NMR and biophysical elucidation of structural effects on extra N-terminal methionine residue of recombinant amphibian RNases from Rana catesbeiana
Journal
Journal of Biochemistry
Journal Volume
148
Journal Volume
148
Journal Issue
2
Journal Issue
2
Pages
209-215
Start Page
209
End Page
215
ISSN
17562651
Date Issued
2010-08
Author(s)
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 β-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 Pyr 1 and Val 95(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 Pyr 1 residue may be crucial for amphibian RNases and the greatest structural differences occur on the network of the Pyr 1 residue and S2 β-sheet region.
Subjects
N-terminal extra methionine
NMR
protein stability
pyroglutamate
ribonuclease
SDGs
Type
journal article