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Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik. (Britt-Marie Sjöberg)
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario,.
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2010 (Engelska)Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 23, nr 8, s. 633-641Artikel i tidskrift (Refereegranskat) 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

Ort, förlag, år, upplaga, sidor
Oxford University Press , 2010. Vol. 23, nr 8, s. 633-641
Nyckelord [en]
Bacillus anthracis, biotin-streptavidin immobilization, inhibitors, interaction constants, nucleotides
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
URN: urn:nbn:se:su:diva-45735DOI: 10.1093/protein/gzq035ISI: 000280316000005OAI: oai:DiVA.org:su-45735DiVA, id: diva2:369442
Tillgänglig från: 2010-11-10 Skapad: 2010-11-10 Senast uppdaterad: 2020-03-04Bibliografiskt granskad
Ingår i avhandling
1. Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
Öppna denna publikation i ny flik eller fönster >>Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2010. s. 57
Nyckelord
Ribonucleotide reductase, allosteric regulation, quaternary structure, subunit interactions, enzyme activity, SPR, GEMMA
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:su:diva-45740 (URN)978-91-7447-186-1 (ISBN)
Disputation
2010-12-16, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Tillgänglig från: 2010-11-24 Skapad: 2010-11-10 Senast uppdaterad: 2010-12-06Bibliografiskt granskad

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Crona, MikaelSjöberg, Britt-Marie
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Institutionen för molekylärbiologi och funktionsgenomik
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Protein Engineering Design & Selection
Biokemi och molekylärbiologi

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Totalt: 331 träffar
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