Polypeptide Research Articles

A proteolytic AAA+ machine poised to unfold protein substrates

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP’s ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX’s ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.

Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.

Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain. Here, we evaluate the increase in sequence coverage obtained by combining activated-ion ETD (AI-ETD) and proton transfer charge reduction (PTCR) in the analysis of 66 kDa human serum albumin, which holds 17 disulfide bridges. We also describe the combination of AI-ETD with supplemental postactivation of the ETD reaction products via higher-energy collisional dissociation─a hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further improvement compared to AI-ETD in both the global number of cleaved backbone bonds and the number of ruptured backbone bonds from disulfide-protected regions. Our results also demonstrate that the full potential of AI-ETD and AI-EThcD is unveiled only when combined with PTCR: reduction in overlap of ion signals leads to a sequence coverage as high as 39% in a single experiment, highlighting the relevance of spectral simplification in top-down mass spectrometry of large proteins.

SOP-MULTI: A Self-Organized Polymer-Based Coarse-Grained Model for Multidomain and Intrinsically Disordered Proteins with Conformation Ensemble Consistent with Experimental Scattering Data.

Multidomain proteins with long flexible linkers and full-length intrinsically disordered proteins (IDPs) are best defined as an ensemble of conformations rather than a single structure. Determining high-resolution ensemble structures of such proteins poses various challenges by using tools from experimental structural biophysics. Integrative approaches combining available low-resolution ensemble-averaged experimental data and in silico biomolecular reconstructions are now often used for the purpose. However, extensive Boltzmann weighted conformation sampling for large proteins, especially for ones where both the folded and disordered domains exist in the same polypeptide chain, remains a challenge. In this work, we present a 2-site per amino-acid resolution SOP-MULTI force field for simulating coarse-grained models of multidomain proteins. SOP-MULTI combines two well-established self-organized polymer models─: (i) SOP-SC models for folded systems and (ii) SOP-IDP for IDPs. For the SOP-MULTI, we introduce cross-interaction terms between the beads belonging to the folded and disordered regions to generate conformation ensembles for full-length multidomain proteins such as hnRNP A1, TDP-43, G3BP1, hGHR-ECD, TIA1, HIV-1 Gag, polyubiquitin, and FUS. When back-mapped to all-atom resolution, SOP-MULTI trajectories faithfully recapitulate the scattering data over the range of the reciprocal space. We also show that individual folded domains preserve native contacts with respect to solved folded structures, and root-mean-square fluctuations of residues in folded domains match those obtained from all-atom molecular dynamics simulation trajectories of the same folded systems. SOP-MULTI force field is made available as a LAMMPS-compatible user package along with setup codes for generating the required files for any full-length protein with folded and disordered regions.

Development and Optimization of a Redox Enzyme-Based Fluorescence Biosensor for the Identification of MsrB1 Inhibitors

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1’s role during inflammation and eventually controlling inflammation in therapeutic approaches.

Biosynthesis of Nonribosomal Peptides Chitinimides Reveal a Special Type of Thioesterase Domains.

Non-ribosomal peptide synthetases (NRPSs) and their tailored enzymes have diverse biological functions. In this study, we investigated the biosynthesis and function of chitinimides, which belong to the non-ribosomal peptide (NRP) subfamily featuring a pyrrolidine-containing part (X part) connected to the polypeptide chain via an ester bond. A conserved gene cassette, chmHIJK, is responsible for oxyacylation of the pyrrolidine moiety in the X part. The thioesterase (TE) domain of ChmC (ChmC-TE) catalyzes transesterification reactions with a free X part or methanol as a nucleophilic reagent to form different chitinimides. The crucial amino acid residues in the ChmC-TE domains responsible for the specific recognition of the X part were identified, and they were conserved in all the biosynthetic pathways of this NRP subfamily to form a signature motif, YNHNR, suggesting a special type of TE domain in NRPSs. Chitinimides demonstrate the biological function of promoting the swarming ability of the native producer. This study provides deep insights into the biosynthesis of this special NRP subfamily, and shows that the special TE domain could be used to generate diverse NRPs by combinatorial biosynthesis.

In Vitro Inhibitory Mechanism of Polyphenol Extracts from Multi-Frequency Power Ultrasound-Pretreated Rose Flower Against α-Glucosidase.

This paper explored the in vitro inhibitory mechanism of polyphenol-rich rose extracts (REs) from an edible rose flower against α-glucosidase using multispectral and molecular docking techniques. Results showed that REs had an inhibitory effect on α-Glu activity (IC50 of 1.96 μg/mL); specifically, the samples pretreated by tri-frequency ultrasound (20/40/60 kHz) exhibited a significantly (p < 0.05) stronger inhibitory effect on α-Glu activity with an IC50 of 1.33 μg/mL. The Lineweaver-Burk assay indicated that REs were mixed-type inhibitors and could statically quench the endogenous fluorescence of α-Glu. REs increased the chance of polypeptide chain misfolding by altering the microenvironment around tryptophan and tyrosine residues and disrupting the natural conformation of the enzyme. Molecular docking results showed that polyhydroxy phenolics had a high fit to the active site of α-Glu, so REs with high polymerization and numerous phenolic hydroxyl groups had a stronger inhibitory effect. Therefore, this study provides new insights into polyphenol-rich REs as potential α-glucosidase inhibitors.

EuDockScore: Euclidean graph neural networks for scoring protein-protein interfaces.

Protein-protein interactions are essential for a variety of biological phenomena including mediating bio-chemical reactions, cell signaling, and the immune response. Proteins seek to form interfaces which reduce overall system energy. Although determination of single polypeptide chain protein structures has been revolutionized by deep learning techniques, complex prediction has still not been perfected. Additionally, experimentally determining structures is incredibly resource and time expensive. An alternative is the technique of computational docking, which takes the solved individual structures of proteins to produce candidate interfaces (decoys). Decoys are then scored using a mathematical function that assess the quality of the system, know as a scoring functions. Beyond docking, scoring functions are a critical component of assessing structures produced by many protein generative models. Scoring models are also used as a final filtering in many generative deep learning models including those that generate antibody binders, and those which perform docking. In this work we present improved scoring functions for protein-protein interactions which utilizes cutting-edge euclidean graph neural network architectures, to assess protein-protein interfaces. These euclidean docking score models are known as EuDockScore, and EuDockScore-Ab with the latter being antibody-antigen dock specific. Finally, we provided EuDockScore-AFM a model trained on antibody-antigen outputs from AlphaFold-Multimer which proves useful in re-ranking large numbers of AlphaFold-Multimer outputs. The code for these models is available at https://gitlab.com/mcfeemat/eudockscore.

Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P.

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

Generation and Selection of Phage Display Antibody Libraries in Fab Format.

Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as "fragment antigen binding" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s−1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB.

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s-1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Key structural role of a conserved cis-proline revealed by the P285S variant of soybean serine hydroxymethyltransferase 8.

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 5'-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site. Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8. P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode. We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution. Crystal structures at 1.9 - 2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an a-helix and shifts a turn region into the active site. This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ~180° with concomitant loss of conserved enzyme-PLP interactions. A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ~10 residues. These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function.

Cryo-EM analysis of Pseudomonas phage Pa193 structural components

The World Health Organization has designated Pseudomonas aeruginosa as a critical pathogen for the development of new antimicrobials. Bacterial viruses, or bacteriophages, have been used in various clinical settings, commonly called phage therapy, to address this growing public health crisis. Here, we describe a high-resolution structural atlas of a therapeutic, contractile-tailed Pseudomonas phage, Pa193. We used bioinformatics, proteomics, and cryogenic electron microscopy single particle analysis to identify, annotate, and build atomic models for 21 distinct structural polypeptide chains forming the icosahedral capsid, neck, contractile tail, and baseplate. We identified a putative scaffolding protein stabilizing the interior of the capsid 5-fold vertex. We also visualized a large portion of Pa193 ~ 500 Å long tail fibers and resolved the interface between the baseplate and tail fibers. The work presented here provides a framework to support a better understanding of phages as biomedicines for phage therapy and inform engineering opportunities.

Diagnostic Utility of Copeptin in Pediatric Patients with Polyuria-Polydipsia Syndrome: A Systematic Review and Meta-Analysis.

Pediatric patients with polyuria polydipsia syndrome (PPS) represent a diagnostic challenge for clinicians because of the technical difficulties in performing the gold standard water deprivation test (WDT). Copeptin, a stable biomarker representing the C-terminal portion of the polypeptide chain of the antidiuretic hormone, is a reliable diagnostic tool. To assess the diagnostic accuracy of baseline copeptin dosing, arginine/hypertonic saline copeptin stimulation tests, and WDT. This study aimed to establish the diagnostic utility of copeptin in pediatric patients by distinguishing between central diabetes insipidus, nephrogenic diabetes insipidus, and primary polydipsia. Comparative and non-comparative primary studies published between January 2018 and August 2024 focusing on children were searched and included in PubMed, Cochrane Library, Web of Science, ScienceDirect, Scopus, and Google Scholar. The QUADAS-2 tool was used to assess the risk of bias and applicability. Meta-analyses used fixed effects models because of low heterogeneity and the HSROC model. Eleven studies were included with an overall low bias and no significant applicability concerns. The mean pooled sensitivity = 0.98 (95% CI: 0.936-1.025), pooled specificity = 0.947 (95% CI: 0.920-0.973), and AUC = 0.972 (95% CI: 0.952-0.992), indicating excellent diagnostic accuracy. Stimulation methods for copeptin dosing represent an effective and less invasive diagnostic test for children with PPS, and future development of standard copeptin testing protocols is needed.

Distance-Dependent Tryptophan-Induced Quenching of Thioflavin T Defines the Amyloid Core Architecture.

Thioflavin T (ThT) is widely employed as a fluorogenic marker for amyloid formation. ThT fluorescence is utilized to detect amyloid fibrils as well as to follow aggregation kinetics. Here, we make a unique case to demonstrate that site-specific tryptophan-induced fluorescence quenching of ThT bound to the α-synuclein amyloid can define the central amyloid core. We show that distance-dependent quenching of amyloid-bound ThT by site-specifically incorporated tryptophan maps the proximal and distal locations of the polypeptide chain within amyloid fibrils. Our studies indicate that tryptophan-induced fluorescence quenching is dominated by the static quenching mechanism. Our findings underscore the utility of site-specific amino acid-based quenching of ThT fluorescence to characterize the core architecture of amyloid derived from a wide range of proteins.

Tumor microenvironment-activated polypeptide nanoparticles for oncolytic immunotherapy

Cationic oncolytic polypeptides have gained increasing attention owing to their ability to directly lyse cancer cells and activate potent antitumor immunity. However, the low tumor cell selectivity and inherent toxicity induced by positive charges of oncolytic polypeptides hinder their systemic application. Herein, a tumor microenvironment-responsive nanoparticle (DNP) is developed by the self-assembly of a cationic oncolytic polypeptide (PLP) with a pH-sensitive anionic polypeptide via electrostatic interactions. After the formation of DNP, the positive charges of PLP are shielded. DNPs can keep stable in physiological conditions (pH 7.4) but respond to acidic tumor microenvironment (pH 6.8) to release oncolytic PLP. As a result, DNPs evoke potent immunogenic cell death by disrupting cell membranes, damaging mitochondria and increasing intracellular levels of reactive oxygen species. In vivo results indicate that DNPs significantly improve the biocompatibility of PLP, and inhibit tumor growth, recurrence and metastasis by direct oncolysis and activation of antitumor immune responses. In summary, these results indicate that pH-sensitive DNPs represent a prospective strategy to improve the tumor selectivity and biosafety of cationic polymers for oncolytic immunotherapy.

B-204 A Real-time Probe-based PCR Assay for Detection of Pneumocystis Pneumonia

Abstract Background Pneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and worldwide distribution that continues to be one of the major opportunistic pathogens causing severe Pneumocystis pneumonia (PcP) in individuals with acquired immune deficiency syndrome (AIDS) and patients with immunosuppression due to other causes. Widespread prophylaxis and treatment for P. jirovecii with sulfa drugs have decreased the incidence of PcP, but concerns have been raised about the possible emergence of P. jirovecii isolates resistant to these drugs. Point mutations in this gene have been associated with prior exposure to sulfa drugs in other microorganisms, the polymorphic positions 165 and 171, which lead to an amino acid change at positions 55 (Thr55Ala) and 57 (Pro57Ser) of the polypeptide chain, respectively. Fluorescent PCR technology can detect the nucleic acid fragments of P. jirovecii in patients' serum, alveolar perfusion fluid (BALF) and tissue samples, and further identify drug-resistant mutations, which is of great significance for early clinical and accurate initiation of antifungal therapy. MycoMDx Pneumocystis Jirovecii PCR Assay is the use of PCR-fluorescence probe method, combined with other methods to achieve the auxiliary diagnosis of P. jirovecii infection and resistance to sulfonamides (Fig. 1). Methods In this study, we did a performance evaluation of the MycoMDx Pneumocystis Jirovecii PCR Assay (CE-approved), including diagnostic sensitivity and specificity, the limit of detection (LoD), precision and stability. The above PCR assay reagents were developed and produced by Dynamiker Biotechnology (Tianjin) Co., Ltd. Results The sensitivity and specificity of the PCR assay reagents were above 90%, the LoD was 1000 copies/mL, and the precision and stability were in accordance with the requirements (CV≤5%). Conclusions The MycoMDx Pneumocystis Jirovecii PCR Assay has great clinical value by providing a rapid and reliable test to aid in early diagnosis.

Enhancing protein aggregation prediction: a unified analysis leveraging graph convolutional networks and active learning.

Protein aggregation (PA) is a critical phenomenon associated with Alzheimer's and Parkinson's disease. Recent studies have suggested that factors like aggregation-prone regions (APRs) and β-strand interactions are crucial in understanding such behavior. While experimental methods have provided valuable insights, there has been a shift towards computational strategies, particularly machine learning, for their efficacy and speed. The challenge, however, lies in effectively incorporating structural information into these models. This study constructs a Graph Convolutional Network (GCN) to predict PA scores with the expanded and refined Protein Data Bank (PDB) and AlphaFold2.0 dataset. We employed AGGRESCAN3D 2.0 to calculate PA propensity and to enhance the dataset, we systematically separated multi polypeptide chains within PDB data into single polypeptide chains, removing redundancy. This effort resulted in a dataset comprising 302 032 unique PDB entries. Subsequently, we compared sequence similarity and obtained 22 774 Homo sapiens data from AlphaFold2.0. Using this expanded and refined dataset, the trained GCN model for PA prediction achieves a remarkable coefficient of determination (R 2) score of 0.9849 and a low mean absolute error (MAE) of 0.0381. Furthermore, the efficacy of the active learning process was demonstrated through its rapid identification of proteins with high PA propensity. Consequently, the active learning approach achieved an MAE of 0.0291 in expected improvement, surpassing other methods. It identified 99% of the target proteins by exploring merely 29% of the entire search space. This improved GCN model demonstrates promise in selecting proteins susceptible to PA, advancing protein science. This work contributes to the development of efficient computational tools for PA prediction, with potential applications in disease diagnosis and therapy.

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Insight into Protein Engineering: From In silico Modelling to In vitro Synthesis.

Protein engineering alters the polypeptide chain to obtain a novel protein with improved functional properties. This field constantly evolves with advanced in silico tools and techniques to design novel proteins and peptides. Rational incorporating mutations, unnatural amino acids, and post-translational modifications increases the applications of engineered proteins and peptides. It aids in developing drugs with maximum efficacy and minimum side effects. Currently, the engineering of peptides is gaining attention due to their high stability, binding specificity, less immunogenic, and reduced toxicity properties. Engineered peptides are potent candidates for drug development due to their high specificity and low cost of production compared with other biologics, including proteins and antibodies. Therefore, understanding the current perception of designing and engineering peptides with the help of currently available in silico tools is crucial. This review extensively studies various in silico tools available for protein engineering in the prospect of designing peptides as therapeutics, followed by in vitro aspects. Moreover, a discussion on the chemical synthesis and purification of peptides, a case study, and challenges are also incorporated.