Abstract
Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling–based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.
[NMR paper] DNA nanotubes for NMR structure determination of membrane proteins.
DNA nanotubes for NMR structure determination of membrane proteins.
DNA nanotubes for NMR structure determination of membrane proteins.
Nat Protoc. 2013 Mar 21;8(4):755-70
Authors: Bellot G, McClintock MA, Chou JJ, Shih WM
Abstract
Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this...
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03-23-2013 06:36 PM
[NMR paper] Solution NMR spectroscopy for the determination of structures of membrane proteins in a lipid environment.
Solution NMR spectroscopy for the determination of structures of membrane proteins in a lipid environment.
Solution NMR spectroscopy for the determination of structures of membrane proteins in a lipid environment.
Methods Mol Biol. 2013;974:389-413
Authors: Arora A
Abstract
Several recent advancements have transformed solution NMR spectroscopy into a competitive, elegant, and eminently viable technique for determining the solution structures of membrane proteins at the level of atomic resolution. Once a good level of cell-based or...
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02-14-2013 02:37 PM
[NMR paper] NMR structure determination of a membrane protein with two transmembrane helices in m
NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system.
Related Articles NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system.
Biochemistry. 2005 Apr 5;44(13):5196-206
Authors: Howell SC, Mesleh MF, Opella SJ
The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that...
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11-25-2010 08:21 PM
[NMR paper] Membrane protein structure determination using solid-state NMR.
Membrane protein structure determination using solid-state NMR.
Related Articles Membrane protein structure determination using solid-state NMR.
Methods Mol Biol. 2004;278:403-73
Authors: Watts A, Straus SK, Grage SL, Kamihira M, Lam YH, Zhao X
Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment...
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11-24-2010 09:25 PM
[NMR paper] Structure determination of aligned samples of membrane proteins by NMR spectroscopy.
Structure determination of aligned samples of membrane proteins by NMR spectroscopy.
Related Articles Structure determination of aligned samples of membrane proteins by NMR spectroscopy.
Magn Reson Chem. 2004 Feb;42(2):162-71
Authors: Nevzorov AA, Mesleh MF, Opella SJ
The paper briefly reviews the process of determining the structures of membrane proteins by NMR spectroscopy of aligned samples, describes the integration of recent developments in the interpretation of spectra of aligned proteins and illustrates the application of these methods...
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11-24-2010 09:25 PM
[NMR paper] NMR solution structure determination of membrane proteins reconstituted in detergent
NMR solution structure determination of membrane proteins reconstituted in detergent micelles.
Related Articles NMR solution structure determination of membrane proteins reconstituted in detergent micelles.
FEBS Lett. 2003 Nov 27;555(1):144-50
Authors: Fernández C, Wüthrich K
As an alternative to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy in solution can be used for three-dimensional structure determination of small membrane proteins, preferably proteins with beta-barrel fold. This paper reviews recent achievements as...
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11-24-2010 09:16 PM
[NMR paper] Simultaneous assignment and structure determination of a membrane protein from NMR or
Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.
Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.
Protein Sci. 2003 Mar;12(3):403-11
Authors: Marassi FM, Opella SJ
A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of (15)N-labeled amino...
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11-24-2010 09:01 PM
[NMR paper] Structure determination of membrane proteins by NMR spectroscopy.
Structure determination of membrane proteins by NMR spectroscopy.
Related Articles Structure determination of membrane proteins by NMR spectroscopy.
Biochem Cell Biol. 2002;80(5):597-604
Authors: Opella SJ, Nevzorov A, Mesleb MF, Marassi FM
Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of...
DNA nanotubes for NMR structure determination of membrane proteins.
Linear Mode
