Biomedical Engineering Reference
In-Depth Information
successfully recorded in A8-35 after exchange from detergent using adsorbing
polystyrene beads.
125
Satisfactory resolution and spectral dispersion in both
1
H and
15
N dimensions was observed, with similar numbers of amide and
indole signals compared to tOmpA in c6-DHPC at pH 7.9, but less than in c6-
DHPC at pH 6.5, and similar chemical shifts indicating no significant
structural perturbations.
125
Linewidths for OmpX-amphipol complexes could
be reduced by addition of EDTA, which likely prevents formation of
interparticle bridges due to interaction of carbonyl groups on A8-35 with
multivalent cations
126
considerably reducing the size difference previously
reported for tOmpA.
125
Comparison of the linewidths between spectra using
hydrogenated amphipol (HAPol), and spectra with deuterated amphipol
(DApol) (isopropylamine and octylamine chains perdeuterated) enabled
mapping of the amphipol-membrane protein interaction to the hydrophobic
transmembrane helices.
125
Other amphipols have been designed which are
zwitterionic, non-ionic or sulfonated and are found to be pH and calcium-
insensitive.
23,127,128
A more recent study used
13
C-detected cross-relaxation
experiments (2D [
1
H,
13
C] HOESY data) to detect intermolecular interactions
between A8-35 and OmpX; spectra recorded for [U-
2
H,
13
C,
15
N]-OmpX in
HAPol and [U-
13
C,
15
N]-OmpX in DAPol enabled identification of inter-
molecular vs. intramolecular contacts demonstrating interactions between
methylene and methyl groups of the surfactant and carbon atoms of aromatic
rings
protein.
129
on
the
H/D
exchange
measurements
have
also
been
reported.
126
These
studies
demonstrate
that
amphipols
are
suitable
for
functional studies.
Amphipol A8-35 can also be used to refold membrane proteins from
bacterial inclusion bodies; refolding and subsequent ligand binding of four
class A GPCRs (BLT1, BLT2, 5-HT
4
, CB1)
130
as well as two eubacterial b-
barrel proteins, OmpA and FomA and the archaebacterial bacteriorhodop-
sin
131
have been reported. These studies demonstrate that amphipols can play
an important part in membrane protein research from refolding, through to
structural studies and are likely to gain in importance as further development is
undertaken.
12.3.4 Problems with Micelles
Detergent micelles have been widely used for studies of membrane proteins via
a range of techniques. They are particularly attractive for NMR studies dueto
their relatively small size. However, many of their properties make them a poor
mimic of the membrane bilayer. Short-chain amphipathic detergents are highly
mobile, existing in equilibrium between the free, monomeric form and the
micelle-bound form. The rapid exchange of molecules in and out of the micelle
can be destabilising for some proteins, leading to aggregation and/or
unfolding, in particular if the CMC is high.
23,24,109
This may be especially
pronounced for protein complexes, and for membrane proteins with large
extracellular portions. The presence of free detergent may also interfere with
