High-Resolution Heteronuclear Multidimensional NMR of Proteins in Living Insect Cells Using a Baculovirus Protein Expression System

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cel...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2013-02, Vol.135 (5), p.1688-1691
Main Authors: Hamatsu, Jumpei, O’Donovan, Daniel, Tanaka, Takashi, Shirai, Takahiro, Hourai, Yuichiro, Mikawa, Tsutomu, Ikeya, Teppei, Mishima, Masaki, Boucher, Wayne, Smith, Brian O, Laue, Ernest D, Shirakawa, Masahiro, Ito, Yutaka
Format: Article
Language:English
Subjects:
Algorithms
Animals
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Baculoviridae - genetics
Calmodulin - chemistry
Calmodulin - genetics
Cell Line
Entropy
Humans
Metallochaperones - chemistry
Metallochaperones - genetics
Nuclear Magnetic Resonance, Biomolecular
Quantum Theory
Rats
Sf9 Cells
Spodoptera - virology
Thermus thermophilus - genetics
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Summary:Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D 1H–15N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D 15N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja310928u