Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.
Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2. We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system. To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside. After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture. The concentrated R1 protein solution had a pinkish red color. Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex. However, all attempts to demonstrate a function of this complex have been inconclusive. A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation. Circular dichroism spectroscopy indicated an alpha-helical content of 50%. A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein. Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein.
Davis
R
R
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Thelander
M
M
Mann
G J
GJ
Behravan
G
G
Soucy
F
F
Beaulieu
P
P
Lavallée
P
P
Gräslund
A
A
Thelander
L
L
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000302:isolation & purification* / Q000378:metabolism; D013455:Sulfur
D000345:Affinity Labels; D002241:Carbohydrates; D010455:Peptides; D011994:Recombinant Proteins; D013455:Sulfur; D007501:Iron; D012264:Ribonucleotide Reductases