight into the mechanism of a ubiquitous protein-modification reaction, and solves the mystery of how the enzyme recognizes certain sequences in proteins. See Article p.350 More than half of the proteins in eukaryotes are glycoproteins, with specific amino-acid side chains linked to oligosaccharides. The most frequent of these chemical modifications is asparagine-linked glycosylation, catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The X-ray structure of a bacterial OST from Campylobacter lari in complex with an acceptor peptide has now been determined. The structure provides the molecular basis for understanding amide nitrogen activation and glycosylation, and offers opportunities for the production of glycoprotein and glycoconjugate therapeutics.

Arginine-specific protein modification using α-oxo-aldehyde functional polymers prepared by atom transfer radical polymerization

The residue-specific modification of peptides and proteins is a powerful strategy for preparing biomolecular–synthetic polymer conjugates with advanced properties. This manuscript aims at expanding the present toolbox of residue-selective protein modification reactions and targets arginine, a residue for which selective polymer coupling chemistry has only recently been established. To this end, a protected, α-oxo-aldehyde functionalized ATRP initiator that can be used for the preparation of a variety of α-oxo-aldehyde functionalized polymethacrylates has been developed. Polymerization kinetics for four different methacrylate monomers have been investigated in detail and optimized conditions for the chain-end deprotection to reveal the α-oxo-aldehyde end-group have been elaborated. As a final proof of concept, the residue-specific modification of a model protein, chicken egg white lysozyme (HEWL), at arginine residues has been demonstrated.