High-Resolution Heteronuclear
Multidimensional NMR
of Proteins in Living Insect Cells Using a Baculovirus Protein Expression
System
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Abstract
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 <sup>1</sup>H–<sup>15</sup>N correlation spectra were observed
for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance
NMR spectra of the <i>Streptococcus</i> 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 <i>ab initio</i>, yielding
approximately 80% of the expected backbone NMR resonances. Well-resolved
NOE cross peaks could be identified in the 3D <sup>15</sup>N-separated
NOESY spectrum, suggesting that structural analysis of this size of
protein will be feasible in sf9 cells