Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ2 conformation by intra-residue NOEs

Article abstract of DOI:10.1007/s10858-011-9568-3

Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U-¹³C,¹⁵N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the ¹³C-¹³C and ¹³C-¹H spin coupling networks (Kainosho et al. in Nature 440:52-57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-²H₂; δ1,ε3,η2- ¹³C₃; ε1-¹⁵N]-indole ring ([¹²C _γ, ¹² C_ε2] SAIL-Trp), which provides a more robust way to correlate the ¹H_β, ¹H_α, and ¹H_N to the ¹H_δ1 and ¹H_ε3 through the intra-residue NOEs. The assignment of the ¹H_δ1/ ¹³C_δ1 and ¹H_ε3/ ¹³C_ε3 signals can thus be transferred to the ¹H_ε1/¹⁵N_ε1 and ¹H_η2/¹³C_η2 signals, as with the previous type of SAIL-Trp, which has an extra ¹³C at the C _γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was ¹H_β2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ₂ angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [¹²C _γ,¹²C_ε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.

Full text of DOI:10.1007/s10858-011-9568-3

J Biomol NMR (2011) 51:425–435
DOI 10.1007/s10858-011-9568-3
ARTICLE
Alternative SAIL-Trp for robust aromatic signal assignment
and determination of the v2 conformation by intra-residue NOEs
•
Yohei Miyanoiri Mitsuhiro Takeda
•
•
•
JunGoo Jee Akira M. Ono Kosuke Okuma
•
•
Tsutomu Terauchi Masatsune Kainosho
Received: 21 July 2011 / Accepted: 5 September 2011 / Published online: 23 September 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Tryptophan (Trp) residues are frequently found
in the hydrophobic cores of proteins, and therefore, their
side-chain conformations, especially the precise locations of
the bulky indole rings, are critical for determining structures
by NMR. However, when analyzing [U–13C,15N]-proteins,
the observation and assignment of the ring signals are often
hampered by excessive overlaps and tight spin couplings.
These difficulties have been greatly alleviated by using
stereo-array isotope labeled (SAIL) proteins, which are
composed of isotope-labeled amino acids optimized for
unambiguous side-chain NMR assignment, exclusively
through the 13C–13C and 13C–1H spin coupling networks
(Kainosho et al. in Nature 440:52–57, 2006). In this paper,
we propose an alternative type of SAIL-Trp with the
experiment, one can determine the side-chain conformation
of the Trp residue including the v2 angle, which is especially
important for Trp residues, as they can adopt three preferred
conformations. We demonstrated the usefulness of
[12Cc,12Ce2] SAIL-Trp for the 12 kDa DNA binding domain
of mouse c-Myb protein (Myb-R2R3), which contains six
Trp residues.
Keywords SAIL-Trp ꢀ Aromatic ring CH assignment ꢀ
Intra-residue NOE ꢀ v2 conformation
Introduction
[f2,f3-2H2; d1,e3,g2-13C3; e1-15N]-indole ring ([12Cc, Ce2]
The side-chain aromatic rings in proteins are important
constituents of the hydrophobic core, and are also involved
in various biological functions through protein–protein and
protein–ligand interactions. The indole ring of the Trp
residue is the largest, as compared to the other aromatic
rings, and thus the side-chain conformations, i.e., v1 and v2
angles, of Trp residues significantly influence the spatial
packing and dynamics of other amino acids in the sur-
rounding hydrophobic core. However, the detailed NMR
analysis of the ring signals of aromatic amino acids, using a
conventional [U–13C,15N]-protein, has often been ham-
pered by the extensive signal overlapping and tight spin
couplings. A conventional, yet still quite useful approach to
overcome this problem is to use aromatic amino acids
isotopically labeled at one or more specific atom(s) in the
rings (Wang et al. 1999; Jacob et al. 2002; Rajesh et al.
2003; Teilum et al. 2006). Especially, the systematic
optimization of the isotope labeling pattern developed in
the stereo-array isotope labeling (SAIL) method has led to
extremely effective assignment schemes, as demonstrated
for the aromatic ring signals of SAIL- phenylalanine (Phe)
12
SAIL-Trp), which provides a more robust way to correlate
the 1Hb, 1Ha, and 1HN to the 1Hd1 and 1He3 through the intra-
1
residue NOEs. The assignment of the Hd1/13Cd1 and
1
1He3/13Ce3 signals can thus be transferred to the He1/15Ne1
1
and Hg2/13Cg2 signals, as with the previous type of SAIL-
Trp, which has an extra 13C at the Cc of the ring. By taking
advantage of the stereospecific deuteration of one of the
1
prochiral b-methylene protons, which was Hb2 in this
Y. Miyanoiri ꢀ M. Takeda ꢀ M. Kainosho (&)
Graduate School of Science, Structural Biology Research Center,
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602,
Japan
e-mail: kainosho@nagoya-u.jp
J. Jee ꢀ A. M. Ono ꢀ K. Okuma ꢀ T. Terauchi ꢀ M. Kainosho
Center for Priority Areas, Tokyo Metropolitan University,
1-1 Minami-ohsawa, Hachioji 192-0397, Japan
A. M. Ono ꢀ K. Okuma ꢀ T. Terauchi
SAIL Technologies Co., Inc., 1-40 Suehiro-cho 1-chome,
Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
123

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J Biomol NMR (2011) 51:425–435
and SAIL-tyrosine (Tyr) residues (Torizawa et al. 2005;
Kainosho et al. 2006; Takeda et al. 2008, 2010). Similarly,
the original SAIL-Trp (Kainosho et al. 2006), which has a
[f2,f3-2H2; c,d1,e2,e3,g2-13C5; e1-15N]-indole ring, can be
modified to improve the observation and assignment of the
ring signals. In doing so, we prepared a modified SAIL-Trp
with the [f2,f3-2H2; d1,e3,g2-13C3; e1-15N]-indole ring
([12Cc,12Ce2] SAIL-Trp), in which the c and e2 positions of
the ring carbons were left unlabeled (Fig. 1). Since one of
the prochiral b-methylene protons (Hb2) is stereo-selec-
tively deuterated, as in the original SAIL-Trp, the
remaining proton (Hb3) provides a high quality signal with
unambiguous stereo-assignment (Kainosho et al. 2006;
Kainosho and Gu¨ntert 2009).
between the aromatic proton and aliphatic/amide protons
can be used to correlate the aromatic and aliphatic protons
(Billeter et al. 1982; Wu¨thrich 1986). The NOE-based
correlation between the aliphatic side-chain and aromatic
ring protons of d-SAIL Phe/Tyr residues in a protein is a
very sensitive and robust alternative assignment method
(Takeda et al. 2010). In the case of Trp, since at least either
1
1
one of the Hd1 and He3 atoms is located in spatial prox-
imity to the a, b-protons or backbone amide protons, as
discussed later, a similar NOE-based assignment method
for the aromatic ring signals should also work well.
Here we present a strategy for assigning the aromatic
signals of Trp residues in proteins, using a new type of
SAIL-Trp, [12Cc,12Ce2] SAIL-Trp (Fig. 1). With this new
1
1
In order to correlate the backbone assignment to the
aromatic ring signals, a variety of scalar coupling-based
experiments via 13Cc were previously developed (Yama-
zaki et al. 1993; Grzesiek and Bax 1995; Carlomagno et al.
SAIL-Trp, Hd1 and He3 can be correlated by the intra-
residue NOEs between the backbone/aliphatic protons (i.e.,
1Hb3, 1Ha and 1HN), as shown in Fig. 1. Another benefit of
[12Cc,12Ce2] SAIL-Trp, as compared to the previous version
of SAIL-Trp, which has two extra 13C atoms at Cc and Ce2
(Kainosho et al. 2006), is that constant-time experiments
are not needed to eliminate the one-bond 13C–13C spin
couplings between Cc and Cd1, leading to much higher
¨
1996; Prompers et al. 1997; Lohr et al. 2005). Similar pulse
sequences based on the one-bond and three-bond 13C–13C
and 13C–1H scalar-couplings have also been used for
assigning the aromatic rings of e- and f-SAIL Phe/Tyr
residues (Torizawa et al. 2005). With the original SAIL-
Trp, which has the [f2,f3-2H2; c,d1,e2,e3,g2-13C5; e1-15N]-
indole ring, this type of assignment scheme should in
1
sensitivity. Moreover, H–13C TROSY HSQC works more
effectively with this new SAIL-Trp (Pervushin et al. 1997,
1998; Sørensen et al. 1997), and thus we can extend the
method for much larger proteins, as compared to conven-
tional through-bond assignment methods (Fesik et al. 1990;
Bax et al. 1990).
13
principle work well for assigning the Cd1/1Hd1 and
15
N /1He1 peaks in a SAIL-Trp residue with a c-carbon
e1
enriched with 13C. However, the intermittent 13C labeling
in the indole ring of SAIL-Trp does not permit the corre-
lation of the NMR resonances of the atoms at the d1/e1
positions to those of the e3/g2 positions by isotropic mixing
type experiments, such as HCCH-TOCSY (Fesik et al.
1990; Bax et al. 1990). Alternatively, intra-residue NOEs
In addition to the advantages described above, the
observation of the intra-residue NOEs also provides an
opportunity to determine the side-chain conformations of
Trp residues. Especially, the stereo-selective mono-deu-
teration at the b-position in [12Cc,12Ce2] SAIL-Trp enabled
us to precisely observe the NOEs between spatially prox-
imal protons, thereby allowing the unambiguous determi-
nation of the v2 angle. When combined with information on
the v1 angle, obtained by another established method
(Cavanagh et al. 2006), the orientation of the indole moiety
relative to the backbone can be determined. In addition,
the systematic 13C enrichment and deuteration allow the
employment of three-bond J coupling, with a size of
7–8 Hz (Torizawa et al. 2005; Takeda et al. 2009, 2010).
We have demonstrated this assignment strategy for six
Trp residues in the minimum DNA binding domain of
mouse c-Myb protein (Myb-R2R3) (amino acids 89–193).
The c-myb gene product is a transcriptional activator that
plays an important role in controlling the proliferation and
differentiation of hematopoietic cells (Graf 1992). Myb-
R2R3 consists of two tandem repeats, R2 and R3, each
containing three Trp residues (R2: Trp-95, Trp-115 and
Trp-134; R3: Trp-147, Trp-166 and Trp-185). The struc-
tures of the unbound and DNA-bound forms have been
determined by NMR spectroscopy (Ogata et al. 1994). The
Fig. 1 Isotope labeling pattern of [12Cc,12Ce2] SAIL-Trp and the
magnetization transfer pathway for the assignment. The 12C atoms are
not shown in the figure. In the original SAIL-Trp, the c- and e2-
carbons (labeled with an asterisk) were 13C (Kainosho et al. 2006). In
this study, the c- and e2-carbons were kept as 12C. The magnetization
transfer pathways for assignment are depicted as arrows, with the
names of the experiments. 3J(C–H) scalar couplings were obtained as
free Trp in D2O, as described previously (van den Berg et al. 1990)
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J Biomol NMR (2011) 51:425–435
427
six Trp residues constitute the hydrophobic core of each
domain, and are highly conserved among homologous
proteins (Ogata et al. 1994).
followed by conversion to SAIL-Trps using tryptophanase
in the presence of SAIL-Ser (Vederas et al. 1978).
Computation of proton–proton distances in the Trp
residue
Materials and methods
The computations of the proton–proton distances between
Hb2 and Hd1, Hb2 and He3, Hb3 and Hd1, and Hb3 and He3 were
performed using the MATLAB software (MathWorks Inc.,
Natick, MA, USA), according to the procedure described by
Synthesis of SAIL-Trps
A number of syntheses concerning isotopically labeled Trp
and indole have been reported (Heidelberger 1949; Bak
et al. 1967; Leete and Wemple 1969; Norton and Bradbury
1976; Lautie 1979; Saito et al. 1984; van den Berg et al.
1988, 1989 and 1990; Yaw and Gawrisch 1999; Unkefer
et al. 1991; Oba et al. 1995; Oba et al. 2002; Boroda et al.
2003; Osborne et al. 2003; Ilic and Cohen 2004; Soledade
et al. 2006; Amir-Heidari et al. 2007; Liu et al. 2009).
However, they dealt with the labeling of only a few posi-
tions in the Trp and are not suitable for 13C-, 2H- and 15N-
multi-labeling, such as in our SAIL method. We therefore,
decided to develop a new general method that allows for
Billeter et al. (1982). The computation used the following
˚
parameters: The bond lengths are Ca–Cb: 1.53 A; Cb–Hb2/3
:
˚
1.08 A; Cb–Cc: 1.50 A; Cc–Cd1: 1.37 A; Cd1–Hd1: 1.08 A;
˚
˚
˚
˚
˚
˚
Cc–Cd3: 1.43 A; Cd3–Ce3: 1.40 A; Ce3–He3: 1.08 A. The bond
angles are Ca–Cb–Hb2/3: 109.0°; Ca–Cb–Cc: 113.3°; Cb–Cc–
Cd1: 126.8°; Cc–Cd1–Hd1: 124.3°; Cb–Cc–Cd3: 126.9°;
Cc-Cd3-Ce3: 133.8°; Cd3-Ce3-He3: 121.3°.
Statistics for torsion angles and distances of Trp
To take the sequence redundancy and inaccuracy in local
geometries into account, we chose the structures in the Top
500 database (http://kinemage.biochem.duke.edu/databases/
top500.php), in which all of the structures have a resolution
the synthesis of any combination of 13C-, H- and 15N-
2
multi-labeling.
From the viewpoints of efficiency and economy, a wide
variety of SAIL amino acids should be synthesized via
common intermediates (Terauchi et al. 2008, 2011).
Recently, we reported the synthesis of isotopomers of Phe
and Tyr (Torizawa et al. 2005; Takeda et al. 2010). A
crucial step in the reports is the hydroxybenzoate synthesis,
which is flexible and accommodates the isotope labeling of
˚
better than 1.8 A. We retrieved data from a total of 1,553 Trp
residues. The pairs of torsion angles, v1 and v2, of these Trp
residues were classified by the K-means algorithm of the
MATLAB software (Press et al. 1986) into seven relatively
well populated conformational clusters, with the means and
standard deviations of the various inter-atom distances for
each cluster, as shown in Table 1.
any combination of 13C and H in the aromatic ring. For-
1
tunately, the labeling pattern used for observing the Hd
2
signals of SAIL-Phe could also be used for observing the
1He3 and 1Hg2 signals of the indole ring of Trp in this study.
Thus, by using the labeled benzoate 1 (Scheme 1), the
common intermediate of SAIL-Phe, we were able to syn-
thesize SAIL-Trps more efficiently.
Expression and purification of Myb-R2R3 selectively
labeled with [12Cc,12Ce2] SAIL-Trp residues
The DNA encoding mouse Myb-R2R3 (amino acids
89–193) was amplified by PCR, and cloned into the pET3a
vector (Novagen). The resulting plasmid, pMybR2R3, was
then transformed into E. coli Rosetta (DE3) pLysS com-
petent cells (Novagen). The cells were grown at 37°C in
M9 minimal medium, containing 15NH4Cl as the sole
nitrogen source. When the A600 value reached about 0.5,
5 mg of [12Cc,12Ce2] SAIL-Trp was added to 1 l of the
culture medium. After 1 h, isopropyl-b-D-1-thiogalacto-
pyranoside (IPTG) was added to a final concentration of
1 mM, and the induction was conducted for 6 h.
Scheme 1 shows the synthesis method of [b2,f2,f3-2H3;
C0,a,b,d1,e3,g2-13C6; UL-15N2]-Trp ([12Cc,12Ce2] SAIL-
Trp) and [b2,f2,f3-2H3; C0,a,b,c,d1,e2,e3,g2-13C8;
UL-15N2]-Trp (SAIL-Trp) in detail. After the conversion of
hydroxyl benzoate 1 to the tetrazolyl ether derivative 2,
deoxygenation was performed using deuterium gas to give
the benzoate 3. The benzoate derivative was then converted
to the benzamide 4, which was, in turn, subjected to
Hoffman rearrangement to yield the aniline 5. Gassman
indole synthesis (Gassman and Van Bergen 1974) followed
by reduction (Yuan and Ajami 1982) was performed to
give the indoline derivatives 8. In this stage, the indoline
derivatives were heated in D2O, because low-intensity H4
The Myb-R2R3 protein was purified by the method
described previously (Ogata et al. 1994), with a slight
modification. Briefly, the lysate was centrifuged at
20,0009g for 30 min at 4°C. Ammonium sulfate was then
added to the supernatant to 50%. After centrifugation at
20,0009g for 30 min, ammonium sulfate was added to the
supernatant to 80%. After centrifugation at 20,0009g for
1
and H6 signals appeared in the H NMR spectrum by H/D
exchange. The indoline 9 was then subjected to oxidation
using trichloroisocyanuric acid (Tilstam et al. 2001),
123

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J Biomol NMR (2011) 51:425–435
Scheme 1 Preparation of
[b2,f2,f3-2H3;
C0,a,b,d1,e3,g2-13C6;
UL-15N2]- and [b2,f2,f3-2H3;
C0,a,b,c,d1,e2,e3,g2-13C8;
UL-15N2]-Trp. Reagents and
Conditions: a 5-chloro-1-
phenyl-1H-tetrazole, K2CO3,
acetone; b 1 Pd/C, Na2CO3, D2,
benzene, 2 NaOH, MeOH; c 1
SOCl2, 2 CH3CO125NH4,
acetone; d NaOCl, D;
e MeS*CH123CO2Et, tert-
BuOCl, Et3N; f Raney-Ni/
EtOH; (g) 1 LiBH4, BF.3Et2O, 2
DCl; h D2O, D;
i trichloroisocyanuric acid,
DBU; j SAIL-Ser,
Tryptophanase
30 min, the precipitate was suspended and dialyzed against
20 mM potassium phosphate buffer (pH 6.8) containing
10 mM dithiothreitol (DTT). The dialyzed sample was loa-
ded onto a cation exchange column, and was eluted with a
NaCl concentration gradient from 0 to 1 M. The eluted
protein samples were applied to a gel filtration column.
Finally, the protein was dialyzed against 100 mM potassium
phosphate buffer (pH 6.8), containing 20 mM DTT and
1 mM sodium azide (NaN3). Approximately 2 mg of puri-
fied [12Cc,12Ce2] SAIL-Trp labeled Myb-R2R3 protein were
obtained per 1 l of the M9 culture medium containing 5 mg
[12Cc,12Ce2] SAIL-Trp, in which more than 90% of the Trp
residues were labeled with [12Cc,12Ce2] SAIL-Trp, according
to the mass spectrometric analysis (data not shown).
volume of 280 ll. The NMR sample buffer contained
100 mM potassium phosphate (pH 6.8), 20 mM DTT,
1 mM NaN3 and either 90% H2O/10% D2O or 100% D2O.
Unless stated otherwise, NMR measurements were per-
formed on a DRX800 spectrometer (Bruker Biospin; 800.
39 MHz for 1H) equipped with a TXI triple resonance
cryogenic probe at 17°C.
The [12Cc,12Ce2] SAIL-Trp-labeled Myb-R2R3: DNA
complex was prepared as described previously (Ogata et al.
1994). The complex sample was concentrated to 0.5 mM in
salt-free solutions (pH 6.8), containing 20 mM DTT, 1 mM
NaN3 and either 90% H2O/10% D2O or 100% D2O. The
protein/DNA molar ratio was 1:1.5. The NMR measure-
ments for the complex sample were performed at 37°C.
The NMR spectra were measured using the samples in
1
Synthesis and purification of the target DNA oligomers
90% H2O/10% D2O, except for the H–13C HSQC spectra
(Bodenhausen and Ruben 1980), which were obtained
using the 100% D2O sample. In the 2D 1H–13C HSQC and
that with the TROSY method on t1 correlation experiments
(Pervushin et al. 1997, 1998; Sørensen et al. 1997), the data
size and the spectral width were 256 (t1) 9 1024 (t2) and
3,200 Hz (x1, 13C) 9 11,200 Hz (x2, 1H), respectively.
The carrier frequencies of 1H and 13C were 4.7 and
122 ppm, respectively. The number of scans/FID was 64,
and the repetition time was 1.1 s. In the 2D 1H–13C HSQC
experiments for correlating the e3 and g2 positions, the
data size and the spectral width were 128 (t1) 9 1024 (t2)
A 16-base DNA oligomer, d(CCTAACTGACACACAT),
and its complementary strand, synthesized with a DNA
synthesizer and purified by HPLC, were purchased from
SIGMA. They were annealed and further purified, as
described previously (Ogata et al. 1994).
NMR spectroscopy
The Myb-R2R3 protein, residue-selectively labeled with
[12Cc,12Ce2] SAIL-Trp, was concentrated to 0.5 mM in a
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J Biomol NMR (2011) 51:425–435
429
Table 1 Possible conformational clusters of the Trp side-chain residues and patterns of observable intra-residue NOEs
˚
Distances between two atoms (A)
No.
%
Torsion angles (°)
v1
v2
Hb2-Hd1
Hb3-Hd1
HN-Hd1
Ha-Hd1
Hb2-He3
Hb3-He3
HN-He3
Ha-He3
1
2
3
4
5
6
7
31.3
17.6
16.6
11.8
9.0
-67.1
(12.0)
-167.5
(18.9)
-167.2
(19.4)
-68.4
(9.4)
98.7
2.67
3.78
3.03
4.28
4.14
2.71
5.12
2.88
(17.2)
74.7
(0.12)
2.79
(0.12)
3.88
(0.39)
4.93
(0.28)
3.05
(0.12)
4.04
(0.21)
2.54
(0.42)
4.70
(0.48)
4.59
(20.7)
-104.7
(15.1)
-5.25
(21.57)
-89.8
(10.7)
88.4
(0.17)
3.58
(0.05)
2.64
(0.23)
5.05
(0.32)
4.35
(0.18)
2.76
(0.12)
4.18
(0.19)
4.55
(0.29)
2.74
(0.11)
3.72
(0.09)
3.60
(0.15)
3.23
(0.23)
2.37
(0.20)
2.81
(0.08)
3.01
(0.33)
4.87
(0.42)
5.13
(0.15)
3.85
(0.18)
2.72
(0.47)
2.77
(0.22)
4.45
(0.26)
2.58
(0.30)
4.11
(0.44)
4.98
(0.14)
4.30
61.5
(11.0)
60.4
(0.06)
2.72
(0.10)
3.85
(0.49)
4.42
(0.12)
4.46
(0.12)
4.10
(0.10)
2.58
(0.36)
3.18
(0.17)
4.27
5.3
(10.1)
-68.8
(14.1)
(12.9)
-88.2
(16.1)
(0.11)
3.86
(0.06)
2.74
(0.39)
5.01
(0.13)
3.13
(0.11)
2.57
(0.12)
4.08
(0.57)
2.73
(0.17)
4.50
4.7
(0.06)
(0.14)
(0.42)
(0.31)
(0.13)
(0.15)
(0.56)
(0.31)
Values in parentheses are SD. Distances shorter than 3.1 A are written in bold. While Hb2 is deuterated in [12Cc,12Ce2] SAIL-Trp, the
˚
corresponding distance is provided, for reference
and 8,100 Hz (x1, 13C) 9 11,700 Hz (x2, 1H), respec-
tively. In the INEPT, which is composed of [(p/2)x(1H)-s-p
(1H,13C)-s-(p/2)y(1H)], the delay s was 6.1 ms when cor-
Results and discussion
1
The assignment of Hd1 and He3 peaks by NOE
1
1
1
1
1
with Hb3, Ha and HN protons
1
relating He3–13Cg2 or Hg2–13Ce3 via three-bond scalar
coupling. Although this delay time was much shorter than
3
the calculated optimal value of ca. 31 ms for the JCH
The assignment of the aromatic peaks in [12Cc,12Ce2]
C /1Hd1 and
1
between He3 and
13
1
13
C
g2
or Hg2–13Ce3, which are both
SAIL-Trp began by correlating the
13
d1
about 8 Hz, we found 6.1 ms to be a good compromise
between the optimal delay and fast relaxation times. The
carrier frequencies of protons and carbons were 4.7 and
125.5 ppm, respectively, and the number of scans/FID was
256. The repetition time was 2 s.
C /1He3 peaks to the backbone/aliphatic peaks (i.e.,
e3
1Hb3, 1Ha and 1HN peaks) previously assigned by the intra-
residue NOEs (Fig. 1).
In the case of Phe and Tyr residues, the v2 angles have a
preference for two regions: *?90° and *-90° (Ponder
and Richards 1987; Schrauber et al. 1993; Dunbrack and
Karplus 1993). On the other hand, the v2 angles of Trp had
preferences for three different regions: *?90°, *0° and
*-90° (Dunbrack and Cohen 1997). In accordance with
this, our survey of the Trp side-chain conformations, using
the TOP500 structure database, revealed that there are
three favorable conformational clusters with respect to the
v2 angle. In addition, when the v2 angles are *?90° and
*-90°, there are three clusters with respect to the v1 angle
(i.e., *?60°, *180° and *-60°). On the other hand,
when the v2 angle is *0°, only the v1 angle of *-60° is
highly populated (Fig. 2).
The 3D 13C-edited NOESY-HSQC experiment (Kay et al.
1989) was performed with a mixing time of 100 ms. The data
size andthe spectral width were 128(t1) 9 20 (t2) 9 2,048(t3)
points and 11,100 Hz (x1, 1H) 9 2,400 Hz (x2, 13C) 9
1
11,100 Hz (x3, H), respectively. The number of scans/FID
was 48, and the repetition time was 1.2 s. The carrier fre-
quencies of 1H and 13C were 4.7 and 122 ppm, respectively.
The NMR chemical shift perturbation of Myb-R2R3
labeled by [12Cc,12Ce2] SAIL-Trp, upon binding to the
target DNA, was performed on a DRX800 spectrometer
equipped with a TXI cryogenic probe at 37°C. The 1H–13C
correlation experiments for the unbound and DNA-bound
1
forms were both acquired using H–13C TROSY HSQC.
In addition, the two preferred angles of *?90° and
*-90° in Phe and Tyr residues are practically identical,
due to the symmetry of the aromatic rings. However, the
Trp residue is unsymmetrical with respect to the 180°
rotation about the Cb–Cc axis, and thus the spatial prox-
imity of the proton pairs of interest is largely affected by
The data size and spectral width were 256 (t1) 9 2,048 (t2)
and 3,200 Hz (x1, 13C) 9 11,200 Hz (x2, 1H), respec-
tively. The number of scans/FID was 256, and the repeti-
1
tion time was 1.2 s. The carrier frequencies of H and 13C
were 4.7 and 121 ppm, respectively.
123

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J Biomol NMR (2011) 51:425–435
the rotameric conformation in the case of Trp, as compared
with those of Phe and Tyr.
*?90°, a strong NOE is observed between 1He3 and 1Hb3.
When the v2 angle is *-90°, a strong NOE between 1Hd1
and 1Hb3 is observed. When v2 is around *0°, a weak NOE
between 1He3 and 1Hb3 and a strong NOE between 1Hd1 and
1Ha are observed. In the case of Myb-R2R3, the first case
corresponds to Trp-95, Trp-115, Trp-147 and Trp-166, the
second case corresponds to Trp-134 and Trp-185, and the last
case is not included, which is in agreement with the v2 value
of Trp in the crystal structure (Fig. 3).
It is therefore, important to understand the relationship
between the spatial proximity of each proton pair and the
rotameric conformation of the Trp residue. Here, we
focused on the seven preferred rotameric conformations,
which correspond to 96.3% of the total conformations, and
surveyed the proton–proton distance of interest for each
conformation (Table 1). In most conformations, at least
1
1
1
˚
one of the Hb3, Ha and HN atoms is located within 3.1 A
If ambiguity remains, then an additional experiment
using another type of [12Cc,12Ce2] SAIL-Trp, in which 1Hb3
is mono-deuterated and 1Hb2 keeps 1H, would be useful. As
shown in Table 1 and Fig. 4, the NOEs observed for the
1Hb2–1Hd1/1Hb2–1He3 pairs are complementary to those for
the 1Hb3–1Hd1/1Hb3–1He3 pairs. By combining the results of
the two [12Cc,12Ce2] SAIL-Trps with different stereo-
selectivities of mono-deuteration, the v2 angle can be
determined unambiguously.
1
of the Hd1 atom, which is also the case for the He3 atom.
1
(Here Hb2 is not considered, because it is deuterated.)
1
1
1
Therefore, the Hd1 and He3 peaks can, in most cases, be
correlated with either of the previously assigned backbone/
aliphatic protons by intra-residue NOEs. For example, in
the most preferred conformation (v1 = - 67 12°,
1
1
v2 = - 99 17°), the Hd1 and He3 protons are spatially
1
proximal to the HN and Hb3 protons, respectively, which
1
1
ensures the observation of NOEs between Hd1 and HN
1
It should be noted that, in the case of proteins labeled
with [U–13C/15N] Trp, an attempt to relate the intra-residue
NOEs between the aromatic and b-protons would not prove
1
and between He3 and Hb3.
1
To demonstrate this, we observed the intra-residue NOEs
using the 12 kDa DNA binding domain of Myb (Myb-
R2R3), selectively labeled with [12Cc,12Ce2] SAIL-Trp. In
this case, we used [12Cc,12Ce2] SAIL-Trp, rather than original
1
fruitful, since the spin diffusion of the NOE via Hb2
generates NOEs between spatially distal protons. In addi-
tion, the stereo-specific assignment of the two methylene
protons in [U–13C/15N] Trp is frequently troublesome. The
use of [12Cc,12Ce2] SAIL-Trp circumvents the laborious
stereo-specific assignment step.
13
SAIL-Trp, to eliminate the Cd1–13Cc spin coupling
1
(70–75 Hz) and observe Hd1/13Cd1 peaks with high inten-
sity and resolution (Fig. 1). As shown in Fig. 3, for all of the
1Hd1 and 1He3 protons of Trp, intra-residue NOEs with either
the 1Ha, 1Hb3 or 1HN proton were observed, which led to the
assignment of the 1Hd1 and 1He3 peaks.
Assignment of the e1/g2 resonance in the [12Cc,12Ce2]
SAIL-Trp residue
In addition, the observed intra-residue NOEs, especially
between1Hd1 and 1Hb3 or 1He3 and 1Hb3, provide information
on the v2 angle. As shown in Fig. 4, when the v2 angle is
Once the 1Hd1 and 1He3 peaks have been assigned, the next
step is the assignment of He1/15Ne1 and Hg2/13Cg2. The
1He1 could readily be correlated to1Hd1 by the vicinal NOE,
without any difficulty (Figs. 1, 3). The assignment of the
1Hg2/13Cg2 resonances was performed by correlating the
1
1
1
1
1He3/13Ce3 resonances to Hg2/13Cg2 via the He3–13Cg2 or
1Hg2–13Ce3 three-bond scalar coupling (*8 Hz). While the
magnetization transfer though such a long range coupling
does not work in the case of [U–13C/15N] Trp, the atom-
specific 2H- and 13C-labeling in [12Cc,12Ce2] SAIL-Trp
permits efficient magnetization transfer (Torizawa et al.
2005; Takeda et al. 2009, 2010). In this magnetization
transfer process, we employed an HSQC pulse scheme,
and in INEPT, composed of [(p/2)x(1H)-s-p (1H,13C)-s-
(p/2)y(1H)], the delay s was adjusted to multiples of
1
1/(21JCH), such that the evolution due to JCH (1He3–13Ce3
and 1Hg2–13Cg2) was refocused. Through the observation of
1
1
the He3–13Cg2 and Hg2–13Ce3 correlation peaks, the
C /1Hg2 peaks of Myb-R2R3 were unambiguous
13
Fig. 2 Distribution of side-chain v1 and v2 dihedral angles in Trp
residues in proteins. These data are derived from the TOP500
structure database (http://kinemage.biochem.duke.edu/databases/top
500.php). The red zone is the major conformation. The clusters are
labeled with numbers, in the order of their population
g2
assigned (Fig. 5). Presumably, conventional hetero-nuclear
multi-bond correlation spectroscopy (HMBC) (Bax and
Summers 1986) should also work in this case.
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J Biomol NMR (2011) 51:425–435
431
Fig. 3 Intra-residue NOEs
between the aromatic and
aliphatic/amide protons with
patterns dependent on the side-
1
chain conformation. a H–1H
1
strips from the 3D H–13
C
NOESY-HSQC spectrum of
Myb-R2R3 residue-selectively
labeled by [12Cc,12Ce2] SAIL-
Trp (0.5 mM sample
concentration), taken at the
13
13
C
of the six Trp residues. Intra-
and
C chemical shifts
e3
d1
1
1
residue NOEs with HN, Ha,
1
1Hb3 and He1 are marked by
green, cyan, red and blue
circles, respectively. Above the
spectra, the values of the v1 and
v2 angles in the corresponding
crystal structure (PDB# 1GV2)
are described. The / angles of
the Trp residues are Trp-95:
-86°; Trp-115: -54°; Trp-134:
-70°; Trp-147: -122°; Trp-
166: -54°; Trp-185: -66°. The
NOESY data were acquired on a
DRX800 spectrometer at 17°C.
The NOE mixing time was
100 ms. As representative cases
of v2 of *?90° and *-90°,
the rotameric conformations are
displayed for Trp-147 (b) and
Trp-185 (c), respectively. In
b and c, the observed intra-
residue NOEs are displayed as
arrows, with colors
corresponding to those in (a)
Myb-R2R3 labeled with [12Cc,12Ce2] SAIL-Trp was
improved, by virtue of the elimination of the one-bond
The NMR spectra of Myb-R2R3 selectively labeled
with [12Cc,12Ce2] SAIL-Trp
1
13C–13C coupling and the deleterious H–1H dipole inter-
Through the aforementioned assignment procedures, the
1
actions (Fig. 6). Finally, we successfully completed the
assignment of the Trp residues of Myb-R2R3 using SAIL-
Trp, a feat that could not have been accomplished using a
conventional, uniformly 13C,15N-double labeled sample
(Ogata et al. 1994). In the case of Myb-R2R3, the signal of
three H–13C aromatic peaks of each [12Cc,12Ce2] SAIL-
Trp residue within the Myb-R2R3 protein were exclusively
assigned. As compared to the spectrum of Myb-R2R3
labeled with UL-Trp (data not shown), the spectrum of
123

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J Biomol NMR (2011) 51:425–435
Fig. 5 Assignment of the 13Cg2/1Hg2 peaks by three bond J coupling
13
with Ce3/1He3. 1H–13C HSQC spectra of Myb-R2R3 selectively
labeled by [12Cc,12Ce2] SAIL-Trp, acquired at different INEPT time
delays. In the INEPT spectra, composed of [(p/2)x(1H)-s-p (1H,13C)-
s-(p/2)y(1H)], the delay s values were 1.5 ms (blue) and 6.1 ms (red).
1
1
13
C
In the spectra, He3/13Ce3 and Hg2
/
peaks are labeled with their
g2
assignments
Fig. 4 The dependence of the distance between the backbone/
aliphatic and aromatic protons on the rotameric conformation of the
Trp residue. The proton–proton distances were simulated for pairs of
Hb2–Hd1, Hb2–He3, Hb3–Hd1 and Hb3–He3. This computation was
performed with fixed bond lengths and bond angles, using the Matlab
software (Mathworks Inc.), according to a previously described
procedure (Billeter et al. 1982). The values of the parameters are
described in the ‘‘Materials and methods’’
Trp-134 1Hd1/13Cd1 was weaker relative to the other peaks,
probably due the exchange-broadening arising from the
previously reported instability of the third helix in R2
(Myrset et al. 1993).
The feasibility of assigning the aromatic peaks in
[12Cc,12Ce2] SAIL-Trp residues generates an opportunity to
proceed to further NMR studies, including the structures,
dynamics and interactions of proteins. As a case study of an
interaction analysis, we performed chemical shift pertur-
bation experiments for the assigned Trp aromatic peaks by
adding the target 16-base-pair DNA oligomer [d(CCTA-
ACTGACACACAT)]2. As shown in Fig. 7a, many of the
signals underwent chemical shift changes upon binding to
the target DNA. As the dissociation constant between the
Fig. 6 NMR spectra of Myb-R2R3 selectively labeled with
1
[
12Cc,12Ce2] SAIL-Trp. H–13C TROSY-HSQC spectrum of 0.5 mM
of Myb-R2R3 residue-selectively labeled by [12Cc,12Ce2] SAIL-Trp.
The peaks are labeled with their assignments. The data were acquired
on a DRX800 spectrometer at 17°C. The peak intensity of the
13
1Hd1
than those of other peaks, probably due to exchange broadening
/
C
d1
signal of Trp-134, marked by a dashed circle, was weaker
Myb-R2R3 and the DNA is on the order of 10-9
M
(Tanikawa et al. 1993), the binding was within the slow
exchange regime on the NMR time scale, and the aromatic
peaks of the DNA-bound form were analyzed in the same
way, independently of the free form (data not shown).
Intriguingly, relatively large chemical shift changes were
observed for the Trp residues distal from the binding site
(e.g., Trp-95 and Trp-147), rather than those located within
the binding site (e.g., Trp-115 and 166) in the complex
structure (PDB code: 1MSE) (Fig. 7b, c). Considering that
the largely perturbed Trp indole ring constitutes the
hydrophobic core in the tandem repeat, the chemical shift
is assumed to be caused by the subtle rearrangement of
helices upon binding to the target DNA, implying the
potential of the aromatic peaks of Trp residues to serve as
probes to detect such subtle conformational rearrangements
occurring in the structural core.
Conclusions
In this study, we present a strategy for assigning the aro-
matic resonances (d1, e1, e3 and g2 peaks) of [12Cc,12Ce2]
SAIL-Trp residues, which has provided new insights into
the mechanism of Myb-R2R3 binding to its target DNA.
123

J Biomol NMR (2011) 51:425–435
433
Acknowledgments We thank Prof. Yoshifumi Nishimura and Dr.
Aritaka Nagadoi, of Yokohama City University, for providing the
a
¨
Myb-R2R3 gene, and Dr. Frank Lohr, Institute of Biophysical
Chemistry and Center of Biological Magnetic Resonance, Goethe-
University, for his kind help in providing the NMR sequence of the
1H–13C TROSY experiments. This work was supported in part by the
Targeted Protein Research Program (MEXT) to M.K., a Grant-in-Aid
for Young Scientists (B) (23770109) to M.T. and a Grant-in-Aid for
Young Scientists (B) (23770111) to Y.M.
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