Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Abstract

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

Structure Determination of a Membrane Protein in Proteoliposomes

“…In the future it may be possible to extend the method to proteins in intact cell membranes. 65 It is increasingly well-recognized that the surrounding membrane environment is important for preserving the structural and functional integrity of membrane proteins. 93 Structure determination of membrane proteins in natural settings provides access to fundamental biological processes that depend on an integral membrane environment for function (e.g., dynamics, conformational changes, and interactions with ligands, drugs, antibody fragments, and lipids).…”

Correlating the structure and activity ofY. pestisAil in a bacterial cell envelope

“…S4). Treatment with cerulenin, an inhibitor of fatty acid biosynthesis has been used to inhibit 13 C labeling of E. coli lipids and simplify 13 C/ 13 C correlation spectra of proteins [21]. Moreover, spectroscopic approaches have been developed to silence lipid signals based on 15 N-filtering [36], and NMR data acquisition above the gel-to-liquid phase transition of the lipids has also been shown to suppress lipid signal intensity [41].…”

“…We have shown that nanodiscs and liposomes can incorporate Ail with various types of LPS, including native Y. pestis LPS [14,19], but while these detergentfree platforms represent important advances in membrane complexity, they remain distant substitutes for the native outer membrane environment, which is highly anisotropic, heterogeneous, and connected to the cytoskeletal peptidoglycan layer. The emerging field of in-situ NMR [20][21][22][23][24][25] presents new opportunities for examining the structural properties of functional Ail in a native bacterial outer membrane.…”

Membrane proteins, magic-angle spinning, and in-cell NMR

“…Dynamic nuclear polarization should improve sensitivity, and its application to native membrane systems is feasible (12,13). New expression systems offer even more powerful ways to suppress background signals (14,15), and membrane fractionation is a tried and true method for improving sample homogeneity and reducing background (16,17). Finally, site-specific labeling with unnatural amino acids provides residuespecific information for protein in real membranes (18).…”