Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

A complete morphological characterization of all life stages of the phorid fly Megaselia scalaris.

Megaselia scalaris, commonly known as the scuttle fly, is a cosmopolitan species in the family Phoridae. It is an easily cultured fly species that is an emerging model organism in the fields of genetics and developmental biology. Its affinity for carrion and its predictable life cycle makes it useful in the field of forensic science for estimating the post-mortem interval (PMI) of human remains. Cases of human myasis caused by M. scalaris have also been reported in the medical literature. Despite its ubiquitous prevalence and its relevance across multiple fields, its morphology has not been adequately characterized. Here, we report the complete morphological characterization of all lifestages of M. scalaris, ranging from egg to adult. Scanning electron microscopy has enabled us to uncover morphological features and developmental processes that have previously not been reported in the literature. Our data lays the groundwork for future genetic studies: a morphological characterization of the wild type must be performed before mutants displaying different phenotypes can be identified. In this vein, we also report the observation of a acephalic, or 'headless', adult phenotype whose study could yield insights into the process of cephalogenesis. Finally, all morphological features observed have been compiled into an 'atlas' that should be of use to all workers in the field.

Blueprinting expandable nanomaterials with standardized protein building blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures 1 . Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

A Simple Radioassay to Detect Nanoscale Membrane Disruption.

Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple 32PO43- release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic 32PO43- as a proxy. Our assay can both detect 32PO43- release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and Ω76: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional 51Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles.

A new blood-based RNA signature (R9), for monitoring effectiveness of tuberculosis treatment in a South Indian longitudinal cohort.

Tuberculosis (TB) treatment involves a multidrug regimen for six months, and until two months, it is unclear if treatment is effective. This delay can lead to the evolution of drug resistance, lung damage, disease spread, and transmission. We identify a blood-based 9-gene signature using a computational pipeline that constructs and interrogates a genome-wide transcriptome-integrated protein-interaction network. The identified signature is able to determine treatment response at week 1-2 in three independent public datasets. Signature-based R9-score correctly detected treatment response at individual timepoints (204 samples) from a newly developed South Indian longitudinal cohort involving 32 patients with pulmonary TB. These results are consistent with conventional clinical metrics and can discriminate good from poor treatment responders at week 2 (AUC 0.93(0.81-1.00)). In this work, we provide proof of concept that the R9-score can determine treatment effectiveness, making a case for designing a larger clinical study.

Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

Tumor necrosis factor-α (TNFα), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody, which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8, was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (glutamine, arginine, glutamic acid) form the core epitope. The observation was supported by bioinformatics analyses of an antigen/antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of infliximab, which is a commercially available anti-TNFα mAb.

Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistant Acinetobacter baumannii.

Drug resistance is a public health concern that threatens to undermine decades of medical progress. ESKAPE pathogens cause most nosocomial infections, and are frequently resistant to carbapenem antibiotics, usually leaving tigecycline and colistin as the last treatment options. However, increasing tigecycline resistance and colistin's nephrotoxicity severely restrict use of these antibiotics. We have designed antimicrobial peptides using a maximum common subgraph approach. Our best peptide (Ω76) displayed high efficacy against carbapenem and tigecycline-resistant Acinetobacter baumannii in mice. Mice treated with repeated sublethal doses of Ω76 displayed no signs of chronic toxicity. Sublethal Ω76 doses co-administered alongside sublethal colistin doses displayed no additive toxicity. These results indicate that Ω76 can potentially supplement or replace colistin, especially where nephrotoxicity is a concern. To our knowledge, no other existing antibiotics occupy this clinical niche. Mechanistically, Ω76 adopts an α-helical structure in membranes, causing rapid membrane disruption, leakage, and bacterial death.

Design of a heme-binding peptide motif adopting a ß-hairpin conformation.

Heme-binding proteins constitute a large family of catalytic and transport proteins. Their widespread presence as globins and as essential oxygen and electron transporters, along with their diverse enzymatic functions, have made them targets for protein design. Most previously reported designs involved the use of α-helical scaffolds, and natural peptides also exhibit a strong preference for these scaffolds. However, the reason for this preference is not well-understood, in part because alternative protein designs, such as those with ß-sheets or hairpins, are challenging to perform. Here, we report the computational design and experimental validation of a water-soluble heme-binding peptide, Pincer-1, composed of predominantly ß-scaffold secondary structures. Such heme-binding proteins are rarely observed in nature, and by designing such a scaffold, we simultaneously increase the known fold space of heme-binding proteins and expand the limits of computational design methods. For a ß-scaffold, two tryptophan zipper ß-hairpins sandwiching a heme molecule were linked through an N-terminal cysteine disulfide bond. ß-Hairpin orientations and residue selection were performed computationally. Heme binding was confirmed through absorbance experiments and surface plasmon resonance experiments (KD = 730 ± 160 nm). CD and NMR experiments validated the ß-hairpin topology of the designed peptide. Our results indicate that a helical scaffold is not essential for heme binding and reveal the first designed water-soluble, heme-binding ß-hairpin peptide. This peptide could help expand the search for and design space to cytoplasmic heme-binding proteins.

N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori.

Many bacterial genomes exclusively display an N4-methyl cytosine base (m4C), whose physiological significance is not yet clear. Helicobacter pylori is a carcinogenic bacterium and the leading cause of gastric cancer in humans. Helicobacter pylori strain 26695 harbors a single m4C cytosine methyltransferase, M2.HpyAII which recognizes 5' TCTTC 3' sequence and methylates the first cytosine residue. To understand the role of m4C modification, M2.hpyAII deletion strain was constructed. Deletion strain displayed lower adherence to host AGS cells and reduced potential to induce inflammation and apoptosis. M2.hpyAII gene deletion strain exhibited reduced capacity for natural transformation, which was rescued in the complemented strain carrying an active copy of M2.hpyAII gene in the genome. Genome-wide gene expression and proteomic analysis were carried out to discern the possible reasons behind the altered phenotype of the M2.hpyAII gene deletion strain. Upon the loss of m4C modification a total of 102 genes belonging to virulence, ribosome assembly and cellular components were differentially expressed. The present study adds a functional role for the presence of m4C modification in H. pylori and provides the first evidence that m4C signal acts as a global epigenetic regulator in H. pylori.

Computational antimicrobial peptide design and evaluation against multidrug-resistant clinical isolates of bacteria.

There is a pressing need for new therapeutics to combat multidrug- and carbapenem-resistant bacterial pathogens. This challenge prompted us to use a long short-term memory (LSTM) language model to understand the underlying grammar, i.e. the arrangement and frequencies of amino acid residues, in known antimicrobial peptide sequences. According to the output of our LSTM network, we synthesized 10 peptides and tested them against known bacterial pathogens. All of these peptides displayed broad-spectrum antimicrobial activity, validating our LSTM-based peptide design approach. Our two most effective antimicrobial peptides displayed activity against multidrug-resistant clinical isolates of Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and coagulase-negative staphylococci strains. High activity against extended-spectrum ß-lactamase, methicillin-resistant S. aureus, and carbapenem-resistant strains was also observed. Our peptides selectively interacted with and disrupted bacterial cell membranes and caused secondary gene-regulatory effects. Initial structural characterization revealed that our most effective peptide appeared to be well folded. We conclude that our LSTM-based peptide design approach appears to have correctly deciphered the underlying grammar of antimicrobial peptide sequences, as demonstrated by the experimentally observed efficacy of our designed peptides.

Design of symmetric TIM barrel proteins from first principles.

BACKGROUND: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/ß)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. METHODS: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. RESULTS: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. CONCLUSIONS: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

PLIC: protein-ligand interaction clusters.

Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein- ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of ∼68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of site attributes are provided as a relational database-protein-ligand interaction clusters (PLIC). Database URL: http://proline.biochem.iisc.ernet.in/PLIC.

ABS-Scan: In silico alanine scanning mutagenesis for binding site residues in protein-ligand complex.

Most physiological processes in living systems are fundamentally regulated by protein-ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are calculated by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide a ranked list of residues to the user in order to obtain loss-of-function mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.