Biomedical Engineering Reference
In-Depth Information
In order to perform this function, membrane transporters usually undergo large
conformational changes during the transport of substrates. For many channels
and membrane transporters, obtaining detailed structural and transport dynamics
information of multiple functional states has been very difficult. However, the use
of solution NMR in combination DNA-nanotube liquid crystal today offers renewed
hope in structural investigations of channels and both active and passive membrane
transporters [
53
].
16.6.2.1
Tetrameric BM2 Channel
The DNA-nanotube liquid crystal has been used to enable measurement of RDCs
for a large membrane protein: a 40 kDa tetrameric BM2 channel (residues 26-109),
which includes a 20-residue membrane anchor and a soluble coiled-coil tetramer-
ization domain. BM2 is the functional homolog of the well-characterized M2 proton
channel from the influenza A virus. Also a pH-gated, proton-selective channel, BM2
comes from the influenza B virus; it plays a central role in equilibrating pH between
membrane-bound compartments and is required in the life cycle of the virus. Using
DNA-nanotube liquid crystals, we have successfully induced a weak alignment
of the BM2 protein in negatively charged detergents (1-myristoyl-2-hydroxy-sn-
glycero-3-[phospho-rao-(1-glycerol)]) (Fig.
16.4
b) [
11
,
20
]. In addition to BM2, in
August 2011, James Chou's lab reported the de novo solution NMR structure deter-
mination of mitochondrial uncoupling protein 2 (UCP2), a six-helix transmembrane
protein of previously unknown structure, by using DNA nanotubes to achieve weak
alignment of the protein in solution [
12
]. Here, UCP2 was reconstituted in mixed
zwitterionic detergents (150 mM DPC, 1 mM cardiolipin and 2 mM DMPC). These
experiments showed the ability of DNA nanotubes to align different molecules
over a large selection of detergents and the potential for the method to extend the
size limit of solution-NMR-based de novo structure determination of membrane
proteins. The detailed strategy for structure determination established on the UCP2
carrier will be outlined in the following paragraph.
16.6.2.2
UCP2 Carrier, a New Approach for Membrane Protein
Structure Determination
For many of membrane carriers, obtaining detailed structures has been difficult.
The only carrier protein with a well-characterized structure is the bovine ADP/ATP
carrier (ANT1), a structure that was solved through X-ray crystallography. However,
James Chou and William Shih's groups have been collaboratively exploring the use
of solution NMR to study passive transporters. Through their efforts, a new solution
NMR method based on previously discussed nanotube alignment medium was
developed for structural characterization of the mitochondrial uncoupling protein
2 (UCP2). UCP2 is a 33 kDa protein with six transmembrane domains, one of
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