Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor
Events
Loading … Spinner

Mendeley | Further Information

{"title"=>"Mapping the homodimer interface of an optimized, artificial, transmembrane protein activator of the human erythropoietin receptor", "type"=>"journal", "authors"=>[{"first_name"=>"Emily B.", "last_name"=>"Cohen", "scopus_author_id"=>"34976400900"}, {"first_name"=>"Susan J.", "last_name"=>"Jun", "scopus_author_id"=>"12240458000"}, {"first_name"=>"Zachary", "last_name"=>"Bears", "scopus_author_id"=>"56147339400"}, {"first_name"=>"Francisco N.", "last_name"=>"Barrera", "scopus_author_id"=>"7003622617"}, {"first_name"=>"Miriam", "last_name"=>"Alonso", "scopus_author_id"=>"56146991300"}, {"first_name"=>"Donald M.", "last_name"=>"Engelman", "scopus_author_id"=>"34769915600"}, {"first_name"=>"Daniel", "last_name"=>"DiMaio", "scopus_author_id"=>"7005547435"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84899743816", "sgr"=>"84899743816", "pui"=>"373008294", "isbn"=>"1932-6203", "pmid"=>"24788775", "doi"=>"10.1371/journal.pone.0095593"}, "id"=>"fabc6a73-ca35-3b27-9524-34ab0885e927", "abstract"=>"Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.", "link"=>"http://www.mendeley.com/research/mapping-homodimer-interface-optimized-artificial-transmembrane-protein-activator-human-erythropoieti", "reader_count"=>15, "reader_count_by_academic_status"=>{"Librarian"=>1, "Researcher"=>3, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>2, "Student > Master"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Librarian"=>1, "Researcher"=>3, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>2, "Student > Master"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>6, "Arts and Humanities"=>1, "Physics and Astronomy"=>2, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Chemistry"=>{"Chemistry"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"Denmark"=>1, "France"=>1}, "group_count"=>0}

Scopus | Further Information

{"@_fa"=>"true", "link"=>[{"@_fa"=>"true", "@ref"=>"self", "@href"=>"https://api.elsevier.com/content/abstract/scopus_id/84899743816"}, {"@_fa"=>"true", "@ref"=>"author-affiliation", "@href"=>"https://api.elsevier.com/content/abstract/scopus_id/84899743816?field=author,affiliation"}, {"@_fa"=>"true", "@ref"=>"scopus", "@href"=>"https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84899743816&origin=inward"}, {"@_fa"=>"true", "@ref"=>"scopus-citedby", "@href"=>"https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=84899743816&origin=inward"}], "prism:url"=>"https://api.elsevier.com/content/abstract/scopus_id/84899743816", "dc:identifier"=>"SCOPUS_ID:84899743816", "eid"=>"2-s2.0-84899743816", "dc:title"=>"Mapping the homodimer interface of an optimized, artificial, transmembrane protein activator of the human erythropoietin receptor", "dc:creator"=>"Cohen E.", "prism:publicationName"=>"PLoS ONE", "prism:eIssn"=>"19326203", "prism:volume"=>"9", "prism:issueIdentifier"=>"4", "prism:pageRange"=>nil, "prism:coverDate"=>"2014-04-30", "prism:coverDisplayDate"=>"30 April 2014", "prism:doi"=>"10.1371/journal.pone.0095593", "citedby-count"=>"4", "affiliation"=>[{"@_fa"=>"true", "affilname"=>"Yale University School of Medicine", "affiliation-city"=>"New Haven", "affiliation-country"=>"United States"}], "pubmed-id"=>"24788775", "prism:aggregationType"=>"Journal", "subtype"=>"ar", "subtypeDescription"=>"Article", "article-number"=>"e95593", "source-id"=>"10600153309", "openaccess"=>"1", "openaccessFlag"=>true}

Facebook

  • {"url"=>"http%3A%2F%2Fjournals.plos.org%2Fplosone%2Farticle%3Fid%3D10.1371%252Fjournal.pone.0095593", "share_count"=>1, "like_count"=>0, "comment_count"=>0, "click_count"=>0, "total_count"=>1}

Counter

  • {"month"=>"4", "year"=>"2014", "pdf_views"=>"7", "xml_views"=>"5", "html_views"=>"95"}
  • {"month"=>"5", "year"=>"2014", "pdf_views"=>"47", "xml_views"=>"7", "html_views"=>"209"}
  • {"month"=>"6", "year"=>"2014", "pdf_views"=>"17", "xml_views"=>"0", "html_views"=>"63"}
  • {"month"=>"7", "year"=>"2014", "pdf_views"=>"16", "xml_views"=>"0", "html_views"=>"43"}
  • {"month"=>"8", "year"=>"2014", "pdf_views"=>"4", "xml_views"=>"2", "html_views"=>"22"}
  • {"month"=>"9", "year"=>"2014", "pdf_views"=>"5", "xml_views"=>"1", "html_views"=>"32"}
  • {"month"=>"10", "year"=>"2014", "pdf_views"=>"5", "xml_views"=>"1", "html_views"=>"28"}
  • {"month"=>"11", "year"=>"2014", "pdf_views"=>"8", "xml_views"=>"1", "html_views"=>"27"}
  • {"month"=>"12", "year"=>"2014", "pdf_views"=>"3", "xml_views"=>"2", "html_views"=>"55"}
  • {"month"=>"1", "year"=>"2015", "pdf_views"=>"13", "xml_views"=>"0", "html_views"=>"41"}
  • {"month"=>"2", "year"=>"2015", "pdf_views"=>"19", "xml_views"=>"0", "html_views"=>"60"}
  • {"month"=>"3", "year"=>"2015", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"26"}
  • {"month"=>"4", "year"=>"2015", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"23"}
  • {"month"=>"5", "year"=>"2015", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"27"}
  • {"month"=>"6", "year"=>"2015", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"35"}
  • {"month"=>"7", "year"=>"2015", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"20"}
  • {"month"=>"8", "year"=>"2015", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"21"}
  • {"month"=>"9", "year"=>"2015", "pdf_views"=>"8", "xml_views"=>"2", "html_views"=>"64"}
  • {"month"=>"10", "year"=>"2015", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"71"}
  • {"month"=>"11", "year"=>"2015", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"63"}
  • {"month"=>"12", "year"=>"2015", "pdf_views"=>"2", "xml_views"=>"0", "html_views"=>"71"}
  • {"month"=>"1", "year"=>"2016", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"86"}
  • {"month"=>"2", "year"=>"2016", "pdf_views"=>"1", "xml_views"=>"0", "html_views"=>"28"}
  • {"month"=>"3", "year"=>"2016", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"66"}
  • {"month"=>"4", "year"=>"2016", "pdf_views"=>"9", "xml_views"=>"0", "html_views"=>"25"}
  • {"month"=>"5", "year"=>"2016", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"11"}
  • {"month"=>"6", "year"=>"2016", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"8"}
  • {"month"=>"7", "year"=>"2016", "pdf_views"=>"1", "xml_views"=>"0", "html_views"=>"14"}
  • {"month"=>"8", "year"=>"2016", "pdf_views"=>"8", "xml_views"=>"0", "html_views"=>"15"}
  • {"month"=>"9", "year"=>"2016", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"25"}
  • {"month"=>"10", "year"=>"2016", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"19"}
  • {"month"=>"11", "year"=>"2016", "pdf_views"=>"2", "xml_views"=>"0", "html_views"=>"21"}
  • {"month"=>"12", "year"=>"2016", "pdf_views"=>"7", "xml_views"=>"0", "html_views"=>"18"}
  • {"month"=>"1", "year"=>"2017", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"14"}
  • {"month"=>"2", "year"=>"2017", "pdf_views"=>"10", "xml_views"=>"1", "html_views"=>"40"}
  • {"month"=>"3", "year"=>"2017", "pdf_views"=>"2", "xml_views"=>"3", "html_views"=>"19"}
  • {"month"=>"4", "year"=>"2017", "pdf_views"=>"7", "xml_views"=>"0", "html_views"=>"35"}
  • {"month"=>"5", "year"=>"2017", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"21"}
  • {"month"=>"6", "year"=>"2017", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"24"}
  • {"month"=>"7", "year"=>"2017", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"21"}
  • {"month"=>"8", "year"=>"2017", "pdf_views"=>"8", "xml_views"=>"1", "html_views"=>"16"}
  • {"month"=>"9", "year"=>"2017", "pdf_views"=>"9", "xml_views"=>"1", "html_views"=>"22"}
  • {"month"=>"10", "year"=>"2017", "pdf_views"=>"4", "xml_views"=>"2", "html_views"=>"33"}
  • {"month"=>"11", "year"=>"2017", "pdf_views"=>"2", "xml_views"=>"0", "html_views"=>"54"}
  • {"month"=>"12", "year"=>"2017", "pdf_views"=>"3", "xml_views"=>"2", "html_views"=>"27"}
  • {"month"=>"1", "year"=>"2018", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"13"}
  • {"month"=>"2", "year"=>"2018", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"8"}
  • {"month"=>"3", "year"=>"2018", "pdf_views"=>"5", "xml_views"=>"2", "html_views"=>"10"}
  • {"month"=>"4", "year"=>"2018", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"8"}
  • {"month"=>"5", "year"=>"2018", "pdf_views"=>"0", "xml_views"=>"0", "html_views"=>"4"}
  • {"month"=>"6", "year"=>"2018", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"5"}
  • {"month"=>"7", "year"=>"2018", "pdf_views"=>"2", "xml_views"=>"3", "html_views"=>"3"}
  • {"month"=>"8", "year"=>"2018", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"7"}
  • {"month"=>"9", "year"=>"2018", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"5"}
  • {"month"=>"10", "year"=>"2018", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"4"}
  • {"month"=>"11", "year"=>"2018", "pdf_views"=>"7", "xml_views"=>"1", "html_views"=>"7"}
  • {"month"=>"12", "year"=>"2018", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"5"}
  • {"month"=>"1", "year"=>"2019", "pdf_views"=>"3", "xml_views"=>"1", "html_views"=>"11"}
  • {"month"=>"2", "year"=>"2019", "pdf_views"=>"1", "xml_views"=>"0", "html_views"=>"2"}
  • {"month"=>"3", "year"=>"2019", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"3"}
  • {"month"=>"4", "year"=>"2019", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"5"}
  • {"month"=>"5", "year"=>"2019", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"3"}
  • {"month"=>"6", "year"=>"2019", "pdf_views"=>"2", "xml_views"=>"0", "html_views"=>"5"}
  • {"month"=>"7", "year"=>"2019", "pdf_views"=>"7", "xml_views"=>"0", "html_views"=>"3"}
  • {"month"=>"8", "year"=>"2019", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"2"}
  • {"month"=>"9", "year"=>"2019", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"2"}
  • {"month"=>"10", "year"=>"2019", "pdf_views"=>"2", "xml_views"=>"0", "html_views"=>"2"}

Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1477825"], "description"=>"<p>(<b>A</b>) The sequence of TC2-3, which was used as a template to generate a retrovirus expression library in which a 19-amino acid transmembrane segment (positions 12 to 30, underlined) was mutagenized. All other residues are derived from the E5 protein and remained unchanged. (<b>B</b>) Equal numbers of BaF3/hEPOR cells expressing RFP alone (vector) or co-expressing TC2-3 and GFP (TC2-3) were co-cultured. Viable cells were analyzed by flow cytometry for GFP and RFP fluorescence immediately after mixing (left panel) and after two days in the absence of growth factors (right panel). (<b>C</b>) BaF3/hEPOR cells were infected with retrovirus expressing TC2-3 from a low expression vector, RVY-hygro (dashed line), or a high expression vector, T2H-F13 (solid line). After selection with hygromycin, viable cells were counted on the indicated days after growth factor removal. (<b>D</b>) Scheme to select optimized small transmembrane activators of the hEPOR. Black lines represent the hEPOR and gray and black X’s represent small transmembrane proteins. Small cells with nuclear blebs represent dead cells.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "confers", "dose-dependent", "hepor"], "article_id"=>1010034, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g001", "stats"=>{"downloads"=>2, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TC2_3_confers_cell_autonomous_dose_dependent_growth_factor_independence_in_hEPOR_cells_/1010034", "title"=>"TC2-3 confers cell-autonomous, dose-dependent growth factor independence in hEPOR cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477826"], "description"=>"<p>(<b>A</b>) (Left) Amino acid sequence of the transmembrane domain (positions 12 to 30) of TC2-3 and the mutants selected from the library. Residues in black indicate amino acid substitutions. (Right) BaF3/HA-hEPOR cells expressing TC2-3 or the selected mutants expressed from the low expression vector, RVY-puro, were tested for their ability to proliferate in the absence of growth factors. Viable cells were counted four days after growth factor removal. (<b>B</b>) Extracts were prepared from BaF3/HA-hEPOR cells expressing empty RVY-puro vector, TC2-3, or the indicated mutant. Samples were immunoprecipitated and immunoblotted with αE5. Size of protein markers (in kDa) is shown on left. (<b>C</b>) Empty MSCV-puro vector, TC2-3, or EBC5-16 were expressed in BaF3 cells expressing no exogenous receptor, murine PDGFβR, murine EPOR, or hEPOR. Cells were then tested for their ability to proliferate in the absence of growth factors. Viable cells were counted three days after growth factor removal. TC2-3 was active with hEPOR in this experiment because it was expressed from MSCV. (<b>D</b>) MSCV-puro/EBC5-16 was expressed in BaF3 cells expressing either HA-hEPOR (solid line) or HA-hEPOR(mPR) (dashed line), and cells were tested for their ability to proliferate in the absence of growth factors. Viable cells were counted on the indicated days.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "mutants", "amino", "substitutions", "compared"], "article_id"=>1010035, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g002", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transmembrane_protein_mutants_with_single_amino_acid_substitutions_display_increased_activity_compared_to_TC2_3_/1010035", "title"=>"Transmembrane protein mutants with single amino acid substitutions display increased activity compared to TC2-3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477828"], "description"=>"<p>(<b>A</b>) Extracts were prepared from parental BaF3 cells or BaF3/HA-hEPOR cells expressing empty RVY-puro vector (V), EBC5-16, or TC2-3. Where indicated, cells were acutely stimulated with EPO. Samples were immunoprecipitated with anti-HA (3F10) antibody and immunoblotted with anti-phosphotyrosine antibody. Size of protein markers (in kDa) is shown on left. (<b>B</b>) Extracts from BaF3/HA-hEPOR cells expressing RVY-puro vector (V), EBC5-16, or TC2-3. Where indicated, cells were acutely stimulated with EPO or RPMI-IL-3 medium. Samples were immunoblotted for phosphorylated JAK2. Blot was reprobed for total JAK2. Size of protein markers (in kDa) is shown on left. (<b>C</b>) Extracts from the cells described in (<b>B</b>) were immunoblotted for phosphorylated STAT5. Blot was reprobed for total STAT5. Size of protein markers (in kDa) is shown on left.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "tyrosine", "phosphorylation"], "article_id"=>1010037, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g003", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_EBC5_16_induces_tyrosine_phosphorylation_of_hEPOR_JAK2_and_STAT5_/1010037", "title"=>"EBC5-16 induces tyrosine phosphorylation of hEPOR, JAK2, and STAT5.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477829"], "description"=>"<p>(A) Primary human CD34<sup>+</sup> cells infected with retrovirus expressing empty CMMP-IRES-GFP vector (green), or CMMP-IRES-GFP expressing TC2-3 (red) or EBC5-16 (blue) were sorted for GFP fluorescence and transferred to differentiation medium in the absence of EPO. A sample of cells expressing vector was also treated with EPO (magenta). After six days in differentiation medium, viable cells were assessed for cell-surface GpA expression by immunostaining and flow cytometry. Similar results were obtained in four independent experiments. (B) Cells were handled as in (A). After six days in differentiation medium, the total number of viable cells expressing GpA (>50 fluorescence units) was determined by immunostaining and flow cytometry. Graph shows average of three independent experiments. Error bars represent the standard error of the mean. A student t-test determined the difference between EBC5-16 and TC2-3 samples to be statistically significant, p<0.05. (C) Cells handled as in (A), but cultured in differentiation medium in methylcellulose to measure erythroid colony formation. EPO was added where indicated. Percent colony forming efficiency is relative to vector plus EPO. Graph shows the average of three independent experiments. Error bars represent the standard error of the mean. A student t-test determined the difference between EBC5-16 and TC2-3 samples not to be statistically significant. (D) After six days in differentiation medium, total RNA was isolated from hHPCs expressing empty vector, TC2-3, or EBC5-16. EPO was added where indicated. Levels of human β-globin mRNA were determined by qRT-PCR relative to GAPDH mRNA. Expression is normalized to vector-infected cells in the absence of EPO. Graph shows average of three independent experiments. Error bars represent the standard error of the mean. A student t-test determined the difference between EBC5-16 and TC2-3 samples not to be statistically significant.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "displays", "erythroid", "differentiation", "hematopoietic", "progenitor"], "article_id"=>1010038, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g004", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_EBC5_16_displays_increased_ability_to_stimulate_erythroid_differentiation_of_human_hematopoietic_progenitor_cells_/1010038", "title"=>"EBC5-16 displays increased ability to stimulate erythroid differentiation of human hematopoietic progenitor cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477830"], "description"=>"<p>(<b>A</b>) Extracts were prepared from BaF3/HA-hEPOR cells expressing empty MSCV-puro vector (V), TC2-3, or EBC5-16. Samples were immunoprecipitated with αE5, electrophoresed in the presence or absence of reducing agents, and immunoblotted with the same antibody. Size of protein markers (in kDa) is shown on left. (<b>B</b>) TOXCAT analysis of EBC5-16 oligomerization. The transmembrane domain of TC2-3 or EBC5-16 was inserted into the maltose binding protein/ToxR fusion protein and expressed in <i>E. coli</i> containing a ToxR-dependent chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured <i>in vitro</i> after normalizing for the amount of fusion protein in the extract. Wild-type GpA and the dimerization-defective GpA G83I mutant were used as controls, and results are normalized to CAT activity induced by the GpA transmembrane domain. Graph shows the average of five independent experiments, each done in triplicate. Error bars represent standard error of the mean. A student t-test determined that the difference between EBC5-16 and TC2-3 samples was statistically significant, p < 10<sup>-5</sup>. (<b>C</b>) The sequences of EBC5-16 and EBC5-16-CCSS (amino acids 12 to the C-terminus) are shown, with position 25 and the cysteine to serine mutations in red. EBC5-16 (solid line) and EBC5-16-CCSS (dashed line) were expressed in BaF3/HA-hEPOR cells from the MSCV-puro vector. After puromycin selection, viable cells were counted on the indicated days after growth factor removal.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "ebc5-16"], "article_id"=>1010039, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g005", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ser25_increases_the_formation_of_EBC5_16_homodimers_/1010039", "title"=>"Ser25 increases the formation of EBC5-16 homodimers.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477831"], "description"=>"<p>(<b>A</b>) Schematic diagram of the fusion proteins constructed between the dimerization domain of Put3 and the transmembrane domain of EBC5-16. The sequences show the point of fusion for each of the chimeras. The different points of fusion cause the relative positions of the amino acids of the EBC5-16 segment to rotate relative to the fixed interface of the Put3 segment. Because inserting seven residues would rotate the EBC5-16 segment by two full turns (720°), inserting a single amino acid would rotate each helix by 103°. Therefore, inserting three or four residues at the point of fusion will rotate the helices by 309° or 412°, respectively, generating structures in which the orientation of the helices is most similar to the original structure. Thus, in the series of seven consecutive insertion constructs, the interfaces can be placed in the following order in terms of their similarity: 0, III, VI, II, V, I, IV, as is listed in panel C. (<b>B</b>) Heptagonal prisms representing α-helical monomers within Put3/EBC5-6 dimers V, II, and VI. The Put3 and EBC5-16 segments are shaded in gray and white, respectively. The dimer interfaces of native Put3 and EBC5-16 are shaded in red. (<b>C</b>) (Left) helical wheel diagrams of the seven Put3/EBC5-16 dimers, with Ser25 shown for orientation, are shown. (Right) BaF3/HA-hEPOR cells expressing these chimeras from the RVY-puro vector were tested for their ability to proliferate in the absence of growth factors. Viable cells were counted six days after growth factor removal. Graph shows results of a representative experiment. Similar results were obtained in three independent experiments. (<b>D</b>) Helical wheel diagram of the predicted EBC5-16 dimer (from Put3/EBC5-16 chimera II), with interface residues shown. Ser25 is highlighted in red. (<b>E</b>) Extracts were prepared from BaF3/HA-hEPOR cells expressing empty RVY-puro vector or a Put3/EBC5-16 chimera from RVY-puro, immunoprecipitated with αE5, separated in the presence or absence of reducing agents, and immunoblotted with an anti-AU1 antibody. Size of protein markers (in kDa) is shown on left.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "ebc5-16", "homodimer", "interface", "put3", "fusion"], "article_id"=>1010040, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g006", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mapping_the_EBC5_16_homodimer_interface_with_Put3_fusion_proteins_/1010040", "title"=>"Mapping the EBC5-16 homodimer interface with Put3 fusion proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477832"], "description"=>"<p>(<b>A</b>) BaF3/HA-hEPOR cells expressing EBC5-16 or the indicated point mutant from MSCV-puro were tested for their ability to proliferate in the absence of growth factors. Viable cells were counted four days after growth factor removal. Graph shows the results of a representative experiment. Similar results were obtained in three independent experiments. (<b>B</b>) BaF3/hEPOR cells expressing empty RVY-hygro vector (blue), TC2-3 (red), EBC5-16 (black), or EBC5-16 S25A point mutant (green) were tested for their ability to proliferate in the absence of growth factors. The amino acid at position 25 is shown, according to the same color code. Viable cells were counted on the indicated days. Two and a half percent heat-inactivated FBS was used instead of 10%.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation"], "article_id"=>1010041, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g007", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mutational_analysis_of_EBC5_16_/1010041", "title"=>"Mutational analysis of EBC5-16.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477833"], "description"=>"<p>(<b>A</b>) The graph shows the interhelical interaction energy of the amino acids in the EBC5-16 homodimer CHI model discussed in the text. The sequence used for the modeling is shown at the bottom. (<b>B</b>) Axial views of helical backbone of the CHI models. First panel shows the view from N-terminus with Val14 side-chain shown in stick figure. Second panel shows view from N-terminus with Gly11 and Gly15 shown as space-filling. The right two panels show the view from C-terminus of EBC5-16 and TC2-3, with Phe29 side-chain shown in stick figure. (<b>C</b>) Left panel: Models of the TC2-3 and EBC5-16 homodimers predicted by CHI simulation, shown in lateral ribbon view with interface residues predicted by the Put3 experiments in red and Val14 in blue. Right panel: Zoomed-in view of an EBC5-16 monomer showing intramolecular H-bonding of the Ser25 side-chain to the backbone carbonyl group of Ile21, represented by the dotted yellow line. Oxygen atoms are shown in pink, hydrogen atoms are shown in white.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "modeling", "tc2-3"], "article_id"=>1010042, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g008", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Molecular_modeling_of_TC2_3_and_EBC5_16_/1010042", "title"=>"Molecular modeling of TC2-3 and EBC5-16.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477834"], "description"=>"<p>(A) Amino acid sequences of the transmembrane domains of EBC5-16, the pL(12-30) poly-leucine construct, and pL(12–30) with predicted interface residues (in black) added back (pL-GIPSF). (B) BaF3/HA-hEPOR cells expressing pL(12–30) (dashed line) or pL-GIPSF (solid line) from MSCV-puro were tested for their ability to proliferate in the absence of growth factors. Viable cells were counted on the indicated days after growth factor removal. Graph shows the results of a representative experiment. Similar results were obtained in four independent experiments. (C) Extracts were prepared from BaF3/HA-hEPOR cells expressing AU1-tagged pL(12–30) or pL-GIPSF from MSCV-puro, immunoprecipitated with αE5, electrophoresed in the presence or absence of reducing agents, and immunoblotted with the same antibody. Size of protein markers (in kDa) is shown on left. Gel was soaked in 200 mM DTT for 30 min prior to transfer. (D) Surface representation of the EBC5-16 dimer with the predicted interface residues in red and Val14 in blue.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "homodimer", "interface", "ebc5-16", "dimerization"], "article_id"=>1010043, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593.g009", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Residues_predicted_to_be_in_the_homodimer_interface_of_EBC5_16_are_sufficient_to_restore_dimerization_and_activity_/1010043", "title"=>"Residues predicted to be in the homodimer interface of EBC5-16 are sufficient to restore dimerization and activity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-04-30 03:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1477835"], "description"=>"<div><p>Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) <i>in trans.</i> This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells <i>in vitro</i>, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.</p></div>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein interactions", "proteomics", "biophysics", "Biophysical simulations", "biotechnology", "Bioengineering", "Protein engineering", "cell biology", "Cellular structures and organelles", "Cell membranes", "Membrane proteins", "Transmembrane proteins", "Signal transduction", "cell signaling", "Membrane receptor signaling", "Transmembrane signaling", "Molecular cell biology", "genetics", "Gene identification and analysis", "Genetic screens", "Molecular genetics", "mutagenesis", "mutation", "homodimer", "interface", "transmembrane", "activator", "erythropoietin"], "article_id"=>1010044, "categories"=>["Biological Sciences"], "users"=>["Emily B. Cohen", "Susan J. Jun", "Zachary Bears", "Francisco N. Barrera", "Miriam Alonso", "Donald M. Engelman", "Daniel DiMaio"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0095593", "stats"=>{"downloads"=>2, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mapping_the_Homodimer_Interface_of_an_Optimized_Artificial_Transmembrane_Protein_Activator_of_the_Human_Erythropoietin_Receptor_/1010044", "title"=>"Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-04-30 03:02:03"}

PMC Usage Stats | Further Information

  • {"unique-ip"=>"17", "full-text"=>"15", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2014", "month"=>"5"}
  • {"unique-ip"=>"19", "full-text"=>"20", "pdf"=>"8", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"6"}
  • {"unique-ip"=>"10", "full-text"=>"11", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"4"}
  • {"unique-ip"=>"8", "full-text"=>"9", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"5"}
  • {"unique-ip"=>"6", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"6"}
  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"7"}
  • {"unique-ip"=>"16", "full-text"=>"9", "pdf"=>"11", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"3"}
  • {"unique-ip"=>"46", "full-text"=>"51", "pdf"=>"24", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"15", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"2"}
  • {"unique-ip"=>"12", "full-text"=>"8", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"8"}
  • {"unique-ip"=>"9", "full-text"=>"8", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"9"}
  • {"unique-ip"=>"15", "full-text"=>"13", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"14", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2015", "month"=>"10"}
  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"7"}
  • {"unique-ip"=>"20", "full-text"=>"19", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"8"}
  • {"unique-ip"=>"11", "full-text"=>"6", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"9"}
  • {"unique-ip"=>"14", "full-text"=>"8", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2014", "month"=>"10"}
  • {"unique-ip"=>"5", "full-text"=>"8", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"2"}
  • {"unique-ip"=>"13", "full-text"=>"10", "pdf"=>"2", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"11"}
  • {"unique-ip"=>"8", "full-text"=>"6", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"12"}
  • {"unique-ip"=>"24", "full-text"=>"26", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"1"}
  • {"unique-ip"=>"14", "full-text"=>"11", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"1", "year"=>"2015", "month"=>"11"}
  • {"unique-ip"=>"7", "full-text"=>"4", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"12"}
  • {"unique-ip"=>"6", "full-text"=>"5", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"1"}
  • {"unique-ip"=>"6", "full-text"=>"2", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"3"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"4"}
  • {"unique-ip"=>"7", "full-text"=>"9", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"5"}
  • {"unique-ip"=>"10", "full-text"=>"5", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"6"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"7"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"8"}
  • {"unique-ip"=>"3", "full-text"=>"4", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"9"}
  • {"unique-ip"=>"9", "full-text"=>"9", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"6", "full-text"=>"8", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
  • {"unique-ip"=>"6", "full-text"=>"6", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"2", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"4"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"6"}
  • {"unique-ip"=>"4", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"7"}
  • {"unique-ip"=>"9", "full-text"=>"13", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"8"}
  • {"unique-ip"=>"2", "full-text"=>"1", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"4", "full-text"=>"2", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"1", "full-text"=>"0", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"4", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"4", "full-text"=>"7", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"7", "full-text"=>"2", "pdf"=>"9", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}

Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[291]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[282]}, {"subject_area"=>"/Biology and life sciences/Developmental biology", "average_usage"=>[285]}, {"subject_area"=>"/Medicine and health sciences/Physiology", "average_usage"=>[278]}, {"subject_area"=>"/Physical sciences", "average_usage"=>[271]}, {"subject_area"=>"/Physical sciences/Chemistry", "average_usage"=>[262]}]}
Loading … Spinner
There are currently no alerts