Current Protocols in Protein Science最新文献

{"title":"De Novo Protein Design Using the Blueprint Builder in Rosetta","authors":"Linna An,&nbsp;Gyu Rie Lee","doi":"10.1002/cpps.116","DOIUrl":"10.1002/cpps.116","url":null,"abstract":"<p>While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo β-barrel proteins as an example to describe the principles and basic procedures of the blueprint builder−based design pipeline. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: The construction of blueprints</p><p><b>Alternate Protocol</b>: Build blueprints based on existing protein <span>.pdb</span> files</p><p><b>Basic Protocol 2</b>: De novo protein design pipeline using the blueprint builder</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38723942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell-Free System","authors":"Rochelle Aw,&nbsp;Alex J. Spice,&nbsp;Karen M. Polizzi","doi":"10.1002/cpps.115","DOIUrl":"10.1002/cpps.115","url":null,"abstract":"<p>Cell-free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell-free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast <i>Pichia pastoris</i> (also known as <i>Komagataella phaffii</i>) is a particularly attractive eukaryotic host from which to generate cell-free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post-translational modifications. Here, we describe methods for conducting cell-free protein synthesis using <i>P. pastoris</i> as the host, from preparing the cell lysates to protocols for both coupled and linked transcription-translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike. © 2020 The Authors.</p><p><b>Basic Protocol 1</b>: Preparation of <i>Pichia pastoris</i> cell lysate</p><p><b>Basic Protocol 2</b>: Coupled in vitro transcription and translation</p><p><b>Basic Protocol 3</b>: Determining luciferase production from cell-free protein synthesis reactions</p><p><b>Alternate Protocol 1</b>: Linked in vitro transcription and translation</p><p><b>Alternate Protocol 2</b>: Quantifying HSA protein concentration</p><p><b>Support Protocol 1</b>: Preparation of mRNA by in vitro transcription for linked transcription and translation</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38531382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans","authors":"Lluís Millan-Ariño,&nbsp;Zuo-Fei Yuan,&nbsp;Marlies E. Oomen,&nbsp;Simone Brandenburg,&nbsp;Alexey Chernobrovkin,&nbsp;Jérôme Salignon,&nbsp;Lioba Körner,&nbsp;Roman A. Zubarev,&nbsp;Benjamin A. Garcia,&nbsp;Christian G. Riedel","doi":"10.1002/cpps.114","DOIUrl":"10.1002/cpps.114","url":null,"abstract":"Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA‐related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post‐translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)−based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co‐occurring PTMs and the discovery of novel PTMs become feasible. The success of MS‐based approaches relies substantially on obtaining pure and well‐preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom‐up MS, and then evaluate the resulting data by a C. elegans−adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins","authors":"Christina J. Welch,&nbsp;Priyanka D. Kadav,&nbsp;Jared L. Edwards,&nbsp;Jessica Krycia,&nbsp;Melanie L. Talaga,&nbsp;Purnima Bandyopadhyay,&nbsp;Tarun K. Dam","doi":"10.1002/cpps.113","DOIUrl":"10.1002/cpps.113","url":null,"abstract":"<p>Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of crude extracts containing the target proteins from soybean flour</p><p><b>Alternate Protocol 1</b>: Preparation of crude extracts from Jack bean meal</p><p><b>Alternate Protocol 2</b>: Preparation of crude extracts from the corms of <i>Colocasia esculenta</i>, <i>Xanthosoma sagittifolium</i>, and from the bulbs of <i>Allium sativum</i></p><p><b>Alternate Protocol 3</b>: Preparation of <i>Escherichia coli</i> cell lysates containing human galectin-3</p><p><b>Alternate Protocol 4</b>: Preparation of crude extracts from chicken egg whites (source of ovalbumin)</p><p><b>Basic Protocol 2</b>: Preparation of 2% (v/v) red blood cell suspension</p><p><b>Basic Protocol 3</b>: Detection of lectin activity of the crude extracts</p><p><b>Basic Protocol 4</b>: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays</p><p><b>Basic Protocol 5</b>: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays</p><p><b>Basic Protocol 6</b>: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts</p><p><b>Basic Protocol 7</b>: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex</p><p><b>Basic Protocol 8</b>: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents</p><p><b>Basic Protocol 9</b>: Verification of the purity of the isolated targets (lectins or glycoproteins)</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.113","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38341341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of Recombinant Human Hemoglobin With NH2-Terminal Acetylation in Escherichia coli","authors":"Chandrasekhar Natarajan,&nbsp;Anthony V. Signore,&nbsp;Vikas Kumar,&nbsp;Jay F. Storz","doi":"10.1002/cpps.112","DOIUrl":"https://doi.org/10.1002/cpps.112","url":null,"abstract":"<p>The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in <i>Escherichia coli</i> host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH₂-terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH₂-terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH₂-terminal acetylation on O₂ binding properties. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Gene synthesis and cloning the cassette to the expression plasmid</p><p><b>Basic Protocol 2</b>: Selection of <i>E. coli</i> expression strains for coexpression</p><p><b>Basic Protocol 3</b>: Large-scale recombinant hemoglobin expression and purification</p><p><b>Support Protocol 1</b>: Measuring O₂ equilibration curves</p><p><b>Support Protocol 2</b>: Mass spectrometry to confirm NH₂-terminal acetylation</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.112","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72159192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Fluorescence-Based Assay to Monitor SUMOylation in Real-Time","authors":"Vasvi Tripathi,&nbsp;Ranabir Das","doi":"10.1002/cpps.111","DOIUrl":"10.1002/cpps.111","url":null,"abstract":"<p>The small ubiquitin-like modifier (SUMO) is an important post-translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS-PAGE and western blots, which makes these methods cumbersome, error-prone, and time-consuming. Here, we describe a fluorescence-based assay for real-time detection of SUMOylation. In the method, a fluorophore-tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τ_c) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real-time SUMOylation assay protocol is elegant, time-saving, and less prone to errors. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Fluorescent anisotropy-based in vitro SUMOylation assay</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38126252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Analysis of Protein Self-Association by Sedimentation Velocity","authors":"Huaying Zhao,&nbsp;Wenqi Li,&nbsp;Wendan Chu,&nbsp;Mary Bollard,&nbsp;Regina Adão,&nbsp;Peter Schuck","doi":"10.1002/cpps.109","DOIUrl":"10.1002/cpps.109","url":null,"abstract":"<p>Sedimentation velocity analytical ultracentrifugation is a powerful classical method to study protein self-association processes in solution based on the size-dependent macromolecular migration in the centrifugal field. This technique can elucidate the assembly scheme, measure affinities ranging from picomolar to millimolar <i>K</i>_d, and in favorable cases provide information on oligomer lifetimes and hydrodynamic shape. The present step-by-step protocols detail the essential steps of instrument calibration, experimental setup, and data analysis. Using a widely available commercial protein as a model system, the protocols invite replication and comparison with our results. A commentary discusses principles for modifications in the protocols that may be necessary to optimize application of sedimentation velocity analysis to other self-associating proteins. ©2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Measurement of external calibration factors</p><p><b>Basic Protocol 2</b>: Sedimentation velocity experiment for protein self-association</p><p><b>Basic Protocol 3</b>: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38109604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PLiMAP: Proximity-Based Labeling of Membrane-Associated Proteins","authors":"Gregor P. Jose,&nbsp;Thomas J. Pucadyil","doi":"10.1002/cpps.110","DOIUrl":"10.1002/cpps.110","url":null,"abstract":"<p>Peripheral membrane proteins participate in numerous biological pathways. Thus, methods to analyze their membrane-binding characteristics have become important. In this report, we detail protocols for the synthesis and utilization of a photoactivable fluorescent lipid as a reporter to monitor membrane binding of proteins. The assay, referred to as proximity-based labeling of membrane-associated proteins (PLiMAP), is based on UV activation of a fluorescent lipid reporter, which in turn crosslinks with proteins bound to membranes and renders them fluorescent. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of BODIPY-diazirine phosphatidylethanolamine (BDPE)</p><p><b>Basic Protocol 2</b>: Preparation of BDPE-containing liposomes</p><p><b>Basic Protocol 3</b>: Performing PLiMAP with a candidate protein</p><p><b>Basic Protocol 4</b>: Quantitation of liposome-binding properties of the candidate protein from analyzing in-gel fluorescence</p><p><b>Support Protocol</b>: Purification of GST-2×P4M domain of SidM protein</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.110","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38100434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads","authors":"Michihiko Tsushima,&nbsp;Shinichi Sato,&nbsp;Keita Nakane,&nbsp;Hiroyuki Nakamura","doi":"10.1002/cpps.108","DOIUrl":"10.1002/cpps.108","url":null,"abstract":"<p>Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer–mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1-methyl-4-aryl-urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst-functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of ruthenium photocatalyst</p><p><b>Basic Protocol 2</b>: Synthesis of azide- or desthiobiotin-conjugated labeling reagents</p><p><b>Basic Protocol 3</b>: Preparation of photocatalyst and ligand-functionalized affinity beads</p><p><b>Basic Protocol 4</b>: Target protein labeling in cell lysate</p><p><b>Basic Protocol 5</b>: Enrichment of labeled proteins with MAUra-DTB for LC-MS/MS analysis</p><p><b>Basic Protocol 6</b>: 2D-DIGE analysis of fluorescence-labeled proteins</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.108","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38099869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of Ciliary Tubulin from Chlamydomonas reinhardtii","authors":"Ron Orbach,&nbsp;Jonathon Howard","doi":"10.1002/cpps.107","DOIUrl":"10.1002/cpps.107","url":null,"abstract":"<p>Cilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule–based structure known as the axoneme, which serves as a scaffold and is crucial for the function of cilia. Despite their key roles, the biochemical and biophysical properties of the ciliary proteins are poorly understood. To address this issue, we have developed a novel method to purify functional tubulins from different parts of the axoneme, namely the central pair and B-tubule. We use the biflagellate green alga <i>Chlamydomonas reinhardtii</i>, a model organism for studying cilia due to the conserved structure of this organelle, availability of genetic tools and a large collection of mutant strains. Our method yields highly purified functional axonemal tubulins in sufficient quantities to be used for <i>in vitro</i> biochemical and biophysical studies, such as microtubule dynamic assays. It takes 7 to 8 days to grow enough cells; the isolation of the flagella and the purification of the axonemal tubulins require an additional two full days.© 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Growth and harvest of large volume of cell culture</p><p><b>Support Protocol</b>: Assembly of homemade concentration apparatus</p><p><b>Basic Protocol 2</b>: Isolation of flagella</p><p><b>Basic Protocol 3</b>: Tubulin extraction and purification</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38070193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}