Isopeptide Research Articles

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

Expanding the Versatility of Microbial Transglutaminase Using α‐Effect Nucleophiles as Noncanonical Substrates

AbstractThe substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine‐containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho‐carbonylphenylboronic acids, to form site‐specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

NMR Spectroscopic Studies Reveal the Critical Role of the Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.

The interplay between protein folding and chemical reaction has been an intriguing subject. In this contribution, we report the study of SpyTag and SpyCatcher reactive mutants using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography and mass spectrometry, circular dichroism, and NMR spectroscopy. It was found that the wild-type SpyCatcher is well-folded in solution and docks with SpyTag to form an intermediate that promotes isopeptide bond formation. By contrast, the double mutant SpyCatcherVA is disordered in solution yet remains reactive toward SpyTag, forming a well-folded covalent complex. Control experiments using the catalytically inactive mutants further reveal the critical role of the isopeptide bond in stabilizing the otherwise loose SpyTag-SpyCatcherVA complex, amplifying the effect of the minute sequence disparity. We believe that the synergy between protein folding and isopeptide bonding is an effective way to enhance protein stability and engineer protein-protein interactions.

Microbial Transglutaminase as a Tool to Improve the Features of Hydrocolloid-Based Bioplastics.

Several proteins from animal and plant origin act as microbial transglutaminase substrate, a crosslinking enzyme capable of introducing isopeptide bonds into proteins between the aminoacids glutamines and lysines. This feature has been widely exploited to modify the biological properties of many proteins, such as emulsifying, gelling, viscosity, and foaming. Besides, microbial transglutaminase has been used to prepare bioplastics that, because made of renewable molecules, are able to replace the high polluting plastics of petrochemical origin. In fact, most of the time, it has been shown that the microbial enzyme strengthens the matrix of protein-based bioplastics, thus, influencing the technological characteristics of the derived materials. In this review, an overview of the ability of many proteins to behave as good substrates of the enzyme and their ability to give rise to bioplastics with improved properties is presented. Different applications of this enzyme confirm its important role as an additive to recover high value-added protein containing by-products with a double aim (i) to produce environmentally friendly materials and (ii) to find alternative uses of wastes as renewable, cheap, and non-polluting sources. Both principles are in line with the bio-economy paradigm.

Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

Identification of Isopeptides Between Human Tissue Transglutaminase and Wheat, Rye, and Barley Gluten Peptides

Celiac disease (CD) is a chronic immune-mediated enteropathy of the small intestine, which is triggered by the ingestion of storage proteins (gluten) from wheat, rye, and barley in genetically predisposed individuals. Human tissue transglutaminase (TG2) plays a central role in the pathogenesis of CD, because it is responsible for specific gluten peptide deamidation and covalent crosslinking, resulting in the formation of Nε-(γ-glutamyl)-lysine isopeptide bonds. The resulting TG2-gluten peptide complexes are assumed to cause the secretion of anti-TG2 autoantibodies, but the underlying mechanisms are only partly known. To gain more insight into the structures of these complexes, the aim of our study was to identify TG2-gluten isopeptides. With the use of discovery-driven as well as targeted nanoscale liquid chromatography tandem mass spectrometry, we detected 29 TG2-gluten isopeptides in total, involving seven selected TG2 lysine residues (K205, K265, K429, K468, K590, K600, K677). Several gluten peptides carried known B-cell epitopes and/or T-cell epitopes, either intact 9-mer core regions or partial sequences, as well as sequences bearing striking similarities to already known epitopes. These novel insights into the molecular structures of TG2-gluten peptide complexes may help clarify their physiological relevance in the initiation of CD autoimmunity and the role of anti-TG2 autoantibodies.

Molecular Basis of Ubiquitination Catalyzed by the Bacterial Transglutaminase MavC.

The Legionella pneumophila effector MavC is a transglutaminase that carries out atypical ubiquitination of the host ubiquitin (Ub)‐conjugation enzyme UBE2N by catalyzing the formation of an isopeptide bond between Gln40Ub and Lys92UBE2N, which leads to inhibition of signaling in the NF‐κB pathway. In the absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self‐ubiquitination. However, the mechanisms underlying these enzymatic activities of MavC are poorly understood at the molecular level. This study reports the structure of the MavC–UBE2N–Ub ternary complex representing a snapshot of MavC‐catalyzed crosslinking of UBE2N and Ub, which reveals the way by which UBE2N–Ub binds to the Insertion and Tail domains of MavC. Based on the structural and experimental data, the catalytic mechanism for the deamidase and transglutaminase activities of MavC is proposed. Finally, by comparing the structures of MavC and MvcA, the homologous protein that reverses MavC‐induced UBE2N ubiquitination, several essential regions and two key residues (Trp255MavC and Phe268MvcA) responsible for their respective enzymatic activities are identified. The results provide insights into the mechanisms for substrate recognition and ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC and MvcA in terms of regulation of UBE2N ubiquitination.

Protein-peptide based assay for the characterization of human blood coagulation factor XIII-A isopeptidase activity

Blood coagulation factor XIII-A (FXIII-A), a member of the transglutaminase enzyme family, is best known for its fibrin clot stabilizing function during blood coagulation. It possesses amine incorporating and protein crosslinking transamidase activities, but it is also able to cleave the previously formed isopeptide bond by its isopeptidase activity. Our aim was to develop a protein-based assay for better characterization of FXIII-A isopeptidase activity. The first attempt applying the crosslinked D-dimer of fibrin as a substrate was not successful because of poor reproducibility. Then, the principle of an earlier published anisotropy based activity assay was adapted for the measurement of FXIII-A isopeptidase activity. After crosslinking the fluorescently labelled α2-antiplasmin derived peptide and S100A4(GST) lysine donor protein, this protease-resistant γ-glutamyl-ε-lysine isopeptide bond containing protein-peptide product was applied as a substrate for FXIII-A. Using this substrate and detecting decreasing anisotropy, kinetic measurement of FXIII-A isopeptidase activity was achieved at high sensitivity even in a complex biological sample and in the presence of inhibitor.

SUMOylation Site Identification Via FRET Analysis

This year’s influenza is estimated to affect more than 20 million people in the United States alone. Although influenza has been extensively researched, it still remains a major public health threat every year. Current methods to cull the spread of the influenza virus are vaccination and anti‐influenza virus drugs. Although these methods can be effective, the infuenza’s capacity to mutate and undergo antigenic shift make these methods fall short. SUMOylation is a post‐translational modification that is important for cellular processes as well as protein function and stability. The dysregulation of SUMOylation has been observed in neurodegenerative diseases, many different types of cancers, and viral infections. SUMOylation has been shown to be an important host factor for the influenza A virus but the mechanism still needs to be better understood. Currently, the methodology to identify SUMOylated lysine residues relies on immunodetection after site‐directed mutagenesis. Although this method has yielded great success, it is prone to false positives due to a lack of sensitivity or the breakdown of the isopeptide bond from endogenous SENP. We have developed a highly‐sensitive FRET‐based methodology to identify the exact lysine residue that is targeted by SUMOylation in the NS1 protein of the influenza virus (Figure 1). Using our method, we found lysine 131 was SUMOylated, contrary to previous findings (Figure 2). Our method can be applied to other proteins and ubiquitin‐like modifications to identify important residues and evaluate posttranslational modifications as druggable targets for cancer and antiviral therapies.Development of in vitro FRET‐based SUMOylation assay of IAV NS1. A) FRET‐based SUMOylation assay of NS1. If a lysine residue is responsible for SUMOylation, the mutation of the lysine residue to alanine will result in a decrease of the FRET emission at 530 nm given a 414 nm excitation. B) The EmFRET RFU is specific to the SUMOylation of NS1, provided the requirements of SUMOylation (SAE1, E2, and ATP).Figure 1Determination of SUMOylated lysine residues of NS1. A) Table of NS1 lysine mutants for the FRET assay. B) Systematic mutation of NS1 lysine residues to alanine.Figure 2

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins.

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

A recent update on the use of microbial transglutaminase for the generation of biotherapeutics.

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.

High Force Magnetic Tweezers Reveal That Bacterial Adhesion Pili Act as Megadalton-scale Schock Absorbers

Gram positive pathogens colonize the oral cavity with their adhesive pili. These organelles are exposed to large mechanical perturbations, such as mastication or teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2nd and the 3rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2nd and 3rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to ∼4·105 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.

Activity and Substrate Specificity of Factor XIII Resulting from Different Activation Pathways

Factor XIII (FXIII) is a transglutaminase that catalyzes the formation of a calcium-dependent isopeptide bond between an acyl donor and a primary amine substrate. Plasma FXIII A2B2 is proteolytically activated by the concerted action of thrombin and low mM Ca2+ (FXIIIA∗) whereas cellular FXIII A2 is activated non-proteolytically by high mM Ca2+ concentration (FXIIIA°). Previous studies demonstrated that these activation pathways result in FXIIIA species that differ conformationally and functionally. The activities and substrate specificities of FXIIIA∗ and FXIIIA° were further examined using monodansyl cadaverine (MDC) incorporation and UV-spectrophotometric assays. In the MDC assay, reactive glutamines within fibrinogen αC (233-425) (Q237, Q328, Q366) were crosslinked by FXIII (A∗ versus A°) to MDC (amine substrate), and results were evaluated by SDS-PAGE. The assay revealed that with αC WT, Q328P (Seoul II), and Q237only, FXIIIA∗ had more transglutaminase activity than FXIIIA°. In contrast, αC E396A and αC 389stop (233-388) exhibited no substantial differences in activities between FXIIIA∗ and A°. This observation may be due to loss of αC E396 and surrounding residues from the putative FXIII anchoring site αC (389-403). From the UV-spectrophotometric assay, kinetic parameters for FXIII A∗ and FXIIIA° were determined using glutamine donor peptides (K9 (1-10), α2 AP (1-15) and α2 AP (1-15, Q4S)) crosslinked with the amine DMPDA. Km values for FXIIIA∗ were modestly lower than FXIIIA° for K9 (1-10) and α2 AP (1-15, Q4S) and higher for α2 AP (1-15). The smaller kinetic differences observed with peptides may be due to amino sequence or the need for larger FXIII substrates that better mimic physiological targets. Overall, FXIIIA activity can be a function of its activation pathway, in addition to substrate size and sequence.

Ubiquitination Modulates a Protein Energy Landscape Site-Specifically with Consequences for Proteasomal Degradation

Ubiquitination is a common posttranslational modification in which the protein ubiquitin is appended to substrate proteins to target them to the proteasome for degradation among a myriad of other cellular functions. In addition to recognition of ubiquitin, successful proteasomal degradation relies upon engagement at an unstructured region within the substrate protein. However, many proteasome clients intrinsically lack these requisite unstructured regions. Previous studies have suggested that ubiquitin modification may destabilize the target protein. However, experimental characterization of the energetics of ubiquitin-conjugated substrates with native isopeptide bonds has been limited by both purification constraints and complication due to the signal from ubiquitin itself. We have developed a generalizable protocol for purification of milligram quantities of homogenously monoubiquitinated single-lysine proteins for experimental measurement of ubiquitin-induced energetic effects. These effects span from no observable changes to dramatic destabilization and depend on the exact site of modification. Further, there exists a trend between ubiquitin-induced substrate destabilization and faster proteasomal degradation rates. We also find that polyubiquitination at destabilizing sites is sufficient to induce the requisite unstructured region required for successful proteasomal degradation. Taken together, these data establish a connection between ubiquitin-induced changes in substrate energetics and proteasomal processing. We propose that ubiquitin's varied and site-specific modulation of substrate energy landscapes plays a consequential role for substrate engagement and successful degradation at the proteasome.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

Interaction between tissue transglutaminase and amyloid-beta: Protein-protein binding versus enzymatic crosslinking

Self-interaction, chaperone binding and posttranslational modification of amyloid-beta (Aβ) is essential in the initiation and propagation of Aβ aggregation. Aggregation results in insoluble Aβ deposits characteristic of Alzheimer's disease (AD) brain lesions, i.e. senile plaques and cerebral amyloid angiopathy. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes posttranslational modifications including the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds (molecular crosslinks), and colocalizes with Aβ deposits in AD. Two independent groups recently found that apart from the induction of Aβ oligomerization, the blood-derived transglutaminase member FXIIIa forms stable protein-protein complexes with Aβ independent of the transamidation reaction. Here, we investigated whether also tTG forms rigid protein complexes with Aβ in the absence of catalytic activation. We found that both Aβ1-40 and Aβ1-42 are substrates for tTG-catalyzed crosslinking. In addition, in the absence of calcium or the presence of a peptidergic inhibitor of tTG, stable tTG-Aβ1-40 complexes were found. Interestingly, the stable complexes between tTG and Aβ1-40, were only found at ‘physiological’ concentrations of Aβ1-40. Together, our data suggest that depending on the Aβ species at hand, and on the concentration of Aβ, rigid protein-complexes are formed between tTG and Aβ1-40 without the involvement of the crosslinking reaction.

Structural Changes and Antibacterial Activity of Epsilon-poly-l-lysine in Response to pH and Phase Transition and Their Mechanisms.

ε-Poly-l-lysine (ε-PL) consists of 25-35 lysine residues which are linked by an isopeptide bond formed by dehydration condensation of α-carboxyl and ε-amino groups and has good antibacterial activity and broad-spectrum inhibition range. However, there is no clear conclusion about the structure and antibacterial mechanism of ε-PL in aqueous solution. Herein, a high purity of ε-PL was prepared using Amberlite IRC-50 ion-exchange resin. Membrane filtration and dynamic light scattering were used to study the variations of ε-PL aggregation in aqueous solution with pH value. The conformational changes and antibacterial activities of ε-PL and carbamoylated ε-PL in different water environments were studied with circular dichroism (CD) and inhibition zone. The structural changes during the spray-drying process were determined by Fourier transform infrared spectroscopy. The results indicated that the side chain amino charge played a decisive role in the ε-PL conformation and aggregation. ε-PL exhibited the properties of a β-sheet during spray drying from acidic liquids to solids. The cation enhanced the antibacterial activity of ε-PL but did not play a key role. Instead, the backbone of ε-PL might determine the mechanism of ε-PL antibacterial.

Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates.

Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.