Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Abstract

While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can be applied towards the determination of 3D structural information. However, there are numerous challenges that need to be overcome to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges with a specific emphasis on obtaining high-resolution structural insights into electron transfer biological processes mediated by membrane-bound proteins like mammalian cytochrome b5, cytochrome P450 and cytochrome P450 reductase. In this study, we demonstrate the feasibility of using the signal-enhancement rendered by dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (ε). Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrates that it is highly possible to suppress the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution 3D structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

Is solid-state NMR enhanced by dynamic nuclear polarization?

“…studied by conventional ssNMR can be down to even $ 30 Hz (with specific isotopic labeling) [48], attributable to high crystallinity or fast conformational averaging. However, dissolution or suspension of the biomolecular analyte in frozen DNP matrices can increase this value to a few hundreds of Hz due to inhomogeneous broadening, leading to a decrease in resolution [49][50][51][52][53][54]. Just cooling the biological sample to low temperatures, in the absence of DNP matrices, can produce extensive inhomogeneous broadening as various conformers are trapped, especially in areas of high solvation level and mobility at RT [55].…”

Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR

“…The in-situ NMR experiments may also serve as a steppingstone toward structural characterization of LHC pigment-protein complexes in their native environment. While in-cell or in-situ NMR spectroscopy for structure characterization is very challenging, several studies have been reported, for example solid-state NMR of recombinant-expressed PagL in E. coli whole cells and cell envelopes, in-situ NMR of the Chl-binding CsmA protein and Dynamic NuclearPolarization NMR of proteins in native cellular membranes[59][60][61]. In-cell NMR studies that are aimed at structural characterization of a target protein generally rely on genetic manipulation to reduce the background signals of other cellular or membrane components.…”

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

“…Indeed, solid-state NMR has a rich history as an analytical tool to study the composition, structure, dynamics and function of solid materials, ranging from coal and earth materials, industrial polymers and catalysts to biomaterials including spider silk, insect exoskeletons, amyloids, membrane proteins, cell walls, whole cells, biofilms and intact tissues [21][22][23][24][25][26][27]. In this contribution, we discuss solidstate NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics in cell walls and whole cells, focusing on examples in S. aureus.…”