[NMR paper] Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.

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Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.

Related Articles Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.

Biochim Biophys Acta. 2014 Jul 10;

Authors: Yamamoto K, Caporini MA, Im SC, Waskell L, Ramamoorthy A

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 (13)C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (?). Further, results obtained from a 2D (13)C/(13)C 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. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces.


PMID: 25017802 [PubMed - as supplied by publisher]



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