Biomedical Engineering Reference
In-Depth Information
which were measured at 600 MHz (
1
H frequency) by interleaving a regular
gradient-enhanced HSQC and a gradient-selected TROSY, both acquired with
80 ms of
15
N evolution. Several programs are available to perform RDC analysis,
including ORDERTEN-SVD [
45
], REDCAT [
46
], PALES [
47
], iDC [
48
], and
CONFORMIST [
49
]. A classic program carrying out RDC calculations typically
requires two sources of information input: the measured residual dipolar-coupling
values with their associated uncertainty, plus an estimation of the order parameter
and a standard coordinate file. From there, there are two commonly used methods
for calculating and best fitting an alignment tensor to experimental RDCs. The
first utilizes tensor eigenvalues and eigenvectors extracted by using least-squares
minimization of the target function's over all couplings. The second relies on
singular value decomposition (SVD) and Monte Carlo sampling as core methods
to solve a system of linear equations relating measured couplings to elements of an
order tensor (Fig.
16.3
b). Order tensor elements are computed by fitting the RDCs
and examine the correlation between the measured and back-calculated RDCs.
Figure
16.4
b show back-calculated RDCs from an ubiquitin sample.
In the last few years, solution NMR studies of
-helical membrane proteins
coupled with the DNA nanotube have contributed substantially towards the structure
determination of monomeric and oligomeric polytopic proteins. These proteins are
illustrated in Fig.
16.4
c and presented in the following section.
'
16.6
Weak Alignment of Membrane or Membrane-
Associated Proteins in DNA-Nanotube Liquid
Crystals
16.6.1
Immunoreceptor Assembly and Structure
Immunoreceptors are cell surface receptors that transmit signals across the mem-
brane. They are molecular sensors that alert the cells of the immune system to the
presence of potential pathogens and injuries. Yet, despite this crucial role played by
the immunoreceptors, their functional mechanisms remain not fully understood, and
only few structures of intact immunoreceptor complexes have been reported [
50
]. In
the last few years, solution NMR spectroscopy has made important contributions to
our current understanding of immunoreceptor-ligand interactions and the formation
of intracellular-signaling complexes [
50
]. To achieve this, traditional NMR analysis
techniques have proven to be insufficient to generate high-resolution structures of
intact, functional immunoreceptors, primarily due to low levels of inter-helical NOE
assignment of the membrane-imbedded helices, a feature that largely determines the
tertiary and quaternary structure of an immunoreceptor. Fortunately, as illustrated
in the coming paragraphs, RDC restraints can help elucidate inter-helical packing,
and DNA-nanotube liquid crystals have successful yielded weak alignment of two
immunoreceptors.
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