Biomedical Engineering Reference
In-Depth Information
acid distributions in membrane proteins differ from soluble proteins, contain-
ing more hydrophobic residues, and spectra of a-helical proteins can suffer
from reduced dispersion since aromatic residues may be less abundant.
Isotopic-labelling approaches need to be tailored with this in mind to suit the
purpose of the envisaged NMR study. The basic labelling strategy involves
15
N-labelling in order to record [
1
H,
15
N] 2D correlation spectra showing the
amides in the protein backbone. If relaxation is too fast, then perdeuteration of
the side-chain moiety is required.
164
The use of deuterated detergent may also
be considered at this stage, taking into account the substantial cost increase.
Subsequently, backbone assignment involves
15
N,
13
C-labelling in order to
record correlations between carbonyl and amide groups via TROSY versions
of triple-resonance experiments such as HNCA, HN(CO)CA, HN(CA)CB,
HNCO, HN(CA)CO etc.
165-170
Fast spin relaxation requires the use of
perdeuterated protein in order to reduce dipolar interactions due to the high
proton density. Frequently, the large size of membrane proteins can lead to
heavily overlapped spectra, even when applying line narrowing TROSY
techniques
171,172
that are used under ideal conditions of higher static field
strengths ($800 MHz). Consequently, 4D experiments may be used in
conjunction with a 3D-based assignment procedure to remove remaining
ambiguities. A particularly challenging task is the collection of inter-residue
connectivity information when using out-and-back type amide-detected
experiments at high static field strengths, e.g., HN(CO)CACB, that involve
multiple magnetization-transfer steps via carbonyl spins. Here, alternative
slower relaxing HNCA-based techniques can be used instead
166
or, in
combination with appropriate hardware,
13
C-detection methods might become
a more competitive choice
173
(Section 12.6.1). In order to record amide-
directed backbone experiments following expression in deuterated media,
back-exchange of amide protons is required. In many cases, this is easily
achievable, but sometimes the stability and close-packing of transmembrane
helices may prevent back-exchange of protons requiring denaturing and
refolding protocols to be developed. This was required in the case of DAGK,
as a result of successful mutagenesis to increase the thermal stability of the
protein,
17,174
and DsbB.
16
For structural studies of large membrane proteins, more sophisticated
isotope-labelling strategies are required. Whilst high levels of backbone
deuteration are required for sufficient resolution and sensitivity, this prevents
recording of NOE distance restraints between side-chain protons.
Consequently, protons must be reintroduced in order to obtain this data. A
highly sensitive approach comes through the use of methyl-proton-selective
ILV labelling, where partially deuterated precursors a-ketobutyrate and a-
ketoisovalerate are used to synthesise U-[
2
H,
12
C],[
13
CH
3
,
13
CD
3
]-valine or
leucine and U-[
2
H,
12
C]-d
1
-[
13
CH
3
]-isoleucine respectively.
175
Labelling of the
hydrophobic core ILV residues can be supplemented by additional incorpora-
tion of methyl-protonated alanine residues.
176,177
Alanine is one of the most
abundant residues, frequently found in positions that are complementary to
