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present, only DPC and SDS are commercially available in this form, although the latter
tends to denature some proteins. [ 35 ] Micelles are formed when the surfactant is in a higher
concentration than its critical micellar concentration (CMC), which can vary from 0.01mM
for nonionic surfactants to 10 nMfor short-chain ionic surfactants, such as SDS. [ 36 ] The equi-
librium shifts frommicellar to monomeric forms of the surfactant when diluted with buffers
that do not contain the detergent and therefore buffers must always contain a concentration
of surfactant above the CMC to prevent micelle disruption and loss of protein conform-
ation. In our hands, there is rapid exchange of surfactant molecules from the micellar to
the monodispersed form, resulting in rapid breakdown of micelle-bound proteins when
the surfactant is not included (see below). Bicelles are micelles which are composed of
phospholipids rather than detergents and are slightly more complex than micelles. Usually
bicelles are composed of long-chain phospholipids such as dimyristoylphosphatidylcholine
(DMPC), forming bilayers, and one shorter chain phospholipid such as dihexanoylphos-
phatidylcholine (DHPC), which lines the hydrophobic edges of the bilayer. [ 37 ] Bicelles,
being mostly planar, represent a better membrane mimic than micelles and should be more
compatible with protein function. The utility of bicelles for functionally solubilizing mem-
brane proteins has recently been demonstrated by their use in crystallization of the GPCR,
2 -adrenergic receptor. [ 38 ] However, we have not yet tested bicelles for compatibility with
TINS. In addition, there are more complex stable bilayer or multilayer vesicles of synthetic
phospholipids which can be used to immobilize and orient membrane proteins on glass
slides in solid-state NMR, [ 39 ] but their usefulness for membrane protein immobilization on
supports that are compatible with static NMR studies is not yet known.
6.3.3
Immobilization
The TINS methodology, by definition, requires immobilized protein to allow flow-through
screening of ligands. Clearly, the choice of the surface upon which the protein will be
immobilized and the choice of the immobilization chemistry have to be made within the
limitations of theTINS equipment. The general requirements for immobilization compatible
with high-resolution NMR have been discussed, so here we focus on issues specifically
related to membrane proteins.
We have taken a pragmatic approach when attempting to apply the TINS methodology
to membrane proteins by beginning with what has worked for soluble proteins. To date we
have immobilized three purified, micelle-solubilized membrane proteins, KcsA, OmpAand
DsbB, all of which are frombacterial sources.All three membrane proteins were solubilized
in dodecylphosphocholine micelles (DPC). [ 40 ] In all three cases we have simply utilized
the same immobilization scheme that has been successfully applied to soluble proteins,
i.e. Schiff's base chemistry, to primary amines. We have found that the yield of immob-
ilized micelle-solubilized protein is nearly identical with that of soluble proteins. Further,
immobilization has not had any detectable effect on the functionality of the immobilized,
micelle-solubilized proteins. This has been checked in twoways. For KcsAa panel of known
ligands was available and we simply assayed for binding using TINS. Since DsbB has an
enzymatic activity, we adapted a spectrophotometric assay [ 41 ] for use with beads contain-
ing immobilized protein. Enzyme inhibition studies were carried out by adding a reduced
partner enzyme and ubiquinone, the reduction of which can be monitored by measuring
the absorption decrease at 275 nm over time. In order to reduce nonspecific interactions to
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