Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Britt-Marie Sjöberg)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario,.
Show others and affiliations
2010 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 23, no 8, 633-641 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors

Place, publisher, year, edition, pages
Oxford University Press , 2010. Vol. 23, no 8, 633-641 p.
Keyword [en]
Bacillus anthracis, biotin-streptavidin immobilization, inhibitors, interaction constants, nucleotides
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-45735DOI: 10.1093/protein/gzq035ISI: 000280316000005OAI: oai:DiVA.org:su-45735DiVA: diva2:369442
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
Open this publication in new window or tab >>Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Deoxyribonucleic acid (DNA) chains in which our genetic blueprint is stored are built from four DNA precursors by DNA polymerases. The enzyme ribonucleotide reductase (RNR) provides the only de novo synthesis pathway of deoxyribonucleotides from ribonucleotides and is essential for nearly all organisms. All four ribonucleotides are substrates for RNR and key to this flexibility is a sophisticated allosteric regulation. Nucleotide effectors (ATP, dATP, dTTP or dGTP) binding to the allosteric specificity site determines substrate specificity for the active site. When present at high concentrations, dATP binds to the allosteric overall activity site and inhibits activity by an unknown mechanism. Three approaches, RNR activity measurements, subunit interaction studies and quaternary structure studies were applied to four different class I RNRs to address the allosteric overall regulation. We found that allosteric overall inhibition was closely linked to formation of tight and large RNR protein complexes; α4β4 complex for the Escherichia coli class Ia RNR and α6β2 for the Dictyostelium discoideum class Ia RNR with functional allosteric inhibitions. The Aeh1 phage class Ia RNR with a non-functional dATP inhibition showed weak remnant inhibition features, while the Bacillus anthracis class Ib RNR without the allosteric overall regulation domain lacked these features. In addition, we presented the first biochemical characterization of a mechanism to restore protein function after gene fragmentation, we showed that the B. anthracis class Ib RNR was most active when reconstituted with manganese and in the presence of a physiological redoxin protein and we found that the class Ia RNR is the principal RNR in D. discoideum, although the coexisting class II RNR could partly compensate class I RNR inhibition during axenic growth. Finally, our improved method for studying RNR interactions has potential for RNR inhibitor screening.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2010. 57 p.
Keyword
Ribonucleotide reductase, allosteric regulation, quaternary structure, subunit interactions, enzyme activity, SPR, GEMMA
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45740 (URN)978-91-7447-186-1 (ISBN)
Public defence
2010-12-16, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2010-11-24 Created: 2010-11-10 Last updated: 2010-12-06Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Crona, MikaelSjöberg, Britt-Marie
By organisation
Department of Molecular Biology and Functional Genomics
In the same journal
Protein Engineering Design & Selection
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 150 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf