Next-Generation GHz
NMR Technology
Focus on Structural Biology and IDPs
Innovation with Integrity
NMR
Improving our Understanding
of the Dark Proteome
Bruker’s next-generation of GHz-class NMR technology is a
combination of major method and instrumentation advances,
which enable even more advanced scientific and translational
research in structural biology, drug discovery and the study of
macromolecular complexes.
The focus of Bruker’s unique, next-generation GigaHertz
NMR spectroscopy technology is to enable breakthrough
fundamental research in molecular and cell biology on
Intrinsically Disordered Proteins (IDPs). In particular, ultrahigh field NMR, in combination with other experimental and
computational methods, has recently been shown to enable
more and more detailed studies of important functions of
Intrinsically Disordered Proteins[1-6]. Due to the scarcity of
understanding of the molecular functions for the vast majority
of IDPs, they are sometimes also referred to as the ‘Dark
Proteome’.
Direct C-13 and N-15 Detection
in Protein NMR
Direct detection of hetero-nuclei, such as C-13 and
N-15 is becoming increasingly popular in studies
of large biomolecules, paramagnetic proteins and
intrinsically disordered proteins [7,8]. This is due to
the significantly larger chemical shift dispersion in
the spectra of hetero-nuclei as compared to proton
NMR spectra that are prone to overlap especially
for unstructured protein segments. Furthermore,
hetero-nuclear direct detection is particularly useful
in situations where proton signals are severely
broadened, sometimes even beyond detection – as in
NMR of paramagnetic proteins or protein sequences
undergoing conformational exchange.
Fig. 1 Comparison of sensitivities of the C-13 detected part of H,C’-N
HSQC spectra of the FABS protein (1 mM in D2O/H2O, 1:9).
The recent advances in CryoProbe technology have
dramatically increased the sensitivity of the direct
detection experiments (see Table 1) making the use of
the direct detection methods in the studies of complex
bio-molecular systems a common practice.
Table 1.
TXI
TCI
TXO
DCH
H-1
8500:1
8500:1
4500:1
4500:1
C13
700:1
1400:1
2800:1
3000:1
CryoProbe
Sensitivity comparison of H-1 and C-13 in various CryoProbes at 600 MHz
The sensitivity of the direct detection becomes
particularly important in parallel NMR experiments [9]
which record several multi-dimensional experiments
simultaneously. This is shown in Figure 1 that
compares the sensitivity improvement of the C-13
detected part of parallel H,C’-N HSQC spectra
recorded for chicken fatty acid binding protein (FABS).
Multiple Receivers and Parallel
NMR Spectroscopy
Parallel acquisition NMR spectroscopy [10] makes
use of the multiple receiver technology that has
been introduced into NMR only recently. Specially
designed experiments allow to record multidimensional experiments with parallel detection of
different nuclei thus offering significant spectrometer
time savings. Not only are such time savings much
appreciated by researchers in charge of precious
ultra-high field NMR systems, but they also allow
reduction of the dimensionality of extensively
coupled spin systems that are common in NMR of
isotope labelled proteins.
The parallel multi-dimensional NMR experiments
typically share one or more frequency axis that
connect the sub-spaces of the hyperdimensional
NMR spectra allowing to obtain higher dimensionality
information by recording two (or more) lower
dimensionality spectra in parallel. For instance, the
2D H,C’-N HSQC experiment can be recorded in
just 10 minutes and provides the same information
as a 3D HNCO experiment (see Fig. 2). Likewise,
a 3D H,C’-detected parallel HNCA experiment [9]
(see Fig. 3) provides the same information as a 4D
HNCA(CO) experiment, but, in a considerably shorter
period of time. This allows the complete assignment
of protein backbone resonances from a single 3D
parallel NMR experiment. Recently several new
parallel NMR experiments have been proposed
by Hosur et al that yield 5D information by
parallel acquisition of 3D HA(CA)
NH and 3D HACACO spectra[11]
or frequencies of up to seven
different types of nuclei from a
single parallel three-dimensional
experiment [12].
Fig. 2 Parallel 2D HN- and C’-detected HSQC spectra of the FABS protein
recorded in 10 minutes on a Bruker Avance III HD spectrometer operating
at 700 MHz and equipped with the TXO CryoProbe optimized for C-13
direct detection. The folded resonances in HN-detected spectrum are
shown in red.
Furthermore, it has been demonstrated that the
higher dimensional spectra can be reconstructed
from the parallel NMR experiments in the same
way as in the Projection Reconstruction (e.g. APSY)
experiments.
Fig. 3 Parallel 3D HN- and C’-detected HNCA spectra of the FABS protein
recorded in 12 hrs on a Bruker Avance III HD spectrometer operation at
700 MHz and equipped with the TXO CryoProbe optimized for C-13 direct
detection (see Table 1).
Customer Driven NMR Methods
5D, 6D and 7D NMR and the
Emerging GHz NMR Technologies
High Dimensionality, Parallel Acquisition
and Non-Uniform Sampling
The projection NMR techniques, such as projection
reconstruction [13] and APSY [14] (Automatic Projection
Spectroscopy) offer further improvements in resolution
that are critical for the studies of highly complex
biological systems, such as the Dark Proteome and
IDPS. Furthermore, it has been shown [15, 16] that not
only such spectra can be recorded automatically in
a reasonably short period of time (5 days) but the
assignment of up to 440 a.a. IDP proteins can be
done fully automatically. Note that is a tremendously
important achievement as it is high dimensionality
experiments that allow to fully exploit the resolution
provided by the new GHz technologies.
The NUS (Non-Uniform Sampling) [12-15] has proven to
be an incredibly important technique for reducing the
acquisition time of multi-dimensional NMR spectra.
Furthermore, it can be combined with other reduced
dimensionality techniques, such as APSY and parallel
acquisition NMR. The newly released TopSpin™
3.5 software provides a routine way to record any
multi-dimensional NMR experiments with the NUS.
Furthermore, the most advanced techniques of
compressed sensing processing are routinely available
for reconstruction of high dimensionality spectra with
high fidelity. The standard TopSpin 3.5 software allows
routine handling of up to 8D NMR experiments.
Disambiguation of highly degenerate
resonances in reduced dimensionality NMR
The technique of using a common frequency axis
to connect cross-peaks in multi-dimensional NMR
spectra is commonly used in bio-molecular NMR.
However, this only works if the resonances are
sufficiently well separated. Therefore the increase
in resolution offered by the introduction of the GHz
technologies in combination with the huge sensitivity
improvements provided by CryoProbe technologies
at these ultrahigh magnetic field strengths will allow
routine use of direct detection and parallel acquisition
NMR techniques that will be of utmost importance for
increasing the productivity and throughput in protein
NMR in the studies of the Dark Proteome and the
IDP-s in particular.
New Techniques – an integral part
of the New GHz Technologies
The NMR techniques described will benefit enormously from the incredibly high
resolution and sensitivity offered by the emerging Giga-Hertz technologies and continuous
developments in the CryoProbe technologies. Such improvements will lead to new ways of
recording the multi-dimensional experiments that will benefit not only the fields of protein
NMR, RNA research and the Human Dark Proteome Initiative (HDPI), but will inevitably
spread to the world of small molecule research, pharma and metabonomics NMR [22]. Most
of the experiments briefly mentioned here are available either from our standard libraries
in TopSpin 3.5 or the online Bruker User Library. TopSpin provides an excellent platform for
development of new experiments and exploring new avenues in parallel experiment design
for the studies of complex biomolecular systems.
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© Bruker BioSpin 04/15 T154700
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