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No more labeled lipids

SBKB [doi:10.1038/sbkb.2011.18]
Technical Highlight - May 2011
Short description: Blocking the production of phospholipids allows for clearer NMR signals in cSPP systems.

Blocking phospholipid biosynthesis with cerulenin (shown) improves NMR resolution of membrane proteins in a cSPP system.

Condensed single-protein-production (cSPP), which relies on RNA interferase activity to suppress expression of other cellular proteins, is incredibly useful for examining the behavior and structure of proteins. By loading the cells with 13C-glucose, the targeted protein is then automatically labeled and suitable for use in NMR experiments, such as whole-cell NMR. The challenge is that other cellular components, such as membrane phosopholipids, also incorporate 13C-glucose, leading to spurious NMR signals that can mask the signals of specific amino acid residues. This is particularly problematic for the analysis of membrane proteins, which would ideally be examined in natural membrane fractions to maintain a physiologically relevant environment.

In a recent study, Mao and colleagues (PSI NESG) hit upon a simple yet very effective solution to the problem: block the synthesis of phospholipids with the antibiotic, cerulenin. By treating bacteria cells subjected to the SPP system with cerulenin, the authors were able to inhibit phospholipid biosynthesis by approximately 95% while maintaining the expression levels of a target protein. Expression and 13C labeling of the c subunit of the Escherichia coli ATP synthase F0 in cells with and without cerulenin allowed for solid-state NMR analysis of the effects of cerulenin on the NMR signal. Cross-peaks due to labeled lipids were significantly reduced in the presence of cerulenin, enabling clear resolution of the signal from subunit c residues. This removal of the interference from labeled phospholipids in cSPP systems has now opened the doors for the rapid examination of the structure of membrane proteins with much improved accuracy.

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  1. L. Mao et al. Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system.
    J. Biomol. NMR 49, 131-137 (2011). doi:10.1007/s10858-011-9469-5

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