A novel cross-linked RNase A dimer with enhanced enzymatic properties.
A new cross-linked ribonuclease A (RNase A) dimer composed of monomeric units covalently linked by a single amide bond between the side-chains of Lys(66) and Glu(9) is described. The dimer was prepared in the absence of water by incubating a lyophilized preparation of RNase, sealed under vacuum, in an oven at 85 degrees C. It was determined that the in vacuo procedure does not induce any significant conformational changes to the overall structure of RNase A, yet the amide cross-link has an increased acid lability, indicating that it is exposed and conformationally strained. Examination of X-ray crystallographic structures indicates that Lys(66) and Glu(9) are not close enough for the in vacuo dimer to adopt any of the known domain-swapped conformations. Therefore, the in vacuo RNase A dimer appears to be a novel dimeric structure. The in vacuo RNase A dimer also exhibits a twofold increase in activity over monomeric RNase A on a per monomer basis. This doubling of enzymatic activity was shown using dsRNA and ssRNA as substrates. In addition to this enhanced ability to degrade RNA, the dimer is not inhibited by the cellular ribonuclease inhibitor protein (cRI).
Simons
Brigitte L
BL
Centre for Biologics Research, Biologics and Genetics Therapies Directorate, Health Canada, Ottawa, Ontario, Canada.
Kaplan
Harvey
H
Fournier
Sylvie M
SM
Cyr
Terry
T
Hefford
Mary Alice
MA
D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D019281:Dimerization; D005612:Freeze Drying; D018698:Glutamic Acid / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D012330:RNA, Double-Stranded; D018698:Glutamic Acid; D012313:RNA; D012259:Ribonuclease, Pancreatic; D008239:Lysine