Anthranilic acid, the new player in the ensemble of aromatic residue labeling precursor compounds

Article abstract of DOI:10.1007/s10858-017-0129-2

The application of metabolic precursors for selective stable isotope labeling of aromatic residues in cell-based protein overexpression has already resulted in numerous NMR probes to study the structural and dynamic characteristics of proteins. With anthranilic acid, we present the structurally simplest precursor for exclusive tryptophan side chain labeling. A synthetic route to ¹³C, ²H isotopologues allows the installation of isolated ¹³C–¹H spin systems in the indole ring of tryptophan, representing a versatile tool to investigate side chain motion using relaxation-based experiments without the loss of magnetization due to strong ¹J_CC and weaker ²J_CH scalar couplings, as well as dipolar interactions with remote hydrogens. In this article, we want to introduce this novel precursor in the context of hitherto existing techniques of in vivo aromatic residue labeling.

Full text of DOI:10.1007/s10858-017-0129-2

J Biomol NMR
DOI 10.1007/s10858-017-0129-2
ARTICLE
Anthranilic acid, the new player in the ensemble of aromatic
residue labeling precursor compounds
Julia Schörghuber1 · Leonhard Geist2 · Marilena Bisaccia1 · Frederik Weber1 ·
Robert Konrat2 · Roman J. Lichtenecker1
Received: 14 June 2017 / Accepted: 28 August 2017
© The Author(s) 2017. This article is an open access publication
Abstract The application of metabolic precursors for
selective stable isotope labeling of aromatic residues in cell-
based protein overexpression has already resulted in numer-
ous NMR probes to study the structural and dynamic char-
acteristics of proteins. With anthranilic acid, we present the
structurally simplest precursor for exclusive tryptophan side
chain labeling. A synthetic route to 13C, 2H isotopologues
allows the installation of isolated 13C–1H spin systems in
the indole ring of tryptophan, representing a versatile tool
to investigate side chain motion using relaxation-based
experiments without the loss of magnetization due to strong
Introduction
The progress in stable isotope labeling has driven the field
of protein NMR spectroscopy, ever since the first spectra of
small randomly deuterated or uniformly 15N-labeled proteins
have been recorded (Campbell 2013). Numerous experi-
mental techniques have been developed in the meantime to
address a wide range of structure-biological questions (Lian
and Middleton 2001; Ohki and Kainosho 2008; Zhang and
van Ingen 2016). However, poor signal resolution and com-
plex magnetization transfer causes severe problems in the
case of high molecular weight target proteins, which can
only be addressed using highly defined patterns of heavy
isotopes (13C, 15N, 2H). Early examples of selective stable
isotope labeling almost exclusively focused on aliphatic side
chains (Goto and Kay 2000; Kerfah et al. 2015). Their high
abundance in the core of globular proteins, as well as their
intense NMR signals due to the threefold symmetry of a
methyl group rotation result in straightforward data acqui-
sition for structure calculation. These aliphatic side chains
play an important role in protein folding, creating a well-
defined hydrophobic microenvironment for substrate binding
and solvent exclusion.
2
1JCC and weaker JCH scalar couplings, as well as dipolar
interactions with remote hydrogens. In this article, we want
to introduce this novel precursor in the context of hitherto
existing techniques of in vivo aromatic residue labeling.
Keywords Isotope labeling · Protein NMR · Tryptophan ·
Anthranilic acid
Although some very early examples can be found in lit-
erature (Wüthrich and Wagner 1975), it was only in recent
years, that studies of aromatic side chains increasingly
attracted the attention of the biomolecular NMR com-
munity. Aromatic side chains provide valuable additional
restraints for structure calculation and exhibit a number of
characteristics, which turn them into important sensors to
study enzymatic mechanisms or binding events. They are
prone to participate in non-covalent π–π or π–cation interac-
tions, which significantly contribute to the protein’s tertiary
structure (Dougherty 1996). Aromatic residues additionally
control enzymatic reaction trajectories by stabilizing charged
Electronic supplementary material The online version
of this article (doi:10.1007/s10858-017-0129-2) contains
supplementary material, which is available to authorized users.
* Roman J. Lichtenecker
roman.lichtenecker@univie.ac.at
1
Institute of Organic Chemistry, University of Vienna,
Währingerstr. 38, 1090 Vienna, Austria
2
Christian Doppler Laboratory for High-Content Structural
Biology and Biotechnology, Department of Structural
and Computational Biology, Max F. Perutz Laboratories,
University of Vienna, Dr-Bohr-Gasse 9, 1030 Vienna,
Austria
Vol.:(0123456789)
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J Biomol NMR
intermediates or direct structural changes to close hydro-
phobic cavities and protect them from water molecules.
Due to their distinct physicochemical properties, aromatic
side chains play a very prominent role at binding interfaces
to other proteins, nucleic acids, lipid membranes or small
ligands (Moreira et al. 2013; Rahman et al. 2015). These
binding events, as well as conformational changes during
enzymatic catalysis, are frequently connected to extensive
aromatic side chain motion in a wide range of time scales
from picoseconds to seconds.
1995), erythrose (Kasinath et al. 2013; Weininger 2017),
pyruvate (Guo et al. 2009; Milbradt et al. 2015), or glycerol
(LeMaster and Kushlan 1996; Ahlner et al. 2015) have been
applied as carbon sources to avoid direct 13C–13C coupling
in the target residues. These compounds are commercially
available and additional organic synthesis is usually not
required. However, the costs are considerable regarding the
concentrations needed for high incorporation levels (up to
3 g/L medium). These methods partially exhibit poor selec-
tivity, as the corresponding isotope sources represent early
metabolic intermediates residing upstream of the shikimate
pathway. This inevitably leads to the undesired loss of heavy
isotopes at metabolic branches. The metabolites of the shi-
kimate pathway 3-dehydroquinate to prephenate feature
multiple chiral centers and are therefore rather too complex
to be considered as isotope containing targets in economic
multistep organic synthesis. In only one example unlabeled
shikimate was thus used to introduce protonated aromatic
residues into an otherwise deuterated protein (Rajesh et al.
2003). As soon as the aromatic ring system is biosyntheti-
cally formed, the corresponding metabolic intermediates
represent much more attractive target molecules for organic
synthesis. In rare cases, aromatic amino acids have been uti-
lized in combination with in vivo overexpression media for
selective labeling/unlabeling (Vuister et al. 1994; Kelly et al.
1999; Wang et al. 1999). However, these examples often
suffer from poor metabolic uptake and increased effects on
metabolic network regulation, since many of the enzymes
involved are inhibited by the final product of the correspond-
ing biosynthetic pathway. These methods additionally lack
the possibility of simple uniform 15N labeling by overex-
pressing the target protein in presence of 15N-ammonium
chloride, which is beneficial for many NMR applications.
α-Ketoacids can be considered as late metabolic intermedi-
ates and their use as precursors for aliphatic residue labe-
ling has a long history due to the effective uptake and the
straightforward aminotransferase-catalyzed conversion to
the target amino acids (Goto et al. 1999; Lichtenecker et al.
2004, 2013a). In a similar approach for aromatic residue
labeling, phenylpyruvate has been shown to be an effective
precursor for phenylalanine, whereas the use of 4-hydroxy-
phenylpyruvate leads to tyrosine labeling (Lichtenecker et al.
2013b). Isotopologues of indole have been used to selec-
tively label tryptophan side chains (Schörghuber et al. 2015;
Rodriguez-Mias and Pellecchia 2003). Alternatively, indole
pyruvate can be applied for backbone Trp-labeling, which is
the first intermediate in the tryptophan degradation pathway,
but is effectively converted to the target amino acid due to
the highly reversible character of the Trp-aminotransferase.
The structurally simplest precursor, which holds prom-
ise to result in exclusive tryptophan labeling is anthranilic
acid. Although 15N-isotopologues of this compound have
been used in the past to produce [1-15N]L-tryptophan by
Side chain dynamics, induced by binding events or
changes in temperature or pressure, have been studied
using different NMR based techniques (Boehr et al. 2006).
The corresponding experiments, such as longitudinal- and
transverse-13C-relaxation (Weininger et al. 2012a, 2014a;
Kasinath et al. 2015), the 13C-TROSY-Carr-Purcell-Mei-
boom-Gill (CPMG) experiment (Weininger et al. 2012b),
the 13C-TROSY-rotating frame relaxation (R1ρ) sequence
(Weininger et al. 2014b), intra-residue NOE interpretation
(Miyanoiri et al. 2011), or the recording of aromatic residual
dipolar couplings (Sathyamoorthy et al. 2013) have been
optimized for applications in aromatic spin systems. These
methods resulted in the calculation of rate constants, energy
barriers, as well as activation volumes. As mentioned above,
the data of such experiments can only be interpreted when
spectra have been simplified and magnetization transfer
pathways optimized using defined isotope distributions in
the protein samples.
Two main strategies can be pursued to generate defined
isotope patterns in a protein of interest: In cell-free overex-
pression methods all components needed for the transcrip-
tion and the translation process are provided to an in vitro
system. This approach is well suited to generate proteins,
which contain posttranslational modifications or modified
amino acids, as well as proteins that are toxic to in vivo
systems. Selective labeling devoid of cross labeling to other
residues is assured, but the method requires the application
of labeled amino acids (Torizawa et al. 2005; Kainosho et al.
2006; Takeda et al. 2010; Miyanoiri et al. 2011). Moreover,
cell-free protein expression exhibits major shortcomings in
providing high protein yields and is considerably expensive
(Staunton et al. 2006).
The second strategy features isotope labeled precursor
compounds as additives in the growth medium of an overex-
pressing microorganism, which has been established as the
method of choice to yield in high concentrations of the cor-
responding labeled molecular targets (Hoogstraten and John-
son 2008). Potential pitfalls include poor precursor uptake
or cross labeling to other residues. Scheme 1 summarizes
the main metabolites, which have been used to overexpress
proteins containing labeled phenylalanine, tyrosine or tryp-
tophan residues. Certain 13C-patterns of D-glucose (Teilum
et al. 2006; Lundström et al. 2007), acetate (Wand et al.
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J Biomol NMR
Scheme 1 Overview of aro-
matic residue labeling precursor
compounds in E. coli overex-
pression media
fermentation (Liu et al. 2009) and protocols for the synthe-
sis of different stable isotope patterns have been reported
(van Liemt et al. 1996), methods to apply anthranilic acid
as a direct metabolic precursor in protein overexpression
media are still not available, at least to our knowledge. We
hypothesized that none of the heavy isotopes present when
using anthranilic acid as a tryptophan precursor should be
metabolized back into the shikimic acid pathway. Since
the second reaction step in the anthranilate synthase (EC
4.1.3.27) catalyzed conversion of chorismate to anthra-
nilate is an irreversible process (elimination of pyruvate,
Scheme 2), we expected exclusive labeling of the trypto-
phan target residues without any cross labeling to pheny-
lalanines and tyrosines.
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J Biomol NMR
of recombinant proteins using a fourfold cell concentration
in M9 minimal medium supplemented with 15NH4Cl (1 g/L)
and 3,5-dideuterio[4,6-13C2]anthranilic acid 11 (50 mg/L)
(Marley et al. 2001). Uniformly 15N / uniformly 13C-labeled
Brd4-BD1 was overexpressed using 1 g/L 15NH4Cl and 3 g/L
13C6-glucose in the minimal medium. Cells were harvested
by centrifugation, lysed by sonication and the lysates were
subsequently centrifuged. Proteins were purified from the
resulting supernatant by Ni2+ affinity chromatography. The
purified protein was treated with TEV protease and again
loaded onto a Ni2+ column to bind the cleaved His6-tag and
the His6-tagged TEV protease. The flow-through contain-
ing Brd4-BD1 was concentrated and purified on a gel filtra-
tion column. NMR samples of Brd4-BD1 were prepared in
sodium phosphate buffer containing 0.4–3 mM protein and
10% D2O.
Scheme 2 Anthranilate synthase catalyzed conversion of chorismate
to anthranilate
Comprehensive information concerning the overexpres-
sion of labeled H6-GB1 and Brd4-BD1 is given in the sup-
porting information.
Materials and methods
Organic synthesis
Protein NMR spectroscopy
Detailed protocols for the synthesis of the anthranilic acid
isotopologues [15N]anthranilic acid 4, 3,5-dideuterio[4,6-
13C2]anthranilic acid 11, and 4,6-dideuterio[5-13C]
anthranilic acid 18, as well as NMR and MS characteriza-
tion of intermediates and final products are provided in the
supporting information.
NMR spectra of H6-GB1 and Brd4-BD1 were acquired at
298 K on a Bruker Avance 3 HD+600 MHz spectrometer.
The system was equipped with a RT 5 mm TXI probe. Spec-
tra were processed using NMR Pipe (Delaglio et al. 1995)
and analyzed using the Sparky software (Goddard and Knel-
ler). Methodological details concerning the quantification of
H6-GB1 labeling using compound 4, as well as an evalua-
tion of Trp-13C-labeling of Brd4-BD1 using compound 11
are reported in the supporting information.
Protein overexpression
His‑tagged GB1
The pET-M11 plasmid containing the sequence for a his-
tagged GB1 protein (H6-GB1) was transformed into an E.
coli BL21(DE3) strain. Bacterial cultures were grown in LB
medium and then transferred to a M9 minimal medium sup-
plemented either with unlabeled NH4Cl (1 g/L) and increas-
ing amounts of [15N]anthranilic acid 4 to acquire selec-
tively labeled protein or with 15NH4Cl (1 g/L) to acquire
uniformly labeled protein (Marley et al. 2001). Protein
overexpression was induced by the addition of isopropyl-β-
D-thiogalactopyranosid and pursued overnight. The crude
proteins were purified using Ni2+ affinity chromatography.
NMR samples contained 1 mM protein and 10% D2O.
Results and discussion
In order to investigate the uptake of anthranilic acid by the
expressing E. coli strain, we prepared [15N]anthranilic acid
4 in a two-step sequence starting from 15NH4Cl 1 or [15N]
urea 2 (Scheme 3) (Hijji and Benjamin 2003). An E. coli
strain, grown in the presence of compound 4, overexpressed
the exclusively tryptophan-15N histidine-tagged G-binding
protein domain (H6-GB1). The resulting 15N-HSQC spec-
trum is devoid of any other signals, except the one caused
by the only 1Hε–15Nε Trp side chain spin system in the target
protein (Fig. 1). [15N]Anthranilic acid 4 was applied to cor-
relate the precursor concentrations in the growth medium
to the protein isotope incorporation rates. The correspond-
ing plot in Fig. 1b displays normalized 15N-HSQC signal
intensities of H6-GB1 overexpressed in presence of different
precursor 4 concentrations. Signal intensities were compared
to the intensity found for the 1Hε–15Nε Trp side chain signal
in uniformly 15N-labeled H6-GB1 (experimental details are
given in the supporting information). A signal intensity of
Brd4‑BD1
Recombinant human Brd4-BD1 (bromodomain 1 of Bro-
modomain containing protein 4) was expressed in E. coli
BL21(DE3) containing an N-terminal TEV-cleavable
His6-tag (the expression plasmid was kindly provided by
Boehringer Ingelheim). Uniformly 15N/selectively 13C,2H-
tryptophan labeled H6-TEV-Brd4-BD1 was expressed fol-
lowing the expression protocol for efficient isotopic labeling
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J Biomol NMR
Scheme 3 Synthesis of [15N]
anthranilic acid 4 and its appli-
cation to selectively label Trp-
residues. a Phthalic anhydride,
250 °C, DMAP, 66%; b phthalic
anhydride, xylenes, 140 °C,
84%; c NaOH, Br2, 53%; d E.
coli protein overexpression,
D-glucose, NH4Cl
we assembled the aromatic system through reaction with
nitromalonaldehyde (Viswanatha and Hruby 1979). A two-
step deoxygenation/reduction sequence yielded [3,5-13C2]
aniline 6 (Musliner and Gates 1966), which was transformed
into anthranilic acid following a literature protocol (Kafka
et al. 2013). Hydrolysis of N-(α-ketoacyl)anthranilic acid
9 in DCl/D2O yielded 3,5-dideuterio[4,6-13C2]anthranilic
acid 11. The 2H-pattern can be installed using mild reaction
conditions, since the electron donating amino substituent
directs the reaction outcome to deuteration in the desired
ortho and para positions.
Using a similar route, but starting from [2-13C]acetone
12 allows for the synthesis of the 4,6-dideuterio[5-13C] iso-
topologue of the target compound (18, Scheme 5). In this
case 2H is introduced by perdeuteration of [13C]aminophe-
nol in acidic D2O at 180 °C at prolonged reaction times
(13→14). The labeling patterns of compounds 11 and 18
are well defined (Fig. 2) and feature isolated 13C–1H spin
systems, which can be effectively applied for the elucida-
tion of tryptophan dynamics using 13C transverse relaxation
dispersion based methods.
3,5-Dideuterio[4,6-13C2]anthranilic acid 11 was supple-
mented to the E. coli growth medium to overexpress the
recombinant human bromodomain 1 of Bromodomain con-
taining protein 4 featuring an N-terminal TEV-cleavable
His6-tag (H6-TEV-Brd4-BD1) (see “Materials and meth-
ods” section for details). BRD4 is linked to diverse human
malignancies and has been shown to regulate the expression
of MYC (Andrews et al. 2017). The MYC gene encodes a
transcription factor, which plays a decisive part in cancer
development and proliferation. The corresponding bromo-
domain was chosen as a model to demonstrate exclusive
Trp labeling by our novel precursor compound, as well as
give a first representative of its potential use. The resulting
13C-HSQC spectra showed a total number of six signals,
caused by the 13C–1Hε3 and 13C–1Hη2 couplings of the three
Fig. 1 a 15N-HSQC spectra overlay of the uniformly 15N-labeled
(black) and tryptophan side chain 15N labeled (red) H6-GB1 protein;
b normalized signal intensity versus precursor concentration plot
showing data for the overexpression of H6-GB1 in presence of 2, 5,
10, 25, 50, 100 and 200 mg/L of precursor 4. The intensities have
been normalized to a uniformly 15N-labeled sample (1 on y-axis)
>90% at a concentration of 10 mg/L indicates the effective
metabolic uptake and conversion of anthranilic acid by the
overexpressing host organism.
These results encouraged us to develop a synthetic route
to anthranilic acid isotopologues possessing 13C in the aro-
matic ring (Scheme 4). Starting from [1,3-13C2]acetone 5,
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J Biomol NMR
Scheme 4 Synthesis of
3,5-dideuterio[4,6-13C2]
anthranilic acid 11 and its
application to selectively label
Trp-residues. a Sodium nitro-
malonaldehyde monohydrate,
aqu. NaOH, then 6N HCl, 63%;
b potassium tert-butoxide,
dimethylformamide, 5-chloro-
1-phenyl-1H-tetrazole, 74%; c
Pd/C, methanol, toluene, H2,
H-cube®, 97%; d diethyl eth-
ylmalonate, 250–270 °C, then
toluene and aqu. NaOH, 77%; e
peroxyacetic acid in acetic acid,
aqu. NaOH; f paraperiodic acid,
ethanol, 75% over steps (ef);
g DCl/D2O, 94%; h HCl/H2O,
91%; i microwave irradiation,
DCl/D2O, 92%; j E. coli protein
overexpression, D-glucose,
15NH4Cl
Scheme 5 Synthesis of
4,6-dideuterio[5-13C]anthranilic
acid 18 and its application to
selectively label Trp-residues.
a Sodium nitromalonaldehyde
monohydrate, aqu. NaOH,
then 6N HCl, 60%; b Pd/C,
H2, H-cube®, methanol,
toluene, 95%; c HCl/D2O, MW
irradiation, 180 °C, 7 h, 90%;
d potassium tert-butoxide,
dimethylformamide, 5-chloro-
1-phenyl-1H-tetrazole, 75%; e
Pd/C 10%, MeOH, toluene, H2,
H-cube®, 95%; f diethyl ethyl-
malonate, heat, then toluene and
aqu. NaOH, 69%; g peroxy-
acetic acid in acetic acid, aqu.
NaOH; h paraperiodic acid,
ethanol, 72% over steps (g, h); i
HCl/H2O, 90%; j E. coli protein
overexpression, D-glucose,
15NH4Cl
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J Biomol NMR
Fig. 2 1H-NMR spectral
region of aromatic protons
showing the corresponding
13C–1H couplings of 3,5-dideu-
terio [4,6-13C2]anthranilic
acid 11, 4,6-dideuterio [5-13C]
anthranilic acid 18 and [15N]
anthranilic acid 4, respectively
tryptophan residues present in the corresponding sequence
(Fig. 3a). Comparing this HSQC with spectra obtained from
a uniformly 13C labeled sample, which was overexpressed in
presence of 13C6-glucose, reveals the value of our labeling
approach in terms of well-defined signals of high intensity in
absence of signal splitting or line shape distortion due to sca-
lar couplings (Fig. 3b). The 1H-15N HSQC of the Brd4-BD1
sample which was labeled using 15NH4Cl and compound
11 showed the complete absence of Trp-Nε2 resonances,
given that the precursor 3,5-dideuterio[4,6-13C2]anthranilic
acid 11 carries an unlabeled amino-group (see supporting
information).
early metabolic intermediates as heavy isotope sources.
Recently, isotopologues of erythrose have been introduced to
direct 13C labeling into defined atomic positions of the aro-
matic side-chains in phenylalanine, tyrosine and tryptophan
(Kasinath et al. 2015; Weininger 2017). In the correspond-
ing protocols, limited isotope incorporation levels in the
range of 50–70% for tryptophan, as well as partial isotope
scrambling to undesired atomic positions in the aromatic
rings have been reported. On the contrary, anthranilic acid
is metabolized to the target amino acid in a well-defined way
without losing labeling selectivity at the major metabolic
intersection points of the shikimate pathway. Our novel pre-
cursor completes our toolbox of isotope labeled compounds
to transfer 13C/2H-patterns onto the aromatic side chains
of phenylalanine and tyrosine (Lichtenecker et al. 2013b),
tryptophan (Schörghuber et al. 2015), as well as histidine
(Schörghuber et al. 2017), with every precursor targeting one
specific type of residue. This residue selective isotope incor-
poration cannot be achieved using the early metabolic pre-
cursors mentioned above, but is of major importance in the
case of high molecular weight protein samples, when signal
overlap in the aromatic region becomes a serious issue.
Efficient metabolic in vivo conversion has also been
observed for the selective tryptophan precursor indol,
which we presented in a recent publication (Schörghuber
et al. 2015). However, the synthetic routes to anthranilic acid
isotopologues from the present study offer clear advantages
in terms of yields and robustness and are thus preferred in
This data confirmed our initial assumption that anthranilic
acid serves as a selective tryptophan precursor with quan-
titative incorporation levels devoid of any cross-labeling to
the other aromatic residues. Addition of a ligand (kindly
provided by Boehringer Ingelheim) targeting the binding site
of Brd4-BD1 for acetylated lysines in histones resulted in an
upfield shift of the Trp81 signals (Fig. 3c). This residue is
positioned in the bromodomain binding-cleft, thus playing
a central role in protein–ligand interactions (Fig. 3d) (Zhang
et al. 2015).
Conclusions
In summary, the E. coli overexpression of model proteins
in presence of labeled anthranilic acid demonstrated both,
high labeling selectivity for the target Trp-residues, as well
as highly effective precursor uptake by the overexpressing
host organism with quantitative isotope incorporation levels
at low precursor concentrations (10–15 mg/L overexpres-
sion medium). Our novel labeling technique represents an
advanced development of hitherto applied methods using
cases where the γ and δ1 positions are not targeted for 13
C
labeling. The synthetic routes to 3,5-dideuterio[4,6-13C2]
anthranilic acid 11 and 4,6-dideuterio[5-13C]anthranilic
acid 18 have been optimized to hold down costs for isotope
sources and reagents, while at the same time minimizing
synthetic efforts. Both precursors are accessible in gram
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J Biomol NMR
Fig. 3 a 13C-HSQC of Brd4-BD1 overexpressed in the presence of
3,5-dideuterio[4,6-13C2]anthranilic acid 11; b 13C-HSQC of Brd4-
BD1 overexpressed in the presence of [13C6]D-glucose; c 13C-HSQC
of selectively Trp-13C/2H labeled Brd4-BD1 (black) and upon addi-
tion of the Brd4-BD1 binding ligand (red); d X-ray structure of apo-
Brd4-BD1 (4LYI) (Lucas et al. 2013) depicting the three tryptophan
residues. Trp81 is part of the Brd4-BD1 binding pocket (red arrow)
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J Biomol NMR
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Acknowledgements Open access funding provided by University
of Vienna. We thank Boehringer Ingelheim for providing the expres-
sion plasmid for H6-TEV-Brd4-BD1, as well as the corresponding
bromodomain ligand. This work was supported by a scholarship of
the uni:docs program from the University of Vienna (J. Schörghuber).
L. Geist was funded by the Christian Doppler Laboratory for High-
Content Structural Biology and Biotechnology, Austria. The financial
support by the Austrian Federal Ministry of Science, Research and
Economy and the National Foundation for Research, Technology and
Development is gratefully acknowledged.
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