orals 5th Asia-Pacific NMR Symposium 2013

Structural analysis of proteins inside living sf9 cells by in-cell NMR spectroscopy (#33)

Takashi Tanaka 1 , Jumpei Hamatsu 1 , Emiko Seiwa 1 , Masaki Mishima 1 , Teppei Ikeya 1 , Yutaka Ito 1
  1. Graduate School of Engineering and Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

  In vivo detection of tertiary structures, structural changes, dynamics or interactions of proteins is required for the understanding of structural bases of their functions in living systems.

  The non-invasive character of NMR spectroscopy makes it ideally suited for detailed investigations of protein behaviours in living environment. Recently, various improvements in hardware, pulse sequences and stable isotope labelling techniques arrows us observation of high-resolution heteronuclear multi-dimensional NMR spectra of proteins inside living cells (in-cell NMR)1,2  . 

  In the case of in-cell NMR of eukaryotic cells, the systems utilising Xenopus laevis oocytos or eggs3,4   or cultured mammalian cells5,6   have been reported. The existing protocols for introducing stable isotope-labelled proteins into the eukaryotic cells for in-cell NMR measurements require relatively large quantity of purified and concentrated stable-isotope enriched proteins, thus restricting the applications to proteins which are difficult to purify and/or unstable. The alternative approaches utilising intrinsic protein expression systems have therefore been awaited in order to extend the range of applications.

  In this presentation, we show a demonstration of eukaryotic in-cell NMR employing the sf9/baculovirus system. Since there is time lag of hours from the inoculation of baculovirus to the expression of target proteins, the timing to introduce 13C/15N-labelled media was optimised so as to achieve the best contrast of signals of target proteins and background. This optimisation provided the significant improvement of the quality of the spectra, and a large majority of backbone resonances of streptcoccus protein G B1 domain (GB1) in sf9 cells were assigned exclusively from the in-cell NMR spectra, which is, to the best of our knowledge, the world's first achievement in eukaryotic cells7 .

  1. Serber, Z. et al. J. Am. Chem. Soc. 123, 2446-2447 (2001).
  2. Serber, Z. et al. J. Am. Chem. Soc. 123, 8895-8901 (2001).
  3. Selenko, P. et al. Proc. Natl. Acad. Sci. USA 103, 11904-11909 (2006).
  4. Sakai, T. et al. J. Biomol. NMR . 36, 179-188 (2006).
  5. Inomata, K. et al. Nature. 458, 106-110 (2009).
  6. Ogino, S. et al. J. Am. Chem. Soc. 131, 10834-10825(2009).
  7. Hamatsu, J. et al. J. Am. Chem. Soc. 135, 1688-1691(2013).