NMR of Membrane Proteins - Recent Developments in Biomolecular NMR - page 287

12.3.6 NMR Studies using Nanodiscs
In the last few years, the suitability of nanodiscs as a membrane-mimicking
medium for NMR spectroscopy has been demonstrated. A variety of
membrane proteins have been embedded in nanodiscs, including the
membrane associated anti-fungal peptide Antiamoebin I (AamI), 143,144 a
single helix of the CD4 receptor, 145 a tetrameric assembly of the KcsA
channel, 143,146 bacteriorhodopsin, 24 VDAC-1 147 and VDAC-2. 25
To date, the full structure determination of a membrane protein in nanodiscs
has not been demonstrated, a situation limited by the comparatively large size
of nanodiscs; with a molecular weight of y200 kDa for a 9.5 nm diameter
particle, a rotational correlation time of 85 ns at 30 uC has been reported. 144
Despite this large size, high-quality 2D [ 1 H, 15 N] TROSY and 2D [ 1 H, 13 C]
HSQC spectra have been recorded. 24,25,145-147 In the case of VDAC-1, the
spectral quality is similar to spectra recorded in detergent micelles enabling
characterisation of ligand-binding effects in a close to native environment. 147
Since the nanodisc boundary is controlled by the scaffold protein, insertion of
a membrane protein requires displacement of a number of lipids equal to its
surface area. Consequently, although the nanodiscs are large, their size will not
increase on addition of a membrane protein, maintaining these particles within
a range of sizes still accessible to NMR.
Based on searching for similarities in the spectral appearance, it has been
proposed to use 2D [ 1 H, 15 N] TROSY data recorded on a protein in nanodiscs
as a reference to help with the selection of membrane-mimicking media that
maintain the native structure of the protein while forming smaller particles
with more favourable tumbling properties. 146 We feel that nanodiscs are likely
to be of most use in NMR for functional and interaction studies once initial
sequential resonance assignment or possibly even structure determination of a
protein has been achieved in a medium of smaller size. The quality of spectra
produced should enable relatively easy transfer of assignments enabling
interactions for example with ligands, 147 potential drug targets, or downstream
signalling molecules to be assessed by NMR in a more representative

membrane environment.

12.3.7 Bicelles
In contrast to detergent-free nanodiscs, bicelles consist of a bilayer of lipid
molecules which is bounded at the edges by a short-chain detergent, providing
the high curvature needed to close the edge of the disc [Figure 12.2(c)]. Similar
to nanodiscs, bicelles also provide a more membrane-like environment and
have also been demonstrated as a suitable environment for a number of crystal
structures 148-150 including GPCRs, 70 although bicelles may not be optimal in
all cases. 151 The size of bicelles can be controlled by varying the ratio of lipid to
detergent, calculated as a 'q' value. Typically for solution-state NMR, small
bicelles using an excess of detergent with q 5 0.25-0.5 are used, avoiding
alignment in the magnetic field found with larger bicelles. Spectra and