Journal of Biomolecular NMR

, Volume 2, Issue 2, pp 137–147

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

  • Yasushi Oda
  • Haruki Nakamura
  • Toshio Yamazaki
  • Kuniaki Nagayama
  • Mayumi Yoshida
  • Shigenori Kanaya
  • Morio Ikehara
Research Papers

Summary

Two-dimensional (2D)1H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the1H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D1H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.

Keywords

Ribonuclease H Selective proton labeling Two-dimensional NMR 

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Copyright information

© ESCOM Science Publishers B.V 1992

Authors and Affiliations

  • Yasushi Oda
    • 1
  • Haruki Nakamura
    • 1
  • Toshio Yamazaki
    • 2
  • Kuniaki Nagayama
    • 2
  • Mayumi Yoshida
    • 3
  • Shigenori Kanaya
    • 1
  • Morio Ikehara
    • 1
  1. 1.Protein Engineering Research InstituteSuita OsakaJapan
  2. 2.Biometrology LaboratoryJEOL Ltd.TokyoJapan
  3. 3.Tokyo Research LaboratoriesKyowa Hakko Kogyo Co. Ltd.TokyoJapan

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