De Novo Design of Immunoglobulin-like Domains

Antibodies and antibody derivatives such as nanobodies contain immunoglobulin-like (Ig) [beta]-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here we develop design rules for the central feature of the Ig fold architecture - the non-local cross-[beta]; structure connecting the two [beta]-sheets - and use these to de novo design highly stable seven-stranded Ig domains, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of a new class of antibody-like scaffolds with tailored structures and superior biophysical properties.

Structural Modelling and in-silico characterization of a Novel Thermophilic β-amylase from Clostridium thermosulfuregenes

β-amylase is a hydrolytic enzyme that is involved in breaking down starch and producing energy. Since the discovery of β-amylase, it has been applied in various applications especially in the food industry. In this study, a novel β-amylase from Clostridium thermosuluregen, a thermophilic anaerobic bacterium that ferments its extracellular emulsion to ethanol at 62 ℃ was modelled and studied using bioinformatics tools and compared with B. cereus β-amylases that functions at mesophilic conditions. The results showed that the overall structural conformations, secondary structures, and important residues involved in active and binding sites were identified in both proteins. The results revealed that the modelled β-amylase of C. thermosulfuregen is very similar with respect to the global conformation, location of active and binding sites. Both proteins showed identical structural domains with the thermophilic variant possessing a high percentage of hydrophobic amino acid residues, polar amino acid residues, and differences in secondary composition such as loops and beta sheets as the potential evolutionary thermal adaptations that make it stable enzyme that functions up to 70 ℃. The results suggest that the thermal stability are not dependent on one single unique mechanism and may use one or a combination of the mechanisms to sustain its structural conformation at a higher operating temperature. Overall, considering the common properties of this modelled protein with the β-amylase of B. cereus, it can be assumed that if the β-amylase of C. thermosulfuregen were expressed in-vitro, it would produce a stable protein that possesses the hydrolysis function for C. thermosulfuregen to break down the starch and sugar formation.

Modalities of Protein Denaturation and Nature of Denaturants

Denaturation of protein is a biological phenomenon in which a protein loses its native shape due to the breaking or disruption of weak chemical bonds and interactions which makes the protein biologically inactive. It is the process where properly folded proteins formed under physiological conditions is transformed to an unfolded protein under non-physiological conditions. The process of denaturation of proteins can occur under different physiological and chemical conditions. Denaturation can be reversible or irreversible. Denaturation mostly takes places when the protein is subjected under external elements like inorganic solutes, organic solvents, acids or bases, and by heat or irradiations. The denaturing agents or denaturants widely used in protein folding or unfolding experiments are urea and guanidinium chloride (GdmCl). In denaturation, the alpha-helix structure and beta sheets structure of the native protein are disrupted and unfolds it into any random shape. We can also say that denaturation occurs due to the disruption of bonding interactions which are responsible for secondary structure and the tertiary structure of the proteins.

Structurally distinct polymorphs of Tau aggregates revealed by nanoscale infrared spectroscopy

Aggregation of the tau protein plays a central role in several neurodegenerative diseases collectively known as tauopathies, including Alzheimers and Parkinsons disease. Tau misfolds into fibrillar beta sheet structures that constitute the paired helical filaments found in Neurofibrillary tangles. It is known that there can be significant structural heterogeneities in tau aggregates associated with different diseases. However, while structures of mature fibrils have been studied, the structural distributions in early stage tau aggregates is not well understood. In the present study, we use AFM-IR to investigate nanoscale spectra of individual tau fibrils at different stages of aggregation and demonstrate the presence of multiple fibrillar polymorphs that exhibit different secondary structures. We further show that mature fibrils contain significant amounts of antiparallel beta sheets. Our results are the very first application of nanoscale infrared spectroscopy to tau aggregates and underscore the promise of spatially resolved infrared spectroscopy for investigating protein aggregation.

Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

On the border of the amyloidogenic sequences: prefix analysis of the parallel beta sheets in the PDB_Amyloid collection

The Protein Data Bank (PDB) today contains more than 174,000 entries with the 3-dimensional structures of biological macromolecules. Using the rich resources of this repository, it is possible identifying subsets with specific, interesting properties for different applications. Our research group prepared an automatically updated list of amyloid- and probably amyloidogenic molecules, the PDB_Amyloid collection, which is freely available at the address http://pitgroup.org/amyloid. This resource applies exclusively the geometric properties of the steric structures for identifying amyloids. In the present contribution, we analyze the starting (i.e., prefix) subsequences of the characteristic, parallel beta-sheets of the structures in the PDB_Amyloid collection, and identify further appearances of these length-5 prefix subsequences in the whole PDB data set. We have identified this way numerous proteins, whose normal or irregular functions involve amyloid formation, structural misfolding, or anti-coagulant properties, simply by containing these prefixes: including the T-cell receptor (TCR), bound with the major histocompatibility complexes MHC-1 and MHC-2; the p53 tumor suppressor protein; a mycobacterial RNA polymerase transcription initialization complex; the human bridging integrator protein BIN-1; and the tick anti-coagulant peptide TAP.

THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain

The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp—DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

Cytochrome (CYP) enzymes catalyze the metabolic reactions of endogenous and exogenous compounds. The superfamily of enzymes is found across many organisms, regardless of type, except for plants. Information was gathered about CYP2D enzymes through protein sequences of humans and other organisms. The secondary structure was predicted using the SOPMA. The structural and functional study of human CYP2D was conducted using ProtParam, SOPMA, Predotar 1.03, SignalP, TMHMM 2.0, and ExPASy. Most animals shared five central motifs according to motif analysis results. The tertiary structure of human CYP2D, as well as other animal species, was predicted by Phyre2. Human CYP2D proteins are heavily conserved across organisms, according to the findings. This indicates that they are descended from a single ancestor. They calculate the ratio of alpha-helices to extended strands to beta sheets to random coils. Most of the enzymes are alpha-helix, but small amounts of the random coil were also found. The data were obtained to provide us with a better understanding of mammalian proteins’ functions and evolutionary relationships.

Silk-based antimicrobial peptide mixed with recombinant spidroin creates functionalized spider silk

Surgical site infection (SSI) from sutures is a global health emergency because of the antibiotic crisis. Methicillin-resistant S. aureus and other emerging strains are difficult to treat with antibiotics, so drug-free sutures with antimicrobial properties are a solution. Functionalized spider silk protein (spidroin) is a candidate for its extraordinary strength because it has a large repetitive region (150Rep) that forms crosslinked beta-sheets. The antimicrobial peptide HNP-1 can be connected to recombinant spidroin to create antimicrobial silk. Ni-NTA purified 2Rep-HNP1 fusion protein was mixed with recombinant NT2RepCT spidroin at 1:25, 1:20, 1:10 ratios, and spun into silk fibers by syringe-pumping protein into a 100% isopropanol bath. Beta-sheet crosslinking of the identical 2Rep regions tagged the 2Rep-HNP1 permanently onto the resultant silk. Silk showed no sign of degradation in an autoclave, PBS, or EtOH. The tagged 2Rep-HNP1 retained broad-spectrum antimicrobial activity >90% against S. aureus and E. coli as measured by log reduction and radial diffusion assay. Furthermore, a modified expression protocol increased protein yield of NT2RepCT 2.8-fold, and variable testing of the spinning process demonstrated the industrial viability of silk production. We present a promising suture alternative in antimicrobial recombinant spider silk.

THAP9 transposase cleaves DNA via conserved acidic residues in an RNaseH-like domain

Background: The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons.Results: In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, has led to the identification of candidate Ds and Es in the RNaseH fold that can be a part of the catalytic triad in hTHAP9.Conclusions: Many DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9’s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterised transposon-derived gene in the human genome.