NMR and biophysical elucidation of structural effects on extra N-terminal methionine residue of recombinant amphibian RNases from Rana catesbeiana.
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 beta-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 beta-sheet region.
Hsu
Chun-Hua
CH
Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan. andyhsu@ntu.edu.tw
Pan
Yun-Ru
YR
Liao
You-Di
YD
Wu
Shih-Hsiung
SH
Chen
Chinpan
C
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry*; D005260:Female; D006860:Hydrogen Bonding; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry*; D016415:Sequence Alignment
D011994:Recombinant Proteins; D008715:Methionine; D004722:Endoribonucleases; D012260:Ribonucleases; C083939:ribonuclease 4; C029494:ribonuclease U