Preparation, characterization, and complete heteronuclear NMR resonance assignments of the glutaredoxin (C14S)-ribonucleotide reductase B1 737-761 (C754S) mixed disulfide

Biochemistry
M J BerardiJ H Bushweller

Abstract

The first committed step in de novo DNA biosynthesis involves the conversion of ribonucleotides to the corresponding deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase. Reduction of disulfides in ribonucleotide reductase is essential and is catalyzed by the protein disulfide reductants glutaredoxin or thioredoxin. The interaction region between Escherichia coli glutaredoxin-1 and E. coli ribonucleotide reductase has been localized to the C-terminal end of the B1 subunit of ribonucleotide reductase. We have demonstrated that a 25-residue peptide corresponding to this C-terminal sequence is a very good substrate for glutaredoxin via a fluorescence assay and that this peptide binds in a specific manner via isothermal titration calorimetric measurements. By selectively mutating the two cysteines in the peptide, we have identified the electrophilic cysteine as C759 (B1 numbering) and prepared a mixed disulfide between E. coli glutaredoxin-1 (C14 --> S) and the C759 monothiol form of the peptide. The peptide and the protein have been labeled with 13C and 15N, and complete heteronuclear NMR resonance assignments have been completed for both the peptide and the protein in the complex. By using half-filtered NOESY spe...Continue Reading

References

May 10, 1995·Archives of Biochemistry and Biophysics·J A ThomasR Honzatko
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Citations

May 26, 2004·Biochimica Et Biophysica Acta·Matthias KolbergK Kristoffer Andersson
Jan 30, 2009·The Journal of Biological Chemistry·Farnaz Zahedi Avval, Arne Holmgren
Mar 3, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·John-Paul BacikBart Hazes
Mar 27, 2001·Protein Science : a Publication of the Protein Society·J W CaveD E Wemmer
Jun 3, 2008·Journal of Magnetic Resonance·Gabriela PasatJeffrey W Peng
Sep 24, 1999·Journal of Molecular Biology·M J Berardi, J H Bushweller
Jan 13, 2015·Biological Chemistry·Marcel Deponte, Christopher Horst Lillig
Mar 13, 1999·The Journal of Biological Chemistry·C H LilligA Holmgren
Jul 3, 1999·The Journal of Biological Chemistry·L VerdoucqY Meyer
Jul 25, 1998·Journal of Molecular Biology·C SunJ H Bushweller

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