Applications of NMR to membrane proteins

Opella, Stanley J.; Marassi, Francesca M.

doi:10.1016/j.abb.2017.05.011

Cited by 71 publications

(47 citation statements)

References 183 publications

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“…Nanodiscs can be also used to analyze oligomer state or interaction with other membrane proteins (Boldog et al 2006;Shih et al 2005). Nanodiscs can be handled like soluble molecules and are effectively used for both solution NMR (Hagn and Wagner 2015;) and solid state NMR Opella and Marassi 2017) in addition to EM studies.…”

Section: Membrane Protein Assembly Into Nanodiscsmentioning

confidence: 99%

Lipid environment of membrane proteins in cryo-EM based structural analysis

Mio

Sato

2017

Biophys Rev

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Cryoelectron microscopy (cryo-EM) in association with a single particle analysis method (SPA) is now a promising tool to determine the structures of proteins and their macromolecular complexes. The development of direct electron detection cameras and image processing technologies has allowed the structures of many important proteins to be solved at near-atomic resolution or, in some cases, at atomic resolution, by overcoming difficulties in crystallization or low yield of protein production. In the case of membrane-integrated proteins, the proteins were traditionally solubilized and stabilized with various kind of detergents. However, the density of detergent micelles diminished the contrast of membrane proteins in cryo-EM studies and made it difficult to obtain high-resolution structures. To improve the resolution of membrane protein structures in cryo-EM studies, major improvements have been made both in sample preparation techniques and in hardware and software developments. The focus of our review is on improvements which have been made in the various techniques for sample preparation for cryo-EM studies, with a specific interest placed on techniques for mimicking the lipid environment of membrane proteins.

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Section: Membrane Protein Assembly Into Nanodiscsmentioning

confidence: 99%

Lipid environment of membrane proteins in cryo-EM based structural analysis

Mio

Sato

2017

Biophys Rev

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“…In globular proteins, it has been found that magic-angle spinning SSNMR can resolve complete protein structures [11][12][13][14][15][16]. This has been adapted to membrane proteins, which are more challenging due to lower sensitivity, spectral degeneracy, and complicated sample preparation [4,6,7,[17][18][19][20][21]. Structures solved using this technique include those of the microbial photosensor Anabaena sensory rhodopsin [22], chemokine receptor CXCR1 [23], Influenza A M2 channel [24], transmembrane protein CrgA from Mycobacterium tuberculosis [25], and outer membrane protein G [26].…”

Section: Introductionmentioning

confidence: 99%

“…The robust expression of milligram quantities of a structurally homogeneous protein and its functional reconstitution into a membrane-like environment are the key requirements for the successful structural and dynamic SSNMR analysis of a membrane protein [20,21,44,45]. Heterologous expression of eukaryotic membrane proteins can be especially challenging due to intrinsic low expression levels as well as native-folding and post-translational modifications that are often not supported by the E. coli expression system [39,46,47].…”

Section: Introductionmentioning

confidence: 99%

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

et al. 2020

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Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.

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“…13 C, 15 N). NMR signals can be measured in solution, ambient temperature, and otherwise native-like conditions, including for membrane proteins [42]. Protocols for NMR isotope labeling are readily available in bacterial, yeast, insect, and mammalian expression systems [43–45], though the latter two can be economically prohibitive, given the relatively high concentrations of pure protein (typically 500 μL of a ≥ 150 μM solution) required.…”

Section: Introductionmentioning

confidence: 99%

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Howard¹,

Carnevale²,

Delemotte³

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

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Applications of NMR to membrane proteins

Cited by 71 publications

References 183 publications

Lipid environment of membrane proteins in cryo-EM based structural analysis

Lipid environment of membrane proteins in cryo-EM based structural analysis

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

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