Authors: Hsu, Chun-Hua¹; Pan, Yun-Ru²; Liao, You-Di³; Wu, Shih-Hsiung⁴; Chen, Chinpan³

Source: The Journal of Biochemistry, Volume 148, Number 2, 11 August 2010 , pp. 209-215(7)

Publisher: Oxford University Press

Abstract:

The stability, structures and steric hindrances of recombinant RNases 2 and 4 expressed in bacteria were studied by circular dichroism (CD) and NMR techniques, and the results were compared with those of their authentic RNases extracted from oocytes of Rana catesbeiana. Although the overall structures of the recombinant and authentic proteins are almost identical, the extra N-terminal Met residue of the recombinant protein remarkably affects catalytic activity and stability. NMR chemical shift comparison of recombinant RNases and the authentic proteins indicated that the structural differences are mainly confined to the N-terminal helical and S2 anti-parallel -sheet regions. Significant shift changes for the residues located on the S2 region indicate that the major influences on the structure around the N terminus is due to the loss of the hydrogen bond between Pyr¹ and Val⁹⁵⁽⁹⁶⁾ in recombinant RNases 2 and 4. We concluded the apparent steric hindrances of the extra Met to the binding pocket. As well, the affected conformational changes of active residues are attributed to the reduced activities of recombinant RNases. The structural integrity exerted by the N-terminal Pyr¹ residue may be crucial for amphibian RNases and the greatest structural differences occur on the network of the Pyr¹ residue and S2 -sheet region.

Keywords: N-terminal extra methionine; NMR; protein stability; pyroglutamate; ribonuclease

Document Type: Regular paper

DOI: http://dx.doi.org/10.1093/jb/mvq058

Affiliations: 1: Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan; , Institute of Biomedical Sciences; , 2: Institute of Biomedical Sciences; , Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 300; and , 3: Institute of Biomedical Sciences; , 4: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan,

Publication date: 2010-08-11

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