Chemistry Reference
In-Depth Information
4.2 Applications to Biomolecules
ssNMR is a powerful technique for the investigation of membrane-associated
peptides and proteins as well as their interactions with lipids, and a variety of
conceptually different approaches have been developed for their study. The tech-
nique is unique in allowing for the high-resolution investigation of liquid disor-
dered lipid bilayers representing well the characteristics of natural membranes.
While magic angle solid-state (MAS) NMR spectroscopy follows approaches that
are related to those developed for solution NMR spectroscopy, the use of static
uniaxially oriented samples results in angular constraints which also provide
information for the detailed analysis of polypeptide structures. Solid-state NMR
spectroscopy has already in the past provided valuable structural information
for biomolecules and major advancement of the technique can be expected when
it is possible to overcome the present lack of sensitivity where recording a two-
dimensional spectrum can take many days, even for samples consisting of several
milligrams of labeled polypeptide. Although high magnetic field spectrometers
have already ameliorated the situation, the novel developments of DNP has
promised to boost tremendously the sensitivity of solid-state NMR spectroscopy
by about two orders of magnitude [
56
]. Notably, solid-state DNP-NMR has already
been applied on oriented bilayer samples [
57
], as well as on membrane proteins
[
56
,
58
]. With such developments the application of three- and four-dimensional
NMR experiments should become possible and much extend the ease of structural
investigations on oriented membranes as well as improving the conformational
details that can be obtained.
Cyanobacteria are widely used as model organisms of oxygenic photosynthesis
due to being the simplest photosynthetic organisms containing both photosystem I
and II. Photochemically induced dynamic nuclear polarization (photo-CIDNP)
13
C
MAS NMR is a powerful tool in understanding the photosynthesis machinery down
to atomic level. Combined with selective isotope enrichment this technique has
now opened the door to study primary charge separation in whole living cells.
Janssen et al
.
[
59
] recently presented the first photo-CIDNP observed in whole cells
of the cyanobacterium Synechocystis.
In the applications of DNP to MAS spectra of biological systems, including
studies of lysozyme [
20
] and bacteriorhodopsin [
60
], the enhancements have been
smaller, with
e ΒΌ
40-50, which has limited the application of DNP-NMR to
biological samples so far. An exception is the amyloidogenic peptide GNNQQNY7
-13 which forms nanocrystals for which the proton T
1
time is long, yielding an
of
~ 100 [
25
]. Almost a decade ago in studies of model systems it was observed that
deuteration of the solvent resulted in significant increases in
e
and subsequently
many DNP experiments have employed
2
H-labeled solvents such as [D
6
]DMSO or
[D
8
]glycerol/D
2
O/H
2
O in an approximately 6:3:1 ratio [
21
,
22
,
61
]. Akbey et al
.
[
62
] recently showed that perdeuteration of a protein has remarkable effects on the
observed DNP enhancements. Superior DNP enhancements are obtained for
perdeuterated SH3 samples of up to 3.9 and 18.5 times for
13
C CP-MAS, and
13
C
e
