Jan 27, · The target proteins used were: Botulinum neurotoxin B HcB expressed as previously described 48, human IgG1 Fc-HisTag (AcroBiosystems, IG1-H) and human HER2-HisTag (AcroBiosystems, HE2-H) Evidence-based updates of best clinical practice across the spectrum of musculoskeletal conditions. Best Practice & Research: Clinical Rheumatology keeps the clinician or trainee informed of the latest developments and current recommended practice in the rapidly advancing fields of . Read more Proceedings of the National Academy of Sciences of the United States of America (often abbreviated PNAS or PNAS USA) is a peer-reviewed multidisciplinary scientific blogger.com is the official journal of the National Academy of Sciences, published since , and publishes original research, scientific reviews, commentaries, and blogger.coming to Journal Citation Reports, the journal has a
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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a botulinum toxin research paper up to date browser or turn off compatibility mode in Internet Explorer.
In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications 1, botulinum toxin research paper. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein botulinum toxin research paper in which the binding of a peptide key triggers biological outputs of interest 2.
The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution.
We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 3we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins.
The former, which incorporates a de novo botulinum toxin research paper spike receptor binding domain RBD binder 4has a limit of detection of 15 pM and a luminescence signal fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
Protein-based biosensors have important roles in synthetic biology and clinical applications, but the design of biosensors has so far been mostly limited botulinum toxin research paper reengineering natural proteins 1. Finding specific analyte-binding domains that undergo conformational changes upon binding is challenging, and even when available, extensive protein engineering efforts are generally required to effectively couple them to a reporter domain 56.
It is therefore desirable to construct modular biosensor platforms that can be easily repurposed to detect different protein targets of interest. Modular systems have been developed to detect antibodies 789 and small molecules 10botulinum toxin research paper, 11but general protein sensors are a bigger challenge given the great diversity of protein structures, sizes and oligomerization states, and approaches such as semisynthetic protein platforms 121314or calmodulin switches 1516usually require considerable screening to find potential candidates owing to limited predictability A protein biosensor can be constructed from a system with two nearly isoenergetic states, the equilibrium between which is modulated by the analyte being sensed.
Desirable properties in such a sensor are: i the conformational change triggered by an analyte should be independent of the details of the analyte, so the same overall system can be used to sense many different targets; ii the system should be tunable so that analytes with different binding energies and different typical concentrations can be detected over a large dynamic range; and iii the conformational change should be coupled to a sensitive output.
We hypothesized that these attributes could be attained by inverting the information flow in de novo designed protein switches in which binding to a target protein of interest is controlled by the presence of a peptide actuator 2, botulinum toxin research paper.
lucCage has two states: a closed state, in which the cage domain binds to the latch and sterically occludes the binding motif from binding the target and SmBiT from combining with LgBit to reconstitute luciferase activity, and an open state, in which these binding interactions are not blocked and lucKey can bind to the cage domain.
The association of lucKey with lucCage results in the reconstitution of luciferase activity Fig. This system satisfies properties i and ii above, as a wide range of binding activities can be caged, and because the switch is thermodynamically controlled, the lucKey and target binding energies can be adjusted to achieve activation at the relevant target concentrations.
Because lucKey and lucCage are always the same, the system is modular—the same molecular association can be coupled to the binding of many different targets. Bioluminescence provides a rapid and sensitive readout of the analyte-driven lucCage—lucKey association, satisfying property iii. aSensor schematic mechanism. The closed form of lucCage left cannot bind to lucKey, thus preventing the split luciferase SmBiT fragment from interacting with LgBit.
The open form right can bind to both the target and the key, enabling the reconstitution of SmBiT and LgBiT for luciferase activity. bThermodynamics of biosensor activation. The free energy cost Δ G open of the transition from closed cage species 1 to open cage species 2 disfavours the association of key species 5 and reconstitution of luciferase activity species 6 in the botulinum toxin research paper of target.
cThermodynamics of biosensor design. The designable parameters are Δ G open and Δ G CK ; Δ G R is the same for all targets, and Δ G LT is pre-specified for each target. For sensitive but low background analyte detection, botulinum toxin research paper, Δ G open and Δ G CK must be tuned such that the closed state species 1 is substantially lower in free energy than the open state species 6 in the absence of target, but higher in free energy than the open state in the presence of target species 7.
The states of this biosensor system are in thermodynamic equilibrium, with the tunable parameters Δ G open and Δ G CK governing the populations of the possible species, botulinum toxin research paper with the free energy of botulinum toxin research paper of the analyte to the binding domain Δ G LT Fig. We simulated the dependence of the sensor system on Δ G open Extended Data Fig.
The sensitivity of analyte detection is a function of Δ G LTbotulinum toxin research paper, with a lower limit of roughly one-tenth of the dissociation constant K d for analyte binding Extended Data Fig. Above this lower limit, varying the concentration of lucCage and lucKey enables the system to respond to different ranges of target concentration Extended Data Fig. Sensitivity can be further modulated by tuning the strength of the intramolecular cage—latch interaction and the intermolecular cage—key interaction Δ G open and Δ G CKrespectively ; for example, too tight cage—latch interaction results in a low signal in the presence of target, and too weak an interaction results in a high background botulinum toxin research paper in the absence of target Extended Data Fig.
Our design strategy aims to find this balance by modulating Δ G open and Δ G CK by varying the length of the latch and key helix and by introducing either favourable hydrophobic or unfavourable buried polar interactions at the cage—latch or cage—key interfaces 2 Extended Data Fig. As a first test, we grafted the SmBiT peptide and the BIM peptide in the closed state of the previously described optimized asymmetric LOCKR switch 2 Extended Data Fig. SmBiT adopts a β-strand conformation within the luciferase holoenzyme, but we assumed that it could adopt a helical secondary structure in the context of the helical bundle scaffold, because secondary structure can be context-dependent We sampled different placements for the two peptide sequences across the latch, selected botulinum toxin research paper lowest energy solutions Extended Data Fig.
We mixed the designs with lucKey in a ratio, then added BCL-2, which binds to BIM with nanomolar affinity 20and observed a rapid increase in luminescence Extended Data Fig, botulinum toxin research paper. The detection range of the analyte could be tuned by varying the concentration of the sensor lucCage plus lucKey Extended Data Fig. lucCageBIM has SmBiT at position in the latch SmBiT Extended Data Fig, botulinum toxin research paper. We next investigated the incorporation of a range of binding modalities for analytes of interest within lucCage by developing methods for computationally caging target-binding proteins, rather than peptides, botulinum toxin research paper, in the closed state Supplementary Methods.
As a test case, we caged the de novo designed influenza A H1 haemagglutinin HA 21 binding protein HB1. Two out of the five designs were functional, and bound HA in the presence but not the absence of key Extended Data Fig. Left, design model of the de novo binder HB1. Middle, all residues of HB1.
The magenta labels indicate the same set of amino acids in the two panels for example, F2 in the botulinum toxin research paper panel corresponds to F in the bottom panel. b — dFunctional characterization of lucCageBot blucCageProA c and lucCageHER2 d. Right, detection over a wide range of analyte concentrations by changing the biosensor lucCage plus lucKey concentration coloured lines. All experiments were performed in triplicate, representative data are shown, and data are mean ± s.
We grafted a de novo designed binder for Botulinum neurotoxin Bot. After screening a few designs for each target Extended Data Figs. The designed sensors respond within minutes after the addition of the target, botulinum toxin research paper, and their sensitivity can be tuned by changing the concentration of lucCage and lucKey Fig. We next designed sensors for cardiac troponin I, which is the standard early diagnostic biomarker for acute myocardial infarction We took advantage of the high-affinity interactions between cardiac troponins T, botulinum toxin research paper, C and I cTnT, cTnC and cTnI, respectively Fig.
The best candidate, lucCageTrop, was able to detect cTnI but not at sufficiently low levels for clinical use as the rule-in and rule-out levels of cTnI assay for the diagnosis of patients with acute myocardial infarction are in the low picomolar range Because the limit of detection LOD of our sensor platform is about 0, botulinum toxin research paper.
We fused cTnC to the C terminus of the sensor to take advantage of the high-affinity interaction between the three cardiac troponins Extended Data Fig. The resulting sensor, lucCageTrop, has a single-digit picomolar LOD that is suitable for the quantification of clinical samples Fig. aDesign of cTnI sensor. Left, structure of cardiac troponin PDB code 4Y cTnT, cTnC and cTnI are shown in cyan, green and magenta, respectively.
Right, design model of lucCageTrop. bLeft, luminescence signal increases after the addition of 1 nM cTnI botulinum toxin research paper 0. Right, wide detection range accessible by changing the concentration of the sensor components coloured lines.
Grey area indicates the cTnI concentration range relevant to the diagnosis of acute myocardial infarction 27 ; the dotted line indicates the clinical cut-off for acute myocardial infarction defined by the World Health Organization WHO 0.
cbotulinum toxin research paper, HBV sensor design models gold, SmBiT; grey, linker; magenta, HBV preS1 epitope. dbotulinum toxin research paper, lucCageHBVα with two epitope copies has higher affinity by biolayer interferometry for the anti-HBV antibody HzKR eLeft, luminescence signal increases after the addition of 50 nM anti-HBV antibody to 1 nM lucCageHBVα plus lucKey.
Right, sensitive anti-HBV antibody detection over a wide concentration range. fMechanism for the detection of preS1 using lucCageHBV. hThe detection of preS1 can be achieved over the relevant post-HBV infection concentration levels grey area by varying the concentration of antibody indicated by coloured labels.
The detection of specific antibodies is important for monitoring the spread of a pathogen in a population 28the success of vaccination 29and levels of therapeutic antibodies 9, botulinum toxin research paper. To adapt our system for serological antibody analyses, we sought to incorporate linear epitopes recognized by the antibodies of interest into lucCage. We first developed a sensor for antibodies against the preS1 domain of the hepatitis B virus HBV surface protein L To further improve the dynamic range and LOD of lucCageHBV Extended Data Fig.
Because the concentrations of most therapeutic antibodies in serum are in the low micromolar to nanomolar range 9this platform should be useful for monitoring the concentrations of therapeutic antibodies in circulation We next sought to use the lucCageHBV sensor to detect HBV surface antigen. Because our sensors are under thermodynamic control, we hypothesized that the pre-assembled sensor—antibody complex would re-equilibrate in the presence of the target HBV surface antigen protein preS1, with antibody redistributing to bind free preS1 instead of the epitope on lucCageHBV Fig, botulinum toxin research paper.
The luminescence of the lucCageHBV plus HzKR The COVID pandemic has created an urgent need for diagnostic tools for both the SARS-CoV-2 virus and antiviral antibodies 3. Among these, we focused on two epitopes in the membrane M and nucleocapsid N proteins that are recognized by sera from patients with SARS and COVID 3536 and for which cross-reactive animal-derived antibodies are commercially available Methods.
We designed sensors for each epitope and identified designs that specifically responded to anti-membrane and anti-nucleocapsid antibodies Extended Data Fig.
aLeft, botulinum toxin research paper sensor incorporates two copies of the SARS-CoV-2 membrane protein 1—17 epitope red connected botulinum toxin research paper a flexible spacer. Middle, kinetics of luminescence activation of 50 nM lucCageSARS2-M plus lucKey after the addition of nM anti-SARS-CoVM rabbit polyclonal antibodies pAb botulinum toxin research paper cross-react with residues 1—17 of the SARS-CoV-2 membrane protein.
Right, botulinum toxin research paper of 5 nM lucCageSARS2-M plus lucKey to botulinum toxin research paper concentrations of target anti-M polyclonal antibody. bLeft, lucCageSARS2-N incorporates two copies of the SARS-CoV-2 nucleocapsid protein — epitope light blue, botulinum toxin research paper. Middle, kinetics of luminescence activation of 50 nM lucCageSARS2-N plus lucKey after the addition of nM anti-SARS-CoVN mouse monoclonal antibody clone 18F Right, response of 50 nM lucCageSARS2-N plus lucKey to varying concentration of anti-N monoclonal antibody.
cLeft, lucCageRBD incorporates a de novo SARS-CoV-2 RBD binder 4 LCB1, magenta. Middle, luminescence intensities increase after the addition of dBiosensor specificity. Each sensor at 1 nM was incubated with 50 nM of its cognate target magenta lines and the targets for the other biosensors grey lines.
Botulinum toxin research paper create sensors that can detect SARS-CoV-2 viral botulinum toxin research paper directly, we integrated a de novo designed picomolar affinity binder to the receptor-binding domain RBD of the SARS-CoV-2 spike protein named LCB1 4 into the lucCage format Fig. In addition to virus detection, the RBD sensor botulinum toxin research paper also be used to monitor antibody generation in response to vaccination using a competition format analogous to that described above for the detection of HBV antibodies Fig.
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Evidence-based updates of best clinical practice across the spectrum of musculoskeletal conditions. Best Practice & Research: Clinical Rheumatology keeps the clinician or trainee informed of the latest developments and current recommended practice in the rapidly advancing fields of . Read more General Hints Regarding C. botulinum Toxin at annual meetings of the Interagency Botulism Research Coordinating Committee (IBRCC). the plate several times on a paper It follows the health and care white paper and an NHS England consultation. 8 July Neurologist James Samuel Risien Russell receives English Heritage London blue plaque
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