Search

Respiratory infections are the major cause of death from infectious disease worldwide. The clinical presentation of many respiratory viruses is indistinguishable; therefore, diagnostic approaches that can identify multiple pathogens are essential for patient management. We aimed to address this challenge with self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid read-out via nanopore sensing. Here, we show this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus, influenza, and parainfluenza. Our nanobait could be reprogrammed to discriminate viral variants, and we identified several key SARS-CoV-2 variants with single-nucleotide resolution. We increased assay specificity with bespoke nanobait that could identify numerous short RNA targets in the same viral sample in a complex background of the human transcriptome. Notably, we found that the sequence position in the viral RNA secondary structure is critical for nanobait design. Lastly, we show that nanobait could discriminate between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients using programmable target cleavage without pre-amplification. Our system allows for multiplexed identification of native RNA molecules, providing a new scalable approach for diagnostics of multiple respiratory viruses in a single assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Obtaining protein crystals suitable for characterizing ligands and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and in this study, we observed it to have a poor success rate. We therefore set out to engineer robust crystallization behaviour into the nanobody scaffold by exploring the nanobody-nanobody interface predominant in the PDB using >200 combinations of surface mutations. This yielded multiple polymorphs, all mediated by this interface, with one providing far superior resolution and reliability of diffraction. We therefore refer to the modified nanobody scaffolds as “Gluebodies” and propose at least 2 variants that should be routinely generated for chaperone experiments. We furthermore show that Gluedodies cannot rescue intrinsically non-crystallizing proteins, but are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals.

Herein we provide a living summary of the data generated during the COVID Moonshot project focused on the development of SARS-CoV-2 main protease (Mpro) inhibitors. Our approach uniquely combines crowdsourced medicinal chemistry insights with high throughput crystallography, exascale computational chemistry infrastructure for simulations, and machine learning in triaging designs and predicting synthetic routes. This manuscript describes our methodologies leading to both covalent and non-covalent inhibitors displaying protease IC50 values under 150 nM and viral inhibition under 5 uM in multiple different viral replication assays. Furthermore, we provide over 200 crystal structures of fragment-like and lead-like molecules in complex with the main protease. Over 1000 synthesized and ordered compounds are also reported with the corresponding activity in Mpro enzymatic assays using two different experimental setups. The data referenced in this document will be continually updated to reflect the current experimental progress of the COVID Moonshot project, and serves as a citable reference for ensuing publications. All of the generated data is open to other researchers who may find it of use.

The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of theCOVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery campaign, all compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

Combined photochemical arylation, "nuisance effect" (SN Ar) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein-ligand structure determination. Reactions were deliberately allowed to run "out of control" in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with SN Ar processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease., The SARS-CoV-2 main protease is an important target for the development of COVID-19 therapeutics. Here, the authors combine X-ray crystallography and mass spectrometry and performed a large scale fragment screening campaign, which yielded 96 liganded structures of this essential viral protein that are of interest for further drug development efforts.

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery., Fragment-based screening of a shape-diverse collection yielded four hits against three proteins. Up to 14 analogues of each hit were rapidly generated, enabling four fragment growth vectors to be explored using inexpensive materials and reliable synthetic transformations.

Filopodia are thin, actin-based membrane protrusions with roles in sensing external mechanical and chemical cues, such as growth factor gradients in tissues. It was proposed that the chemical sensing role of filopodia is achieved through clearance of activated signaling receptors from filopodia. Type I insulin-like growth factor receptor (IGF1R) is a key regulator of normal development and growth, as well as tumor development and progression. Its biological roles depend on its activation upon IGF1 binding at the cell membrane. IGF1R behavior at the cell membrane and in particular in filopodia, has not been established. We found that IGF1 activation led to a gradual reduction in IGF1R puncta in filopodia, and that this clearance depended on actin, non-muscle myosin II, and IGF1R kinase activity. Using single particle tracking of filopodial IGF1R, we established that ligand-free IGF1R undergoes non-directional unidimensional diffusion along the filopodium. Moreover, after initial diffusion, the ligand-bound IGF1R is actively transported along the filopodium towards the filopodium base, and consequently cleared from the filopodium. Our results show that IGF1R can move directionally on the plasma membrane protrusions, supporting a sensory role for filopodia in interpreting local IGF1 gradients.

[Image: see text] Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems.

Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.

Conventional cross-linking of proteins involves the use of toxic chemicals. Here, cross-linking of gelatine and gelatine hydrolysates with tyrosinases from Botryosphaeria obtusa (BoT1 and BoT2), Agaricus bisporus (AbT) and from Verrucomicrobium spinosum (VsT) and with laccases from Trametes hirsuta (ThL) and T. versicolor (TvL) was demonstrated. Enzymatic oxidation of tyrosine residues was indicated by UV/VIS and fluorescence spectroscopy and further confirmed by oxygen consumption measurements. Using a model substrate (Tyr-Ala) dimerization was demonstrated by using RP-HPLC and LC-MS. Enzymatic cross-linking significantly increased the molecular weight of the soluble material up to the point of precipitation as demonstrated by both SDS-PAGE and size exclusion chromatography. The effect of cross-linking was further enhanced in the presence of phenolic molecules such as catechin.

Detection methods for immunoblot analysis are often based on peroxidase conjugates. However, molecular weight markers directly detectable for general use in such systems are not available. Here, we describe the preparation of a direct molecular weight marker consisting of heme-tagged proteins, whose enzymatic activities make them detectable simultaneously with the antigen in peroxidase-based immunoblot systems. The peroxidase activity results from the covalent attachment of heme to selected engineered periplasmic proteins, catalyzed by the cytochrome c maturation system of Escherichia coli. The newly designed heme-tagged proteins were combined with a previously constructed heme-tagged maltose-binding protein and cytochrome c. The resulting heme ladder was shown to be suitable as a protein standard for direct molecular weight estimation in immunoblot analysis due to the peroxidase activity of its constituents. The heme ladder consists of proteins between 12 and 85 kDa and can be produced at low cost. The marker was stable when kept at 4, −20, and −80 °C for >6 months.

The well studied bacterial tyrosinases from the Streptomyces sp. bacteria are distinguishable from their eukaryotic counterparts by the absence of a C-terminal extension. In the present study, we report that the tyrosinase from the bacterium Verrucomicrobium spinosum also has such a C-terminal extension, thus making it distinct from the Streptomyces enzymes. The entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full-length unprocessed form), which has a twin arginine translocase type signal peptide, the two copper-binding motifs typical of the tyrosinase protein family and the aforementioned C-terminal extension. We expressed various mutants of the recombinant enzyme in Escherichia coli and found that removal of the C-terminal extension by genetic engineering or limited trypsin digest of the pro-form results in a more active enzyme (i.e. 30-100-fold increase in monophenolase and diphenolase activities). Further studies also revealed the importance of a phenylalanine residue in this C-terminal domain. These results demonstrate that the V. spinosum tyrosinase is a new example of this interesting family of enzymes. In addition, we show that this enzyme can be readily overproduced and purified and that it will prove useful in furthering the understanding of these enzymes, as well as their biotechnological application.

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1–BMR, contains the N-terminally modified residues 22–493 of the human P450 2E1 fused at the C-terminus to residues 473–1049 of the P450 BM3 reductase (BMR). The P450 2E1–BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (K M=1.84±0.09 mM and k cat of 2.98±0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (K M=0.65±0.08 mM and k cat of 0.95±0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2E1) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP-FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form, displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2E1 enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1-BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1-BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates, such as pesticides and polyaromatic hydrocarbons were identified, providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology.

The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.

The pathway of ligand dissociation and how binding sites respond to force are not well understood for any macromolecule. Force effects on biological receptors have been studied through simulation or force spectroscopy, but not by high resolution structural experiments. To investigate this challenge, we took advantage of the extreme stability of the streptavidin-biotin interaction, a paradigm for understanding non-covalent binding as well as a ubiquitous research tool. We synthesized a series of biotin-conjugates having an unchanged strong-binding biotin moiety, along with pincer-like arms designed to clash with the protein surface: 'Love-Hate ligands'. The Love-Hate ligands contained various 2,6-di-ortho aryl groups, installed using Suzuki coupling as the last synthetic step, making the steric repulsion highly modular. We determined binding affinity, as well as solving 1.1-1.6 Å resolution crystal structures of streptavidin bound to Love-Hate ligands. Striking distortion of streptavidin's binding contacts was found for these complexes. Hydrogen bonds to biotin's ureido and thiophene rings were preserved for all the ligands, but biotin's valeryl tail was distorted from the classic conformation. Streptavidin's L3/4 loop, normally forming multiple energetically-important hydrogen bonds to biotin, was forced away by clashes with Love-Hate ligands, but Ser45 from L3/4 could adapt to hydrogen-bond to a different part of the ligand. This approach of preparing conflicted ligands represents a direct way to visualize strained biological interactions and test protein plasticity.

Tyrosinases are copper-containing dioxygen activating enzymes found in many species of bacteria and are usually associated with melanin production. These proteins have a strong preference for phenolic and diphenolic substrates and are somewhat limited in their reaction scope, always producing an activated quinone as product. Despite this fact they have potential in several biotechnological applications, including the production of novel mixed melanins, protein cross-linking, phenolic biosensors, production of l-DOPA, phenol and dye removal and biocatalysis. Although most studies have used Streptomyces sp. enzymes, there are several other examples of these proteins that are also of potential interest. For instance a solvent tolerant enzyme has been described, as well as an enzyme with both tyrosinase and laccase activities, enzymes with altered substrate preferences, an enzyme produced as an inactive zymogen as well as examples which do not require auxiliary proteins for copper insertion (unlike the Streptomyces sp. enzymes which do require such a protein). This article will summarise the reports on the biotechnological applications of bacterial tyrosinases as well as the current information available on the different types of this enzyme.

This paper reports on the cross-linking and immobilisation of various proteins by the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase). In general it is found that Vs-tyrosinase can readily cross-link proteins with a low degree of complexity, such as casein, but that the enzyme cannot readily cross-link well folded protein substrates such as lysozyme, myoglobin, cytochrome c or Candida antarctica lipase B (CALB). However, the inclusion of phenolic compounds (phenol or caffeic acid) to reaction mixtures of these proteins can greatly enhance the levels of cross-linking. For example it is possible to prepare cross-linked aggregates of industrially applicable enzymes such as CALB by simply incubating it with Vs-tyrosinase and phenol. The resulting aggregates can be collected by centrifugation and retain high levels of activity and may find applications in biocatalysis.

The well studied bacterial tyrosinases from the Streptomyces sp. bacteria are distinguishable from their eukaryotic counterparts by the absence of a C-terminal extension. In the present study, we report that the tyrosinase from the bacterium Verrucomicrobium spinosum also has such a C-terminal extension, thus making it distinct from the Streptomyces enzymes. The entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full-length unprocessed form), which has a twin arginine translocase type signal peptide, the two copper-binding motifs typical of the tyrosinase protein family and the aforementioned C-terminal extension. We expressed various mutants of the recombinant enzyme in Escherichia coli and found that removal of the C-terminal extension by genetic engineering or limited trypsin digest of the pro-form results in a more active enzyme (i.e. 30-100-fold increase in monophenolase and diphenolase activities). Further studies also revealed the importance of a phenylalanine residue in this C-terminal domain. These results demonstrate that the V. spinosum tyrosinase is a new example of this interesting family of enzymes. In addition, we show that this enzyme can be readily overproduced and purified and that it will prove useful in furthering the understanding of these enzymes, as well as their biotechnological application.

Porous silica particles are emerging as complementary systems to polyester microspheres for the encapsulation and controlled delivery of small-organic drugs. Their recent application in pharmaceutics is strengthened by well-established characterization and synthetic routes from the chemical engineering sciences. Silica is an interesting scaffold material for the encapsulation of organic molecules. It can be formed into hierarchical structures over a wide range of length scales and interconnectivities. Encapsulation can therefore be tailored not only to the drug but the desired release properties. In addition to surfactant-templating of hierarchical silica structures, polypeptides from marine organisms may offer biological routes to novel silica materials. Silica sol-gels have also been evaluated as delivery vehicles, particularly with regard to generating hybrid systems with mesoporous silica or composite xerogels. This review will first focus on the detailed characterisation of pore size and structure of mesoporous silica with regards water penetration and drug diffusion. We then describe the pharmaceutical applications of silica materials with regard to improving oral bioavailability, multiparticulate systems for gastroretention or sustained release, composite xerogels and in vivo biocompatibility.

Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 A crystal structure of one of these chimeras allows us to rationalise the catalytic mechanism of silicic acid condensation.

Differences evident in the sequence alignment of human cathepsin-L with shrimp cathepsin-L and silicatein-alpha suggest the indirect involvement of the heavy to light chain loop (E 286 to E 289) in the function of these enzymes. Deletion of the loop and adjacent residues S 290 to N 293, decreased specific protease activity by 81% and 63%, respectively; complete substitution for the corresponding silicatein-alpha loop decreased activity by 35%. In all cases the Km was largely unchanged. The conformational stability of human procathepsin-L was not altered by deletion of E 286 to E 289 but increased on deletion of S 290 to N 293. Therefore, shortening the loop does not change substrate affinity but does influence activity, in part via conformational change.

The molecular mechanism by which heparin modulates the processing of procathepsin L in the extracellular environment is proposed. We show that heparin reduces the stability of the pro form of cathepsin L at pH 5 by binding to a putative heparin binding motif (BBXB) in the pro-domain. Mutations to this motif on procathepsin L reduce heparin binding affinity and heparin-induced destabilization; in contrast, heparin only slightly destabilizes the mature cathepsin L domain. Gel analysis further shows that heparin makes procathepsin L a much better substrate for cathepsin L. Thus, heparin enhances the rate of zymogen activation by destabilization upon binding to the BBXB motif. Determining the mechanism by which procathepsin L is activated in the extracellular matrix is important to the understanding of the role that cathepsin L plays in tumour invasion.

Our understanding of protein emulsifying properties is largely based on analysis of emulsifiers found in milk and seed. The 9th-10th type III fibronectin domain pair retains full biological activity following emulsification-encapsulation into polyester microspheres, for controlled delivery, but the conformational criteria determining emulsification efficiency (EE) are unknown. Here, we have generated a series of mutants of this beta-sandwich protein, changing the hydrophobic moment and conformational stability, to investigate the structure-emulsification relationship. Predictive modelling of the hydrophobic moment of beta-strands and mutations known to increase conformational stability were used to generate the series. The proteins were tested for their emulsion stability and EE for oil-in-water mixtures. We show that the stabilization of emulsions by beta-sandwich proteins is best predicted by conformational stability during equilibrium denaturation in ionic surfactant. In contrast, the EE of these proteins is inversely related to an increase in their surface hydrophobicity following unfolding in surfactant. We also describe a novel beta-sandwich emulsifier with strong EE. The requirement for interdomain flexibility to achieve maximum emulsion stability and EE is also shown. This work increases our understanding of the mechanisms involved in protein emulsification and will be of use to the microencapsulation of proteins into polyester microspheres via emulsion-extraction protocols.

This communication reports the first electrochemical study of the human P450 2E1 either absorbed or covalently linked to different electrode surfaces. Glassy-carbon and gold electrodes gave reversible electrochemical signals of an active P450 2E1. Molecular modeling of the enzyme helped to rationalize the results. A monolayer coverage was obtained on gold modified with cystamine/maleimide that covalently linked surface accessible cysteines of P450 2E1. The midpoint potential measured for the oriented P450 2E1 was -177 +/- 5 mV comparable to that of the FeIII/FeII of other P450 enzymes. The observed electron-transfer rate for this electrode was 10 s-1. The turnover of the active enzyme was measured with the P450 2E1 specific substrate p-nitrophenol, resulting in a KM of 130 +/- 3 muM and the formation of 2.2 muM of the p-nitrocatechol product upon application of a -300 mV bias.

Integrin α5β1 binds the human 9th–10th type III fibronectin domain pair (FIII9–10) to mediate cell attachment and spreading. FIII9–10 mutants with increased conformational stability (FIII9′10) or highly restricted interdomain mobility (FIII9′10-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9–10 was 14% compared to 31% for FIII9′10 and 100% for FIII9′10-CC. For FIII9′10-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9–10 and only a transition to bilayer was observed for FIII9′10. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9′10-CC were fitted with an integrin layer thickness of 130 A. This indicates a movement of the integrin α5β1 headpiece away from its position in the compact ‘bent’ conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.

A comparison between engineered and commercially available L-lactate oxidases from Aerococcus viridans was conducted for biosensing applications. Enzymes were adsorbed onto the surfaces of graphite electrodes modified with multi-walled carbon nanotubes. Thermostable L-lactate oxidases were cloned with a (i) N-, (ii) a C-terminal His-tag and (iii) a wild-type enzyme. Subsequently to the heterologous expression in Escherichia coil and purification, we determined the kinetic parameters of these enzymes in solution. The kinetics of the wild-type, of the N-terminally His-tagged enzyme and of the commercial L-lactate oxidase from A. viridans were studied with a classical Michaelis-Menten as well as with a substrate inhibition model, while the enzyme carrying a C-terminal His-tag showed no activity. The active enzymes were used to fabricate and comparatively investigate multi-walled carbon nanotubes-based biosensors. The enzyme kinetic results were compared with electrochemical studies. By using both spectrophotometric and amperometric techniques, the inhibition phenomenon fits better to the data especially those data related with Lox-His-N. The electrochemical data of the fabricated enzymatic biosensors showed that the N-terminally His-tagged L-lactate oxidase performed best on carboxyl-modified carbon nanotubes. The sensor based on this engineered enzyme showed the highest sensitivity and lowest detection limit in the range of L-lactate concentration 0-1 mM as well as long term stability over one month. (C) 2013 Elsevier Ltd. All rights reserved.