Biomedical Engineering Reference
In-Depth Information
complementary, labelling of certain amino acid combinations, combinatorial
labelling approaches aim to reduce the complexity of spectra, whilst providing
unambiguous determination of amino acids using a minimum number of
samples. The technique has been applied for both
15
N-labelling where only five
HSQC-type spectra, each displaying approximately one-third of the total
number of amide groups were required
187
or for combinations of
15
N/
13
C- and
15
N/
14
N-labelled samples.
188,189
Software is also available to predict optimal
labelling patterns for backbone assignment.
190
12.5 Structure Determination
The traditional approach to NMR structure determination involves determin-
ing a dense network of long-range
1
H-
1
H NOE distance information in order
to restrain the 3D structure. This approach can be applied to b-barrel proteins
where hydrogen bonds between amide groups in adjacent strands of the barrel
domain enable relatively easy collection of these restraints. However, for a-
helical membrane proteins, whilst many short-range NOEs between amide
groups are available to build the helices to good precision, long-range NOEs
between the helices are much more limited, leading to difficulties in the global
assembly of the helix bundle. Side-chain assignments are often difficult to
complete and extensive deuteration dramatically reduces the number of NOEs
available between side-chain protons. This difficulty may be overcome by
using ILV methyl-protonated samples on a deuterated background for
example, DsbB,
16
OmpX,
12
VDAC,
13
and pSRII.
19
In the case of pSRII,
long-range inter-methyl NOEs were used to restrain the global fold. Whilst this
strategy yielded a relatively high-resolution initial structure, additional side-
chain NOE restraints were then collected to result in a very high-quality final
structure, thus providing a proof of principle for the structure determination of
seven-helical proteins based solely on NOEs. Initially 140 out of 141 ILV side-
chain methyl groups were assigned and additional restraints provided from3D
13
C-separated NOESY, 4D
13
C-separated NOESY and methyl-to-amide
NOEs from 3D
15
N-separated NOESY spectra. In some cases, signal overlap
could be reduced through exchanging the protein into c6-DHPC, which has a
shorter carbon chain. Further distance restraints were obtained by assigning
the alanine, threonine, methionine and isoleucine c2 methyl groups. For larger
proteins with broader linewidths, similar restraints are also available using the
methyl-protonated forms of these residues in a deuterated back-
ground.
176,177,191
Further expansion of the side-chain assignments including
those of phenylalanine, tyrosine and tryptophan residues critically improved
the results of the structure calculations on pSRII enabling a successful packing
of the structure which used, overall, 1131 and 1536 medium and long-range
NOEs, respectively.
19
The approach was time-consuming and its intention was
to emphasise the high quality of side-chain packing obtainable based on
experimental restraints. Such an approach based on a large number of NOEs
is, in many cases, unlikely to be feasible for larger membrane proteins or
