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Improved in-cell structure determination of proteins at near-physiological concentration.

ABSTRACT: Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250??M concentration in living E. coli cells.

SUBMITTER: Ikeya T

PROVIDER: S-EPMC5133543 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Improved in-cell structure determination of proteins at near-physiological concentration.

Ikeya Teppei T Hanashima Tomomi T Hosoya Saori S Shimazaki Manato M Ikeda Shiro S Mishima Masaki M Güntert Peter P Ito Yutaka Y

Scientific reports 20161202

Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cel ...[more]

PMID: 27910948

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Project description:Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) ?-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) ?-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly ?-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, ?-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of ?-synuclein.

Dataset Information

Improved in-cell structure determination of proteins at near-physiological concentration.

Publications

Improved in-cell structure determination of proteins at near-physiological concentration.

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