Biomedical Engineering Reference
In-Depth Information
the locations of ILV residues. All four types of amino acid can be incorporated
at the same time including varying types of
13
C-labelling patterns that are
either optimised for through-bond sidechain assignment (linearised side-chain
13
C-labelling) or NOESY spectra (methyl-only
13
C-labelling). The incorpora-
tion of isolated
13
CH
3
methyl groups into ILV and A residues has been shown
to allow studies of high molecular weight globular proteins
178,179
and this
approach has also been used to great success for NOE analysis in a number of
recent membrane protein structures including pSRII,
19
DsbB,
16
VDAC-1,
13
and OmpX.
12
Methyl protonation in a deuterated environment can generate different
isotopomers as has been shown when using pyruvate as a
13
C source.
180
An
alternative approach to the use of the
13
CH
3
isotopomer has been recently
proposed which involves labelling with [
1
H,
13
C]-glucose and
15
NH
4
Cl on a
background of 100% D
2
O.
181
As a result of the small number of residual
protons from the protonated glucose, the E. coli biosynthetic pathway for
glucose results in the majority of methyl groups being labelled as CHD
2
or
CH
2
D, with very low levels of CH
3
incorporation. Detection via CHD
2
-
detected CT-[
1
H-
13
C] HSQC was combined with a 3D C-TOCSY-CHD
2
spectrum and assignments for
13
C
a
and
13
C
b
from backbone assignment
experiments to enable assignment of methyl groups but at the expense of the
methyl-TROSY
182
effect.
181
For the 34 kDa protein FebB, 91% of possible
assignments (i.e., previously assigned in backbone experiments) was achieved
and the authors predict this method will be suitable for single-chain proteins
up to y300 amino acids, including for membrane proteins.
181
While Ile c2
183
and methionine methyl-selective protonation
184
have been introduced, label-
ling of the Thr methyl groups is also highly desirable.
The SAIL (Stereo-Array Isotope Labelling) methodology has been proposed
as a method to selectively reintroduce protons allowing NOE assignments,
whilst retaining the benefits of increased T
2
values. This involves stereo-
selective replacement of one
1
H in methylene groups by
2
H, replacement of two
1
H in each methyl group by
2
H, modification of the prochiral methyl groups of
Leu and Val to give
12
C(
2
H)
3
and
13
C
1
H(
2
H)
2
methyl groups and labelling of
six-membered aromatic rings by alternating
12
C-
2
Hand
13
C-
1
H moi-
eties.
185,186
The reduced proton density leads to fewer NOE restraints that
are of better quality. The approach is used in combination with cell-free
protein expression and has been demonstrated as suitable for solving protein
structures,
185
including determination of the side-chain positions. In principle,
the SAIL approach is also perfectly suited to membrane proteins. As sample
costs are very high, combined use of all the available specifically labelled
amino acids is less advised. However, a particularly well-suited application
seems to be the use of labelled aromatic amino acids in order to enable their
side-chain assignment and to exploit NOE distance restraints to these residues.
Complementary techniques have been proposed to reduce spectral crowding,
which is likely to be particularly prevalent in studies of large membrane
proteins,
possibly
including
GPCRs.
Through
controlled
selective,
but
