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

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 (1)H-(15)N 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 (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

A Unique Tool for Cellular Structural Biology: In-cell NMR

“…In-cell NMR on proteins expressed directly in the cells can also be performed in yeast, as shown by Bertrand et al (Fig. 1b), and in insect cells, as shown by Hamatsu et al (31) (Fig. 1c).…”

Isotope Labeling of Proteins in Insect Cells

“…Uniform 13 C or 15 N, 13 C labeling can be performed using commercial Bioexpress 2000 (CIL; Hamatsu et al, 2013;Strauss et al, 2005) or homemade media (Sitarska et al, 2015;Walton, Kasprzak, Hare, & Logan, 2006). For carbon labeling, algal extracts are the most economic source of amino acids, as they can be produced from 13 CO 2 instead of more complex and more expensive carbohydrates.…”

“…In-cell NMR was successfully demonstrated using insect cells as the expression host (Hamatsu et al, 2013). Four proteins, ranging in length from 57 to 148 amino acids, were expressed uniformly 13 C, 15 N-labeled in Sf9 cells using the Bioexpress 2000 medium (CIL).…”

Protein–nucleic acids interactions: new ways of connecting structure, dynamics and function

“…While an understanding of nucleoprotein architecture at atomic resolution and how it changes in response to regulatory events can be achieved using structural biology techniques, singlemolecule techniques allow us to visualize and interrogate biologically important molecules individually, in real time. For both structural biology and single-molecule techniques, the ability to validate observations made in vitro under physiological conditions is both more important and more feasible (Sakakibara et al 2009;Ito et al 2012;Hamatsu et al 2013). The ability to observe and directly manipulate individual protein and nucleic acid molecules provides further unprecedented insight into the kinetics of the molecular recognition steps, which are often obscured in bulk biochemical and biophysical studies (Cornish and Ha 2007;Boehm et al 2016).…”