EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels

Lade...
Vorschaubild
Dateien
Zu diesem Dokument gibt es keine Dateien.
Datum
2015
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
URI (zitierfähiger Link)
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
ACS Chemical Biology. 2015, 10(12), pp. 2764-2771. ISSN 1554-8929. eISSN 1554-8937. Available under: doi: 10.1021/acschembio.5b00512
Zusammenfassung

The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
540 Chemie
Schlagwörter
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690SCHMIDT, Moritz J., Artem FEDOSEEV, Dennis BÜCKER, Julia BORBAS, Christine PETER, Malte DRESCHER, Daniel SUMMERER, 2015. EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels. In: ACS Chemical Biology. 2015, 10(12), pp. 2764-2771. ISSN 1554-8929. eISSN 1554-8937. Available under: doi: 10.1021/acschembio.5b00512
BibTex
@article{Schmidt2015Dista-32652,
  year={2015},
  doi={10.1021/acschembio.5b00512},
  title={EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels},
  number={12},
  volume={10},
  issn={1554-8929},
  journal={ACS Chemical Biology},
  pages={2764--2771},
  author={Schmidt, Moritz J. and Fedoseev, Artem and Bücker, Dennis and Borbas, Julia and Peter, Christine and Drescher, Malte and Summerer, Daniel}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/32652">
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dc:creator>Fedoseev, Artem</dc:creator>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2016-01-20T13:53:49Z</dcterms:available>
    <dc:contributor>Summerer, Daniel</dc:contributor>
    <dc:contributor>Fedoseev, Artem</dc:contributor>
    <dcterms:abstract xml:lang="eng">The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.</dcterms:abstract>
    <dc:contributor>Borbas, Julia</dc:contributor>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:creator>Summerer, Daniel</dc:creator>
    <dc:contributor>Peter, Christine</dc:contributor>
    <dc:contributor>Schmidt, Moritz J.</dc:contributor>
    <dcterms:title>EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels</dcterms:title>
    <dc:language>eng</dc:language>
    <dcterms:issued>2015</dcterms:issued>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/32652"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2016-01-20T13:53:49Z</dc:date>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dc:creator>Borbas, Julia</dc:creator>
    <dc:creator>Drescher, Malte</dc:creator>
    <dc:creator>Peter, Christine</dc:creator>
    <dc:creator>Schmidt, Moritz J.</dc:creator>
    <dc:contributor>Drescher, Malte</dc:contributor>
    <dc:contributor>Bücker, Dennis</dc:contributor>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dc:creator>Bücker, Dennis</dc:creator>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Diese Publikation teilen