Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions. 1 , 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly₅ on the peptidoglycan of bacterial cell walls by sortase A. 1 The enzyme is an important anti‐virulence and anti‐infective drug target for resistant strains of Gram‐positive bacteria. 2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science. 3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase A_ΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size‐exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.     

Flow‐Based Enzymatic Ligation by Sortase A

Sortase‐mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase‐mediated side reactions. A flow‐based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations. The platform was tested with several reaction partners and used to generate a protein bioconjugate inaccessible by solution‐phase batch sortagging.     

Sortase A-catalyzed site-specific coimmobilization on microparticles via streptavidin

A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG)was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.     

3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three‐dimensional (3D) structure of an unstable, low‐abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site‐specific cysteine‐reactive paramagnetic tag that does not react with the active‐site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non‐equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.     

Directed evolution of sortase A mutants with altered substrate selectivity profiles

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.     

A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full‐length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε‐amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site‐specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site‐specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.     

In Situ Cyclization of Native Proteins: Structure‐Based Design of a Bicyclic Enzyme

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface‐exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability.     

Making and breaking peptide bonds: protein engineering using sortase

Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.     

蛋白质修饰的电化学研究

蛋白质的翻译后修饰对于生命体执行正常的生理功能具有十分重要的作用,是蛋白质科学研究的重要内容.目前研究蛋白质修饰的方法主要有质谱法、亲和层析等,然而由于蛋白质修饰研究的复杂性,迫切需要发掘新的技术手段.电化学方法理论成熟、应用广泛,在生命科学许多领域发挥着越来越重要的作用.蛋白质的体外修饰必将导致蛋白质特定位点基团的变化,可以利用巧妙设计的电化学方法予以表征和分析,以期探明修饰对蛋白质结构和功能的影响.此外,又可以利用电化学定量分析的独特优势快速准确地测定蛋白质修饰中涉及的相关酶活.正因为如此,蛋白质体外修饰的电化学研究已引起越来越多的关注.本文以作者课题组近期研究工作为主,结合国内外同行的相关代表性工作,介绍电化学方法在蛋白质修饰方面的近期研究进展,并探讨了今后的发展方向和趋势.     

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Staphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.     

Targeted Antithrombotic Protein Micelles

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site‐specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one‐pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single‐chain antibody (scFv), which binds to the ligand‐induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.     

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

Enzyme‐mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N‐terminally labeled with lithocholic acid (LA)—an inexpensive bile acid that exhibits strong binding to β‐cyclodextrin (βCD). Capture and recycling of the LA‐Pro‐SrtA 7M conjugate was achieved using βCD‐modified sepharose resin. The LA‐Pro‐SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Strategies for protein-based nanofabrication: Ni-NTA as a chemical mask to control biologically imposed symmetry

Background: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties.Results:Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affinity resin Ni²⁺-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of ‘short’ dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni²⁺-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines.Conclusions: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins. Ni²⁺-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional Histags. This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents.     

Light-Controlled Chemoenzymatic Immobilization of Proteins towards Engineering of Bioactive Papers

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point-of-care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo-triggered cycloaddition followed by site-specific sortase A-catalyzed transamidation. This site-directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper-based diagnostics.     

Mapping N6-Methyladenosine (m6A) in RNA: Established Methods,Remaining Challenges,and Emerging Approaches

N⁶-Methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic mRNA. Specific m⁶A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m⁶A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m⁶A mapping. Starting with the seminal m⁶A-sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo-cross-linking and remaining challenges. As an alternative, antibody-free approaches will be presented. These include wild-type or engineered m⁶A-sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m⁶A. Finally, single-molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.     

The Xanthate Route to Indolines,Indoles, and their Aza Congeners

Convergent routes to a variety of indolines, indoles, oxindoles, and their aza analogues involving radical additions of xanthates are described. Three approaches are summarized. The first is the least general and relies on the generation of aryl or heteroaryl radicals starting from diazonium salts. The second involves radical addition to N-allylanilines followed by ring-closure onto the aromatic core. A large variety of indolines and azaindolines can thus be obtained and, in many cases, converted into the corresponding indoles and azaindoles by various methods. The synthesis of novel fluoroazaindolines and fluoroazaindoles by a rare homolytic ipso-substitution of fluorine atoms is particularly noteworthy. The last approach hinges on the direct modification of indoles by radical addition to the pyrrole subunit of the indole nucleus. Application of this methodology to the total synthesis of melatonin and the alkaloids mersicarpine, caulerpine, and the pentacyclic skeleton of tronocarpine is briefly discussed. Most of the compounds described herein would be difficult to obtain by more traditional routes.     

Chemical approaches to the study of nucleoprotein structures

The main chemical approaches to the study of macromolecular structure and dynamics and to the elucidation of interbiopolymer contact points are considered and illustrated by particular examples. Primary attention is paid to the chemical footprinting and affinity modification methods. The use of bifunctional reagents for the study of nucleoprotein structure architecture is described. The ways of enhancing the selectivity of affinity modification available from the literature are analyzed with an emphasis on catalytically competent (superselective) labeling. The identification of proteins responsible for replication of the tickborne encephalitis virus by this method is described to demonstrate the possibility of the application of the method to multicomponent systems such as the nucleus fraction of infected cells. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1225–1231, July, 1999.     

Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides,Glycopeptides, and Proteins

Sortases are a family of transpeptidases found in gram-positive bacteria responsible for covalent anchoring of cell surface proteins to bacterial cell walls. It has been discovered that sortase A (SrtA) of Staphylococcus aureus origin is rather promiscuous and can accept various molecules as substrates. As a result, SrtA has been widely used to ligate peptides and proteins with a variety of nucleophiles, and the ligation products are useful for research in chemical biology, proteomics, biomedicine, etc. This review summarizes the recent applications of SrtA with special emphasis on SrtA-catalyzed ligation of carbohydrates with peptides and proteins.     

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site‐Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid A

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed‐batch fermentation of E. coli, followed by its structural modification through tailored, site‐selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.