Towards structure determination of self-assembled peptides using dynamic nuclear polarization enhanced solid-state NMR spectroscopy

Article abstract of DOI:10.1002/anie.201210093

Supra-sensitivity: Dynamic nuclear polarization (DNP) enhanced solid-state NMR spectroscopy was performed on self-assembled peptide nanotubes. This approach yields significant experimental time savings (about five orders of magnitude; see picture) and was

Full text of DOI:10.1002/anie.201210093

Angewandte
Chemie
DOI: 10.1002/anie.201210093
Peptide Nanoassemblies
Towards Structure Determination of Self-Assembled Peptides Using
Dynamic Nuclear Polarization Enhanced Solid-State NMR
Spectroscopy**
Hiroki Takahashi, Bastien Viverge, Daniel Lee, Patrice Rannou, and Gaꢀl De Paꢀpe*
[
9]
Bio-inspired self-assemblies made of peptide building blocks
have great potential for nanotechnology ranging from bio-
logical and pharmaceutical applications to (opto)electron-
minutes has recently been reported on crystalline cellulose.
This illustrated the realistic feasibility of studying atomic-
scale structures of unlabeled organic systems with NMR
spectroscopy. Since self-assembling systems possess ordered
structures, they are also a priori suitable candidates for DNP
experiments.
Herein, we demonstrate the possibility of structural study
on challenging nanoassemblies. In particular, we chose to
tackle systems based on the diphenylalanine (FF) dipep-
[
1–3]
ics.
With these goals, a variety of peptide nanoassemblies
[4]
have been studied and designed over the last few decades.
Inevitably, structural studies at an atomic scale are crucial to
unravel the mechanisms that drive nanoassembly formation
as well as to relate these structures to their physical and
chemical properties. However, structure determination at an
atomic level is challenging essentially because of the difficulty
associated with using X-ray crystallography on such nano-
[
2,11–13]
tide
organic semiconductors.
Alzheimerꢀs amyloid-b and plays a key role in the self-
which are currently emerging as a new class of
[
14,15]
FF is also a core motif of
[3]
assemblies.
[16]
Solid-state NMR (SSNMR) spectroscopy is a powerful
and promising technique for structural analysis of nano-
assemblies. In principle, SSNMR spectroscopy can be used for
any form of solid sample from well-ordered crystals to
assembly of the amyloid.
Amongst FF derivatives, we
particularly focus on the cyclic form of FF (cyclo-FF). Recent
[17]
[18]
articles by Adler-Abramovich et al.
and Lee et al.
reported that FF can be self-assembled into cyclo-FF-based
nanotubes (NTs)/nanowires (NWs) prepared by a vapor-
phase transport method.
Here, we introduce an efficient method to prepare self-
assembled peptide NTs suitable for MF-DNP measure-
[
5]
disordered powders. Furthermore, the recent development
of high-field magic-angle-spinning dynamic nuclear polar-
ization (MAS-DNP) allows one to compensate the inherent
[
6]
low sensitivity of NMR experiments.
[
9]
13
13
The main concern regarding the DNP technique is the
relative line broadening typically encountered in biomolec-
ments. We perform 2D C– C correlation experiments on
cyclo-FF NTs that provide fundamental structural informa-
tion such as hydrogen-bonding and p-stacking interactions.
Furthermore, the naturally low isotopic abundance allows one
to detect both intra- and intermolecular long-range interac-
tions during dipolar mediated polarization transfer experi-
ments. This is the first and major step in determining de novo
3D structures of nanoassemblies.
For DNP experiments, polarizing agents need to be
distributed uniformly in a sample. Thus, it is highly ineffective
to add them after NT formation because of rapid aggregation
of NTs (see Section S2 in the Supporting Information).
Alternatively, we used a solution-based method to obtain
cyclo-FF NTs with a uniform distribution of polarizing agents.
This sample preparation technique is original and extremely
useful for “DNP-ready” samples, especially self-assembling
systems that tend to aggregate. Adding anti-solvents (water
and methanol in this case) to a cyclo-FF solution in 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) creates NTs that aggregate
[7]
ular systems at low temperatures (LT). This broadening is
induced by a change of dynamics at LT which leads to the
detection of conformational disorder. This drawback can be
circumvented when MAS-DNP is applied to crystalline
materials that keep their ordered structures and yield
[
8–10]
narrow linewidths even at LT.
favorable for DNP experiments. In fact, a natural-abundance
Therefore, they are a priori
1
3
13
2
D
C– C correlation experiment using matrix-free (MF)
sample preparation with an experimental time of only tens of
[
*] Dr. H. Takahashi, B. Viverge, Dr. D. Lee, Dr. G. De Paꢀpe
Laboratoire de Chimie Inorganique et Biologique
UMR-E3 (CEA/UJF) and CNRS
Institut Nanosciences et Cryogꢁnie, CEA, 38054 Grenoble (France)
E-mail: gael.depaepe@cea.fr
Dr. P. Rannou
Laboratoire d’Electronique Molꢁculaire, Organique et Hybride
UMR5819-SPrAM, Institut Nanosciences et Cryogꢁnie, CEA
immediately after their formation. Here, the polarizing agent,
38054 Grenoble (France)
[19]
TOTAPOL,
was added simultaneously with the anti-
[**] This work was supported by the French National Research Agency
solvents so that TOTAPOL biradicals were trapped inside
the NTs when they formed and outside the NT walls when
aggregation occurred. Details of the procedure are described
in the Experimental Section.
Formation of cyclo-FF NTs was confirmed by scanning
electron microscopy (SEM; Figure 1). The diameters of the
obtained NTs are not uniform but typically fall in the range of
through the “programme blanc” (grant number ANR-12-BS08-0016-
01) and the “programme Labex” (ARCANE project number ANR-11-
LABX-003) and funding from the RTB. SEM images were taken at
the PFNC platform at MINATEC (CEA Grenoble). P. A. Bayle is
acknowledged for solution NMR measurements.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201210093.
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1
3
13
fore it is infeasible to perform 2D C– C correlation
1
3
experiments even on a C-enriched system. On the other
hand, in the DNP sample where radicals are uniformly
1
distributed, the T of H spins is shortened to 2.6 s because of
1
the presence of paramagnetic spins. This alone is already
leading to a substantial time-saving factor greater than 230 for
SSNMR measurements.
Signal-to-noise ratios (S/N) of DNP-enhanced NMR and
conventional NMR spectroscopy were 390 with 4 scans and
Figure 1. SEM images of the cyclo-FF NTs prepared by a solution-
based method. Image of NTs (left) and a magnified image of one of
the NTs from the left image (right).
a recycle delay of 3.4 s (S/N = 562 at 5T ) and 73 with 168
1
scans and a recycle delay of 300 s, respectively. Overall, an
[9]
absolute sensitivity ratio (ASR), determined by comparing
the S/N per unit time of DNP-enhanced NMR and conven-
tional NMR spectroscopy, of 320 was found. This corresponds
1
2
00–1000 nm. The NTs have wall thicknesses smaller than
00 nm. This is thin enough to perform efficient DNP
5
to a remarkable time-saving factor of 10 . The ASR repre-
[8]
experiments. Larger objects require more time for DNP
build-up and thus need to possess a long spin-lattice
sents the “real” gain of performing DNP and includes positive
contributions (DNP enhancement factor, Boltzmann factor
(including thermal noise), reduced repetition rate) as well as
negative contributions such as signal bleaching, changes in
linewidths and reduced sample volumes. The negative con-
tributions from the latter two are minimized using our
approach. Note that a 4 mm rotor (80 mL) was used for
conventional NMR spectroscopy whereas a 3.2 mm rotor
(42 mL) was used for the DNP experiments.
relaxation time constant (T ) for substantial polarization
1
[
10]
transfer.
1
3
A DNP-enhanced 1D C-CPMAS spectrum is shown in
Figure 2 and compared to that obtained by conventional
SSNMR. A DNP enhancement factor (eDNP) of 8.8 was
observed (by comparing microwaves-on and -off spectra). No
line-broadening is observed at LT, since the cyclo-FF
molecules are well-ordered and rigid, which prevents any
conformational distribution in the frozen state. Furthermore,
since we utilized the MF approach, there is no deleterious
dilution effect of the sample of interest unlike other sample
The S/N per unit time returned from the DNP experi-
1
3
13
ments makes it possible to perform 2D C– C correlation
1
3
experiments without C enrichment. Thus, one such through-
[
20]
bond experiment was recorded at natural abundance on the
cyclo-FF NTs (Figure 3). All one-bond correlations were
preparation methods.
1
13
13
The T of H spins in cyclo-FF is exceedingly long because
observed within 5 h. It is worth noting that no C– C
correlations were seen in a solution-state NMR correlation
experiment recorded in 60 h even though the amount of
sample used was 1.5 times larger than that for DNP-enhanced
SSNMR spectroscopy.
1
of its rigidity (T > 10 minutes at room temperature). There-
1
All signals for the cyclo-FF NTs were assigned and
compared to the chemical shifts of cyclo-FF molecules
dissolved in HFIP and obtained by solution-state NMR
spectroscopy (Table 1). The two phenylalanine residues show
the same chemical shifts in solution indicating that the
chemical environments are identical for both residues. This is
no more the case in the solid state because of the absence of
1
3
molecular motions. Most of the C resonances show signifi-
cant deviations compared to solution-state NMR data; this
being a clear signature of intra- and intermolecular inter-
d1
actions. For instance, C of Phe 2 (d ) experiences a down-
2
1
field shift indicating a p–p interaction between the aromatic
ring of Phe 1 and d21 (T-shaped interaction). Furthermore, the
narrow linewidths of the aromatic peaks at LT suggest the
presence of multiple p–p interactions that can prevent ring
flips. Upfield shifts on the carbonyl and alpha carbons
(
compared to empirical values in proteins) suggest that
1
3
Figure 2. C-CPMAS spectra of natural-abundance cyclo-FF NTs
recorded using a DNP equipped spectrometer with a) microwaves-on
and b) -off at 105 K and 9.4 T (3.2 mm zirconia rotor) and using c) a
conventional NMR spectrometer at room temperature and 9.4 T
cyclo-FF forms b-sheet-type hydrogen bonds with neighbor-
ing molecules. These interactions are fully consistent with the
model of cyclo-FF NWs obtained by Lee et al. using X-ray
[18]
powder diffraction.
(
4 mm zirconia rotor). The MAS frequency was 13 kHz. Experimental
To further demonstrate the feasibility of supramolecular
structure determination using this approach, through-space
times were 14 s (a recycle delay of 3.4 s, 4 scans) and 14 h (a recycle
delay of 300 s, 168 scans) for the DNP experiments and the conven-
tional NMR experiment, respectively. Asterisks indicate the signals
from glucose, glycerol, and remaining methanol.
1
3
13
C– C dipolar correlation experiments were performed
[
21]
using SPC5 recoupling at various mixing times (Figure S6).
2
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identified (Figure 3c). Among
a
e
a
z
b
them the C ÀC , C ÀC , and C –
z
C cross-peaks (indicated by cir-
cles) mainly result from intermo-
lecular contacts (for details, see
Section S4 in the Supporting Infor-
mation). These results clearly pave
the way towards supramolecular
structure determination.
In conclusion, we have shown
a
novel sample preparation
method targeting DNP experi-
ments on self-assembling peptides.
Using this approach, polarizing
agents can be uniformly added
even into samples that inherently
tend to strongly aggregate. Com-
bining this approach and the MF
method enabled us to perform 2D
1
3
13
C– C correlation experiments
on an unlabeled self-assembled
peptide. All signals were assigned
and supramolecular structural
information such as hydrogen
bonding and p–p stacking was
obtained. Furthermore, the feasi-
bility of detection of intermolecu-
lar contacts was demonstrated.
Self-assembling supramolec-
ular systems exhibiting narrow
linewidths even at LT proved to
be particularly pertinent for DNP
experiments (especially with the
MF approach). As such, DNP-
1
3
13
[20]
Figure 3. a) A DNP-enhanced C– C 2D refocused INADEQUATE spectrum of natural-abundance
cyclo-FF NTs recorded at 105 K. The spectrum was obtained in 4.8 h with a recycle delay of 1 s and
6 ms (in total) of echo periods at a MAS frequency of 12.5 kHz. The C –C cross-peaks are folded and
appear between 240–250 ppm in the DQ frequency dimension. b) The aromatic region was enlarged to
show all correlations within the aromatic rings. An external reference (1D C-CPMAS) is shown on the
top. c) A DNP-enhanced 2D DQ-SQ C– C dipolar correlation spectrum. The MAS frequency was
0.5 kHz and the mixing time was set to 7.62 ms. For other experimental conditions, see Section S4 in
the Supporting Information. DQ: double quantum, SQ: single quantum.
a
b
1
1
3
1
3
13
1
enhanced SSNMR spectroscopy
is a perfect tool for de novo struc-
Owing to the spin dilution (only 1.1% of the carbon nuclei are
C spins), the spin dynamics involved during C– C polar-
ization transfer are much simpler than in the case of
a uniformly labeled system where strong dipolar couplings
dominate and strongly quench long-distance transfer (dipolar
truncation). As shown in Figure S6, short mixing times
tural determination of nanoassemblies that are unattainable
using X-ray crystallography. Finally, it is important to note
that this work is useful not only for unlabeled nanoassemblies
but also has a great impact on self-assemblies that consist of
larger and/or more complex molecules such as amyloid fibrils
which are usually C- and/or N-enriched for SSNMR
studies. Signal overlapping is a major issue in such systems.
The results demonstrated here will facilitate higher-dimen-
sional NMR experiments that lead to separation of over-
lapped signals. This study exemplifies that DNP-enhanced
SSNMR spectroscopy has the potential to become a key
1
3
13
13
1
3
15
(
1.5 ms) favor mostly one-bond correlations (1.5 ꢁ) whereas
longer mixing (4.6 ms) clearly yields two-bond (2.5 ꢁ) and
three-bond (2.7 ꢁ for the cis- and 3.8 ꢁ for the trans-
conformer) correlations. For even longer mixing times
(
7.6 ms), multiple long distance contacts can clearly be
Table 1: Chemical shifts (ppm) of cyclo-FF NTs obtained by DNP-enhanced SSNMR and cyclo-FF monomers obtained by solution-state NMR
spectroscopy.
a
C
b
C
g
C
d1
d2
e1
e2
z
C
CO
C
C
C
C
[
[
a]
b]
Phe 1
Phe 2
168.8
168.0
168.8
55.7
55.2
56.5
43.3
38.7
39.5
137.0
135.4
134.1
130.8
132.4
129.8
130.4
130.0
129.8
129.5
128.8
129.2
129.1
127.9
129.2
126.0
125.5
128.0
[
c]
Monomer
[a,b] Phenylalanine 1 (a) and 2 (b) of cyclo-FF NTs measured by DNP-enhanced SSNMR spectroscopy. Chemical shifts were calibrated indirectly
through the carbonyl peak of glycine (176.03 ppm relative to TMS) at room temperature. [c] The cyclo-FF monomer was dissolved in HFIP and
measured by solution-state NMR spectroscopy using TMS as an external standard. The assignment of the chemical shifts was performed using
empirical data.
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8
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[
9]
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Received: December 18, 2012
Revised: March 11, 2013
Published online: && &&, &&&&
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Keywords: dynamic nuclear polarization · nanostructures ·
NMR spectroscopy · peptides · self-assembly
.
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Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Peptide Nanoassemblies
Supra-sensitivity: Dynamic nuclear
polarization (DNP) enhanced solid-state
NMR spectroscopy was performed on
self-assembled peptide nanotubes. This
approach yields significant experimental
time savings (about five orders of mag-
nitude; see picture) and was used to
exemplify the feasibility of supramolec-
ular structural studies of organic nano-
assemblies at an atomic scale using
DNP-enhanced solid-state NMR spec-
troscopy.
H. Takahashi, B. Viverge, D. Lee,
P. Rannou, G. De Paꢁpe*
&&&& — &&&&
Towards Structure Determination of Self-
Assembled Peptides Using Dynamic
Nuclear Polarization Enhanced Solid-
State NMR Spectroscopy
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!