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Glutamate transporters play key roles in nervous physiology by modulating excitatory neurotransmitter levels, when malfunctioning, involving in a wide range of neurological and physiological disorders. However, integral transmembrane proteins including the glutamate transporters remain notoriously difficult to study, due to their localization within the cell membrane. Here we present the structural bioinformatics studies of glutamate transporters and their water-soluble variants generated through QTY-code, a protein design strategy based on systematic amino acid substitutions. These include 2 structures determined by X-ray crystallography, cryo-EM, and 6 predicted by AlphaFold2, and their predicted water-soluble QTY variants. In the native structures of glutamate transporters, transmembrane helices contain hydrophobic amino acids such as leucine (L), isoleucine (I), and phenylalanine (F). To design water-soluble variants, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, namely glutamine (Q), threonine (T) and tyrosine (Y). The QTY variants exhibited water-solubility, with four having identical isoelectric focusing points (pI) and the other four having very similar pI. We present the superposed structures of the native glutamate transporters and their water-soluble QTY variants. The superposed structures displayed remarkable similarity with RMSD 0.528Å-2.456Å, despite significant protein transmembrane sequence differences (41.1%—>53.8%). Additionally, we examined the differences of hydrophobicity patches between the native glutamate transporters and their QTY variants. Upon closer inspection, we discovered multiple natural variations of L->Q, I->T, F->Y and Q->L, T->I, Y->F in these transporters. Some of these natural variations were benign and the remaining were reported in specific neurological disorders. We further investigated the characteristics of hydrophobic to hydrophilic substitutions in glutamate transporters, utilizing variant analysis and evolutionary profiling. Our structural bioinformatics studies not only provided insight into the differences between the hydrophobic helices and hydrophilic helices in the glutamate transporters, but they are also expected to stimulate further study of other water-soluble transmembrane proteins. [ABSTRACT FROM AUTHOR]

Brought about by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease (COVID-19) pandemic has resulted in large numbers of worldwide deaths and cases. Several SARS-CoV-2 variants have evolved, and Omicron (B.1.1.529) was one of the important variants of concern. It gets inside human cells by using its S1 subunit's receptor-binding domain (SARS-CoV-2-RBD) to bind to Angiotensin-converting enzyme 2 receptor's peptidase domain (ACE2-PD). Using peptides to inhibit binding interactions (BIs) between ACE2-PD and SARS-CoV-2-RBD is one of promising COVID-19 therapies. Employing computational protein design (CPD) as well as molecular dynamics (MD), this study used ACE2-PD's α1 helix to generate novel 25-mer peptide binders (SPB25) of Omicron RBD that have predicted binding affinities (ΔGbind (MM‑GBSA)) better than ACE2 by increasing favorable BIs between SPB25 and the conserved residues of RBD. Results from MD and the MM-GBSA method identified two best designed peptides (SPB25T7L/K11A and SPB25T7L/K11L with ΔGbind (MM‑GBSA) of −92.4 ± 0.4 and −95.7 ± 0.5 kcal/mol, respectively) that have better ΔGbind (MM‑GBSA) to Omicron RBD than ACE2 (−87.9 ± 0.5 kcal/mol) and SPB25 (−71.6 ± 0.5 kcal/mol). Additionally, they were predicted to have slightly higher stabilities, based on their percent helicities in water, than SBP1 (the experimentally proven inhibitor of SARS-CoV-2-RBD). Our two best designed SPB25s are promising candidates as omicron variant inhibitors. [ABSTRACT FROM AUTHOR]

Biomolecular self-assembly is an emerging bottom-up approach for the synthesis of novel nanomaterials. DNA and viruses have both been used to create scaffolds but the former lacks chemical diversity and the latter lack spatial control. To date, the use of protein scaffolds to template materials on the nanoscale has focused on amyloidogenic proteins that are known to form fibrils or two-protein systems where a second protein acts as a cross-linker. We previously developed a unique approach for self-assembly of nanomaterials based on engineering β-solenoid proteins (BSPs) to polymerize into micrometer-length fibrils. BSPs have highly regular geometries, tunable lengths, and flat surfaces that are amenable to engineering and functionalization. Here, we present a newly engineered BSP based on the antifreeze protein of the beetle Rhagium inquisitor (RiAFP-m9), which polymerizes into stable fibrils under benign conditions. Gold nanoparticles were used to functionalize the RiAFP-m9 fibrils as well as those assembled from the previously described SBAFP-m1 protein. Cysteines incorporated into the sequences provide site-specific gold attachment. Additionally, silver was deposited on the gold-labelled fibrils by electroless plating to create nanowires. These results bolster prospects for programable self-assembly of BSPs to create scaffolds for functional nanomaterials.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker's yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions. [ABSTRACT FROM AUTHOR]

Protein tandem repeats (TRs) are motifs comprised of near-identical contiguous sequence duplications. They are found in approximately 14% of all proteins and are implicated in diverse biological functions facilitating both structured and disordered protein-protein and protein-DNA interactions. These functionalities make protein TR domains an attractive component for the modular design of protein constructs. However, the repetitive nature of DNA sequences encoding TR motifs complicates their synthesis and mutagenesis by traditional molecular biology workflows commonly employed by protein engineers and synthetic biologists. To address this challenge, we developed a computational protocol to significantly reduce the complementarity of DNA sequences encoding TRs called TReSR (for Tandem Repeat DNA Sequence Redesign). The utility of TReSR was demonstrated by constructing a novel constitutive repressor synthesized by duplicating the LacI DNA binding domain into a single-chain TR construct by assembly PCR. Repressor function was evaluated by expression of a fluorescent reporter delivered on a single plasmid encoding a three-component genetic circuit. The successful application of TReSR to construct a novel TR-containing repressor with a DNA sequence that is amenable to PCR-based construction and manipulation will enable the incorporation of a wide range of TR-containing proteins for protein engineering and synthetic biology applications. [ABSTRACT FROM AUTHOR]

Protein thermostability engineering is a powerful tool to improve resistance of proteins against high temperatures and thereafter broaden their applications. For efficient protein thermostability engineering, different thermostability-classified data sources including sequences and 3D structures are needed for different protein families. However, no data source is available providing such data easily. It is the first release of ProtDataTherm database for analysis and engineering of protein thermostability which contains more than 14 million protein sequences categorized based on their thermal stability and protein family. This database contains data needed for better understanding protein thermostability and stability engineering. Providing categorized protein sequences and structures as psychrophilic, mesophilic and thermophilic makes this database useful for the development of new tools in protein stability prediction. This database is available at http://profiles.bs.ipm.ir/softwares/protdatatherm. As a proof of concept, the thermostability that improves mutations were suggested for one sample protein belonging to one of protein families with more than 20 mesophilic and thermophilic sequences and with known experimentally measured ΔT of mutations available within ProTherm database.

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies.

Protein thermostability engineering is a powerful tool to improve resistance of proteins against high temperatures and thereafter broaden their applications. For efficient protein thermostability engineering, different thermostability-classified data sources including sequences and 3D structures are needed for different protein families. However, no data source is available providing such data easily. It is the first release of ProtDataTherm database for analysis and engineering of protein thermostability which contains more than 14 million protein sequences categorized based on their thermal stability and protein family. This database contains data needed for better understanding protein thermostability and stability engineering. Providing categorized protein sequences and structures as psychrophilic, mesophilic and thermophilic makes this database useful for the development of new tools in protein stability prediction. This database is available at http://profiles.bs.ipm.ir/softwares/protdatatherm. As a proof of concept, the thermostability that improves mutations were suggested for one sample protein belonging to one of protein families with more than 20 mesophilic and thermophilic sequences and with known experimentally measured ΔT of mutations available within ProTherm database.

In vitro protein display methods can access extensive libraries (e.g., 1012–1014) and play an increasingly important role in protein engineering. However, the preparation of large libraries remains a laborious and time-consuming process. Here we report an efficient one-pot ligation & elongation (L&E) method for sizeable synthetic library preparation free of PCR amplification or any purification steps. As a proof of concept, we constructed an ankyrin repeat protein templated synthetic library with 1011 variants in 150 μL volume. The entire process from the oligos to DNA template ready for transcription is linearly scalable and took merely 90 minutes. We believe this L&E method can significantly simplify the preparation of synthetic libraries and accelerate in vitro protein display experiments. [ABSTRACT FROM AUTHOR]

One of the most straightforward and commonly used chemical modifications of proteins is to react surface amino groups (lysine residues) with activated esters. This chemistry has been used to generate protein-polymer conjugates, many of which are now approved therapeutics. Similar conjugates have also been generated by reacting activated ester atom transfer polymerization initiators with lysine residues to create biomacromolecular initiators for polymerization reactions. The reaction between activated esters and lysine amino groups is rapid and has been consistently described in almost every publication on the topic as a "random reaction". A random reaction implies that every accessible lysine amino group on a protein molecule is equally reactive, and as a result, that the reaction is indiscriminate. Nonetheless, the literature contradicts itself by also suggesting that some lysine amino groups are more reactive than others (as a function of pKa, surface accessibility, temperature, and local environment). If the latter assumption is correct, then the outcome of these reactions cannot be random at all, and we should be able to predict the outcome from the structure of the protein. Predicting the non-random outcome of a reaction between surface lysines and reactive esters could transform the speed at which active bioconjugates can be developed and engineered. Herein, we describe a robust integrated tool that predicts the activated ester reactivity of every lysine in a protein, thereby allowing us to calculate the non-random sequence of reaction as a function of reaction conditions. Specifically, we have predicted the intrinsic reactivity of each lysine in multiple proteins with a bromine-functionalised N-hydroxysuccinimide initiator molecule. We have also shown that the model applied to PEGylation. The rules-based analysis has been coupled together in a single Python program that can bypass tedious trial and error experiments usually needed in protein-polymer conjugate design and synthesis. [ABSTRACT FROM AUTHOR]

Rapid advancements in automated genomic technologies have uncovered many unique findings about the turtle genome and its associated features including olfactory gene expansions and duplications of toll-like receptors. However, despite the advent of large-scale sequencing, assembly, and annotation, about 40–50% of genes in eukaryotic genomes are left without functional annotation, severely limiting our knowledge of the biological information of genes. Additionally, these automated processes are prone to errors since draft genomes consist of several disconnected scaffolds whose order is unknown; erroneous draft assemblies may also be contaminated with foreign sequences and propagate to cause errors in annotation. Many of these automated annotations are thus incomplete and inaccurate, highlighting the need for functional annotation to link gene sequences to biological identity. In this study, we have functionally annotated two genes of the red-bellied short-neck turtle (Emydura subglobosa), a member of the relatively understudied pleurodire lineage of turtles. We improved upon initial ab initio gene predictions through homology-based evidence and generated refined consensus gene models. Through functional, localization, and structural analyses of the predicted proteins, we discovered conserved putative genes encoding mitochondrial proteins that play a role in C21-steroid hormone biosynthetic processes and fatty acid catabolism—both of which are distantly related by the tricarboxylic acid (TCA) cycle and share similar metabolic pathways. Overall, these findings further our knowledge about the genetic features underlying turtle physiology, morphology, and longevity, which have important implications for the treatment of human diseases and evolutionary studies. [ABSTRACT FROM AUTHOR]

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems. [ABSTRACT FROM AUTHOR]

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.

We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast. [ABSTRACT FROM AUTHOR]

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins. [ABSTRACT FROM AUTHOR]

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies. [ABSTRACT FROM AUTHOR]

α-helices are deformable secondary structural components regularly observed in protein folds. The overall flexibility of an α-helix can be resolved into constituent physical deformations such as bending in two orthogonal planes and twisting along the principal axis. We used Principal Component Analysis to identify and quantify the contribution of each of these dominant deformation modes in transmembrane α-helices, extramembrane α-helices, and α-helices in soluble proteins. Using three α-helical samples from Protein Data Bank entries spanning these three cellular contexts, we determined that the relative contributions of these modes towards total deformation are independent of the α-helix's surroundings. This conclusion is supported by the observation that the identities of the top three deformation modes, the scaling behaviours of mode eigenvalues as a function of α-helix length, and the percentage contribution of individual modes on total variance were comparable across all three α-helical samples. These findings highlight that α-helical deformations are independent of cellular location and will prove to be valuable in furthering the development of flexible templates in de novo protein design. [ABSTRACT FROM AUTHOR]

Finding optimal parameters for force fields used in molecular simulation is a challenging and time-consuming task, partly due to the difficulty of tuning multiple parameters at once. Automatic differentiation presents a general solution: run a simulation, obtain gradients of a loss function with respect to all the parameters, and use these to improve the force field. This approach takes advantage of the deep learning revolution whilst retaining the interpretability and efficiency of existing force fields. We demonstrate that this is possible by parameterising a simple coarse-grained force field for proteins, based on training simulations of up to 2,000 steps learning to keep the native structure stable. The learned potential matches chemical knowledge and PDB data, can fold and reproduce the dynamics of small proteins, and shows ability in protein design and model scoring applications. Problems in applying differentiable molecular simulation to all-atom models of proteins are discussed along with possible solutions and the variety of available loss functions. The learned potential, simulation scripts and training code are made available at https://github.com/psipred/cgdms. [ABSTRACT FROM AUTHOR]

A wide range of de novo design of αβ-proteins has been achieved based on the design rules, which describe secondary structure lengths and loop torsion patterns favorable for design target topologies. This paper proposes design rules for register shifts in βαβ-motifs, which have not been reported previously, but are necessary for determining a target structure of de novo design of αβ-proteins. By analyzing naturally occurring protein structures in a database, we found preferences for register shifts in βαβ-motifs, and derived the following empirical rules: (1) register shifts must not be negative regardless of torsion types for a constituent loop in βαβ-motifs; (2) preferred register shifts strongly depend on the loop torsion types. To explain these empirical rules by physical interactions, we conducted physics-based simulations for systems mimicking a βαβ-motif that contains the most frequently observed loop type in the database. We performed an exhaustive conformational sampling of the loop region, imposing the exclusion volume and hydrogen bond satisfaction condition. The distributions of register shifts obtained from the simulations agreed well with those of the database analysis, indicating that the empirical rules are a consequence of physical interactions, rather than an evolutionary sampling bias. Our proposed design rules will serve as a guide to making appropriate target structures for the de novo design of αβ-proteins. [ABSTRACT FROM AUTHOR]

Rational protein design aims at the targeted modification of existing proteins. To reach this goal, software suites like Rosetta propose sequences to introduce the desired properties. Challenging design problems necessitate the representation of a protein by means of a structural ensemble. Thus, Rosetta multi-state design (MSD) protocols have been developed wherein each state represents one protein conformation. Computational demands of MSD protocols are high, because for each of the candidate sequences a costly three-dimensional (3D) model has to be created and assessed for all states. Each of these scores contributes one data point to a complex, design-specific energy landscape. As neural networks (NN) proved well-suited to learn such solution spaces, we integrated one into the framework Rosetta:MSF instead of the so far used genetic algorithm with the aim to reduce computational costs. As its predecessor, Rosetta:MSF:NN administers a set of candidate sequences and their scores and scans sequence space iteratively. During each iteration, the union of all candidate sequences and their Rosetta scores are used to re-train NNs that possess a design-specific architecture. The enormous speed of the NNs allows an extensive assessment of alternative sequences, which are ranked on the scores predicted by the NN. Costly 3D models are computed only for a small fraction of best-scoring sequences; these and the corresponding 3D-based scores replace half of the candidate sequences during each iteration. The analysis of two sets of candidate sequences generated for a specific design problem by means of a genetic algorithm confirmed that the NN predicted 3D-based scores quite well; the Pearson correlation coefficient was at least 0.95. Applying Rosetta:MSF:NN:enzdes to a benchmark consisting of 16 ligand-binding problems showed that this protocol converges ten-times faster than the genetic algorithm and finds sequences with comparable scores. [ABSTRACT FROM AUTHOR]

Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.

Lysine acetylation (Kac) plays a critical role in the regulation of many important cellular processes. However, little is known about Kac in Solenopsis invicta, which is among the 100 most dangerous invasive species in the world. Kac in S. invicta was evaluated for the first time in this study. Altogether, 2387 Kac sites were tested in 992 proteins. The prediction of subcellular localization indicated that most identified proteins were located in the cytoplasm, mitochondria, and nucleus. Venom allergen Sol i 2, Sol i 3, and Sol i 4 were found to be located in the extracellular. The enriched Kac site motifs included Kac H, Kac Y, Kac G, Kac F, Kac T, and Kac W. H, Y, F, and W frequently occurred at the +1 position, whereas G, Y, and T frequently occurred at the –1 position. In the cellular component, acetylated proteins were enriched in the cytoplasmic part, mitochondrial matrix, and cytosolic ribosome. Furthermore, 25 pathways were detected to have significant enrichment. Interestingly, arginine and proline metabolism, as well as phagosome, which are related to immunity, involved several Kac proteins. Sequence alignment analyses demonstrated that V-type proton ATPase subunit G, tubulin alpha chain, and arginine kinase, the acetylated lysine residues, were evolutionarily conserved among different ant species. In the investigation of the interaction network, diverse interactions were adjusted by Kac. The results indicated that Kac may play an important role in the sensitization, cellular energy metabolism, immune response, nerve signal transduction, and response to biotic and abiotic stress of S. invicta. It may be useful to confirm the functions of Kac target proteins for the design of specific and effective drugs to prevent and control this dangerous invasive species. [ABSTRACT FROM AUTHOR]

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability. [ABSTRACT FROM AUTHOR]

Mannanase 19287 enzyme is an engineered β-mannanase that can be added to diets for animals raised for human consumption to hydrolyze β-mannans. Established toxicological analyses were conducted with the enzyme preparation to ensure the safety of this product for the intended use. The mannanase 19287 preparation was produced with Thermothelomyces thermophilus strain DSM 33149. In vitro toxicity studies presented here used dosages of the mannanase 19287 test articles up to 5000 μg/plate. For in vivo toxicity studies in Wistar rats, test articles were administered at 5.1 mg/L for inhalation toxicity and up to 15,000 mg/kg rat feed for oral toxicity, based on the Total Organic Solids (TOS) content in each test article. No treatment related adverse effects were reported in any study. The No Observed Adverse Effect Levels in the high dose group of the subchronic oral toxicity study were calculated as 1117–1298 mg TOS/kg bw/day in rats. Comparing these values to an Estimated Daily Intake for poultry demonstrated safety factors larger than 5000. Our results confirm that T. thermophilus fulfills the recognized safety criteria for the manufacture of food enzyme preparations and represent the first peer-reviewed safety evaluation of an enzyme preparation by T. thermophilus. The results of the toxicity studies presented herein attest to the safety of the mannanase 19287 enzyme for its intended use. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase (MMP) is a class of metalloenzyme that cleaves peptide bonds in extracellular matrices. Their functions are important in both health and disease of animals. Here using quantum mechanics simulations of the MMP8 protein, the coordination chemistry of different metal cofactors is examined. Structural comparisons reveal that Jhan-Teller effects induced by Cu(II) coordination distorts the wild-type MMP8 active site corresponding to a significant reduction in activity observed in previous experiments. In addition, further analysis suggests that a histidine to glutamine mutation at residue number 197 can potentially allow the MMP8 protein to utilize Cu(II) in reactions. Simulations also demonstrates the requirement of a conformational change in the ligand before enzymatic cleavage. The insights provided here will assist future protein engineering efforts utilizing the MMP8 protein. [ABSTRACT FROM AUTHOR]

Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease.

The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction. [ABSTRACT FROM AUTHOR]

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported. [ABSTRACT FROM AUTHOR]

Milling properties and flour color are essential selection criteria in soft wheat breeding. However, high phenotypic screening costs restrict selection to relatively few breeding lines in late generations. To achieve marker-based selection of these traits in early generations, we performed genetic dissection of quality traits using three doubled haploid populations that shared the high-quality soft wheat variety Kitahonami as the paternal parent. An amplicon sequencing approach allowed effective construction of well-saturated linkage maps of the populations. Marker-based heritability estimates revealed that target quality traits had relatively high values, indicating the possibility of selection in early generations. Taking advantage of Chinese Spring reference sequences, joint linkage maps of the three populations were generated. Based on the maps, multifamily quantitative trait locus (QTL) analysis revealed a total of 86 QTLs for ten traits investigated. In terms of target quality traits, 12 QTLs were detected for flour yield, and 12 were detected for flour redness (a* value). Among these QTLs, six for flour yield and nine for flour a* were segregating in more than two populations. Some relationships among traits were explained by QTL collocations on chromosomes, especially group 7 chromosomes. Ten different ideotypes with various combinations of favorable alleles for the flour yield and flour a* QTLs were generated. Phenotypes of derivatives from these ideotypes were predicted to design ideal genotypes for high-quality wheat. Simulations revealed the possibility of breeding varieties with better quality than Kitahonami. [ABSTRACT FROM AUTHOR]

The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities. [ABSTRACT FROM AUTHOR]

Significant advancements in the field of protein structure prediction have necessitated the need for objective and robust evaluation of protein structural models by comparing predicted models against the experimentally determined native structures to quantitate their structural similarities. Existing protein model versus native similarity metrics either consider the distances between alpha carbon (Cα) or side-chain atoms for computing the similarity. However, side-chain orientation of a protein plays a critical role in defining its conformation at the atomic-level. Despite its importance, inclusion of side-chain orientation in structural similarity evaluation has not yet been addressed. Here, we present SPECS, a side-chain-orientation-included protein model-native similarity metric for improved evaluation of protein structural models. SPECS combines side-chain orientation and global distance based measures in an integrated framework using the united-residue model of polypeptide conformation for computing model-native similarity. Experimental results demonstrate that SPECS is a reliable measure for evaluating structural similarity at the global level including and beyond the accuracy of Cα positioning. Moreover, SPECS delivers superior performance in capturing local quality aspect compared to popular global Cα positioning-based metrics ranging from models at near-experimental accuracies to models with correct overall folds—making it a robust measure suitable for both high- and moderate-resolution models. Finally, SPECS is sensitive to minute variations in side-chain χ angles even for models with perfect Cα trace, revealing the power of including side-chain orientation. Collectively, SPECS is a versatile evaluation metric covering a wide spectrum of protein modeling scenarios and simultaneously captures complementary aspects of structural similarities at multiple levels of granularities. SPECS is freely available at http://watson.cse.eng.auburn.edu/SPECS/. [ABSTRACT FROM AUTHOR]

The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines. [ABSTRACT FROM AUTHOR]

Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology.

The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s) signaling in vivo. We used naturally occurring human mutations to develop two G(s)-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our G(s)-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s) pathway in vivo. These RASSLs can be used to activate G(s) signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.

Although the Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 μM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 (3-(2,6-dichlorophenyl)-5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 μM) with minimal effect on the human orthologue class I (GLUTs 1–4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 μM and EC50 of 5.5 ± 0.6 μM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 μM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs. [ABSTRACT FROM AUTHOR]

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form. [ABSTRACT FROM AUTHOR]

In chronic kidney disease both renal insufficiency and chronic inflammation trigger elevated hepcidin levels, which impairs iron uptake, availability. and erythropoiesis. Here we report the two first-in-human phase 1 trials of PRS-080#22, a novel, rationally engineered Anticalin protein that targets and antagonizes hepcidin. A single intravenous infusion of placebo or PRS-080#22 was administered to 48 healthy volunteers (phase 1a) and 24 patients with end stage chronic kidney disease (CKD) on hemodialysis (phase 1b) at different doses (0.08-16mg/kg for the phase 1a study and 2-8mg/kg for the phase 1b study) in successive dosing cohorts. The primary endpoint for both randomized, double-blind, phase 1 trials was safety and tolerability. Following treatment, all subjects were evaluable, with none experiencing dose limiting toxicities. Most adverse events were mild. One serious adverse event occurred in the phase 1b (CKD patient) study. There were no clinically significant changes in safety laboratory values or vital signs. PRS-080#22 showed dose-proportional pharmacokinetics (PK), with a terminal half-life of approximately three days in healthy volunteers and 10 to 12 days in CKD patients. Serum hepcidin levels were suppressed in a dose dependent manner and remained low for up to 48 hours after dosing. PRS-080#22 dose-dependently mobilized serum iron with increases in both serum iron concentration and transferrin saturation. No consistent changes were observed with regard to ferritin, reticulocytes, hemoglobin, and reticulocyte hemoglobin. Low titer anti-drug-antibodies were detected in five healthy volunteers but in none of the CKD patients. PRS-080#22, a novel Anticalin protein with picomolar affinity for hepcidin, was safe and well-tolerated when administered to healthy volunteers and CKD patients at all doses tested. The drug exhibited linear pharmacokinetics, longer half-life in CKD patients in comparison to healthy volunteers as well as expected pharmacodynamic effects which hold promise for further clinical studies. [ABSTRACT FROM AUTHOR]

Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2’s catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG‡) and maximal rate constant (k’ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of ‘kinetic’ disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds. [ABSTRACT FROM AUTHOR]

Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5–antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders. [ABSTRACT FROM AUTHOR]

Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase’s DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites. [ABSTRACT FROM AUTHOR]

Attaining recombinant thermostable proteins is still a challenge for protein engineering. The complexity is the length of time and enormous efforts required to achieve the desired results. Present work proposes a novel and economic strategy of attaining protein thermostability by predicting site-specific mutations at the shortest possible time. The success of the approach can be attributed to Analytical Hierarchical Process and the outcome was a rationalized thermostable mutation(s) prediction tool- RankProt. Briefly the method involved ranking of 17 biophysical protein features as class predictors, derived from 127 pairs of thermostable and mesostable proteins. Among the 17 predictors, ionic interactions and main-chain to main-chain hydrogen bonds were the highest ranked features with eigen value of 0.091. The success of the tool was judged by multi-fold in silico validation tests and it achieved the prediction accuracy of 91% with AUC 0.927. Further, in vitro validation was carried out by predicting thermostabilizing mutations for mesostable Bacillus subtilis lipase and performing the predicted mutations by multi-site directed mutagenesis. The rationalized method was successful to render the lipase thermostable with optimum temperature stability and Tm increase by 20°C and 7°C respectively. Conclusively it can be said that it was the minimum number of mutations in comparison to the number of mutations incorporated to render Bacillus subtilis lipase thermostable, by directed evolution techniques. The present work shows that protein stabilizing mutations can be rationally designed by balancing the biophysical pleiotropy of proteins, in accordance to the selection pressure. [ABSTRACT FROM AUTHOR]

Selecting amino acids to design novel protein-protein interactions that facilitate catalysis is a daunting challenge. We propose that a computational coevolutionary landscape based on sequence analysis alone offers a major advantage over expensive, time-consuming brute-force approaches currently employed. Our coevolutionary landscape allows prediction of single amino acid substitutions that produce functional interactions between non-cognate, interspecies signaling partners. In addition, it can also predict mutations that maintain segregation of signaling pathways across species. Specifically, predictions of phosphotransfer activity between the Escherichia coli histidine kinase EnvZ to the non-cognate receiver Spo0F from Bacillus subtilis were compiled. Twelve mutations designed to enhance, suppress, or have a neutral effect on kinase phosphotransfer activity to a non-cognate partner were selected. We experimentally tested the ability of the kinase to relay phosphate to the respective designed Spo0F receiver proteins against the theoretical predictions. Our key finding is that the coevolutionary landscape theory, with limited structural data, can significantly reduce the search-space for successful prediction of single amino acid substitutions that modulate phosphotransfer between the two-component His-Asp relay partners in a predicted fashion. This combined approach offers significant improvements over large-scale mutations studies currently used for protein engineering and design. [ABSTRACT FROM AUTHOR]

Amino acid mutations in proteins are random and those mutations which are beneficial or neutral survive during the course of evolution. Conservation or co-evolution analyses are performed on the multiple sequence alignment of homologous proteins to understand how important different amino acids or groups of them are. However, these traditional analyses do not explore the directed influence of amino acid mutations, such as compensatory effects. In this work we develop a method to capture the directed evolutionary impact of one amino acid on all other amino acids, and provide a visual network representation for it. The method developed for these directed networks of inter- and intra-protein evolutionary interactions can also be used for noting the differences in amino acid evolution between the control and experimental groups. The analysis is illustrated with a few examples, where the method identifies several directed interactions of functionally critical amino acids. The impact of an amino acid is quantified as the number of amino acids that are influenced as a consequence of its mutation, and it is intended to summarize the compensatory mutations in large evolutionary sequence data sets as well as to rationally identify targets for mutagenesis when their functional significance can not be assessed using structure or conservation. [ABSTRACT FROM AUTHOR]

Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β--glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α--arabinofuranoside in these ginsenosides. To determine candidate residues involved in α--arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α--arabinofuranosidase activity. A L213A variant β-glycosidase with increased α--arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α--arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α--arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides. [ABSTRACT FROM AUTHOR]

Redesigning protein surface topology to improve target binding holds great promise in the search for highly selective therapeutics. While significant binding improvements can be achieved using natural amino acids, the introduction of non-canonical residues vastly increases sequence space and thus the chance to significantly out-compete native partners. The potency of protein inhibitors can be further enhanced by synthesising mirror image, D-amino versions. This renders them non-immunogenic and makes them highly resistant to proteolytic degradation. Current experimental design methods often preclude the use of D-amino acids and non-canonical amino acids for a variety of reasons. To address this, we build an in silico pipeline for D-protein designs featuring non-canonical amino acids. For a test scaffold we use an existing D-protein inhibitor of VEGF: D-RFX001. We benchmark the approach by recapitulating previous experimental optimisation with canonical amino acids. Subsequent incorporation of non-canonical amino acids allows designs that are predicted to improve binding affinity by up to -7.18 kcal/mol. [ABSTRACT FROM AUTHOR]

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms. [ABSTRACT FROM AUTHOR]

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters. [ABSTRACT FROM AUTHOR]

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts. [ABSTRACT FROM AUTHOR]