Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.
Nat Protoc. 2018 Jan;13(1):79-98
Authors: Hagn F, Nasr ML, Wagner G
Abstract
Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.
[NMR paper] An adaptable phospholipid membrane mimetic system for solution NMR studies of membrane proteins.
An adaptable phospholipid membrane mimetic system for solution NMR studies of membrane proteins.
An adaptable phospholipid membrane mimetic system for solution NMR studies of membrane proteins.
J Am Chem Soc. 2017 Oct 09;:
Authors: Chien CH, Helfinger LR, Bostock MJ, Solt A, Tan YL, Nietlispach D
Abstract
Based on the saposin-A (SapA) scaffold protein we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins under close-to-native conditions. The Salipro...
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10-11-2017 10:37 AM
[NMR paper] Reconstitution of the Cytb5 -CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy.
Reconstitution of the Cytb5 -CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy.
Reconstitution of the Cytb5 -CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy.
Angew Chem Int Ed Engl. 2016 Feb 29;
Authors: Zhang M, Huang R, Ackermann R, Im SC, Waskell L, Schwendeman A, Ramamoorthy A
Abstract
Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full-length membrane-bound P450 and its redox...
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03-01-2016 05:59 PM
[NMR paper] Optimizing nanodiscs and bicelles for solution NMR studies of two ?-barrel membrane proteins.
Optimizing nanodiscs and bicelles for solution NMR studies of two ?-barrel membrane proteins.
Related Articles Optimizing nanodiscs and bicelles for solution NMR studies of two ?-barrel membrane proteins.
J Biomol NMR. 2015 Apr;61(3-4):261-74
Authors: Kucharska I, Edrington TC, Liang B, Tamm LK
Abstract
Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in...
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04-15-2015 04:40 PM
Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins
Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins
Abstract
Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore,...
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02-10-2015 10:56 AM
Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs
Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs
Abstract
NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR...
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11-28-2014 11:37 AM
Nanodiscs versus Macrodiscs for NMR of Membrane Proteins
Nanodiscs versus Macrodiscs for NMR of Membrane Proteins
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/0/bichaw.ahead-of-print/bi201289c/aop/images/medium/bi-2011-01289c_0002.gif
Biochemistry
DOI: 10.1021/bi201289c
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09-30-2011 08:01 PM
Nanodiscs vs. Macrodiscs for NMR of Membrane Proteins.
Nanodiscs vs. Macrodiscs for NMR of Membrane Proteins.
Nanodiscs vs. Macrodiscs for NMR of Membrane Proteins.
Biochemistry. 2011 Sep 21;
Authors: Park SH, Berkamp S, Cook GA, Chan MK, Viadiu H, Opella SJ
Abstract
It is challenging to find membrane mimics that stabilize the native structure, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. Increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation...
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09-23-2011 05:30 PM
[NMR paper] An evaluation of detergents for NMR structural studies of membrane proteins.
An evaluation of detergents for NMR structural studies of membrane proteins.
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J Biomol NMR. 2004 Jan;28(1):43-57
Authors: Krueger-Koplin RD, Sorgen PL, Krueger-Koplin ST, Rivera-Torres IO, Cahill SM, Hicks DB, Grinius L, Krulwich TA, Girvin ME
Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment....
Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.
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