NMR assignment methods for the aromatic ring resonances of phenylalanine and tyrosine residues in proteins

Article abstract of DOI:10.1021/ja051386m

The unambiguous assignment of the aromatic ring resonances in proteins has been severely hampered by the inherently poor sensitivities of the currently available methodologies developed for uniformly ¹³C/ ¹⁵N-labeled proteins. Especi

Full text of DOI:10.1021/ja051386m

Published on Web 08/20/2005
NMR Assignment Methods for the Aromatic Ring Resonances
of Phenylalanine and Tyrosine Residues in Proteins
Takuya Torizawa,† Akira Mei Ono,† Tsutomu Terauchi,† and
Masatsune Kainosho*,†,‡
CREST-JST and Graduate School of Science, Tokyo Metropolitan UniVersity,
1-1 Minami-ohsawa, Hachioji, 192-0397, Japan
Received March 4, 2005; E-mail: kainosho@nmr.chem.metro-u.ac.jp
Abstract: The unambiguous assignment of the aromatic ring resonances in proteins has been severely
hampered by the inherently poor sensitivities of the currently available methodologies developed for uniformly
13C/15N-labeled proteins. Especially, the small chemical shift differences between aromatic ring carbons
and protons for phenylalanine residues in proteins have prevented the selective observation and
unambiguous assignment of each signal. We have solved all of the difficulties due to the tightly coupled
spin systems by preparing regio-/stereoselectively 13C/2H/15N-labeled phenylalanine (Phe) and tyrosine
(Tyr) to avoid the presence of directly connected 13C-1H pairs in the aromatic rings. The superiority of the
new labeling schemes for the assignment of aromatic ring signals is clearly demonstrated for a 17 kDa
calcium binding protein, calmodulin.
Introduction
As aromatic amino acid residues are often located in the
hydrophobic core regions of proteins, structural data, such as
the nuclear Overhauser effect (NOE) cross-peaks associated with
the ring protons, are crucial for determining protein structures
by NMR.1-4 However, the insufficient chemical shift dispersions
of the 1H and/or 13C resonances of the aromatic ring resonances
have frequently made the detection of the individual resonances
of the aromatic ring very difficult or even impossible for
conventional, uniformly 13C,15N-labeled proteins.5 Especially,
the NMR resonances of the δ, ꢀ, and ú of Phe and the δ of Tyr
tend to overlap each other, according to the NMR database
(BioMagResBank URL: http://www.bmrb.wisc.edu/).6 In ad-
dition, another difficulty lies in analyses of the aromatic
resonances. Many measurement methods to assign them, using
through-bond connectivity from the aliphatic resonances, have
been reported.7-11 However, an uncertain assignment method
employing the NOE between aliphatic and aromatic resonances12
Figure 1. Stable isotope labeling patterns for Phe and Tyr, and magnetiza-
tion transfer pathways for HBCBCGHE and HBCBCGCZHZ measurements.
(1) 13Cγ-13Cꢀ-1Hꢀ pattern for [1Hâ-13Câ-13Cγ-13Cꢀ-1Hꢀ]-Tyr (1a)/Phe
(1b). (2) 13Cγ-13Cú-1Hú pattern for [1Hâ-13Câ-13Cγ-13Cú-1Hú]-Phe (2).
12C atoms are not shown in the figures. Arrows indicate the magnetization
1
transfer pathways. In both cases, the transfer started from Hâ.
is still generally used because some of the developed methods
either are not sensitive enough or adopt the evolution of the
poorly dispersed chemical shifts. The two problems described
above have been factors that limit the application of NMR to
structural determination because they become more serious as
the molecular weight of the targeted protein increases.
† CREST-JST.
‡ Graduate School of Science.
(1) Smith, B. O.; Ito, Y.; Raine, A.; Teichmann, S.; Ben-Tovim, L.; Nietlispach,
D.; Broadhurst, R. W.; Terada, T.; Kelly, M.; Oschkimat, H.; Shibata, T.;
Yokoyama, S.; Laue, E. D. J. Biomol. NMR 1996, 8, 360-368.
(2) Clore, G. M.; Starich, M. R.; Bewely, C. A.; Cai, M.; Kuszewski, J. J.
Am. Chem. Soc. 1999, 121, 6513-6514.
To make the NMR spectra for aromatic resonances simple
(3) Aghazadeh, B.; Zhu, K.; Kubiseski, T. J.; Liu, G. A.; Pawson, T.; Zheng,
Y.; Rosen, M. K. Nat. Struct. Biol. 1998, 5, 1098-1107.
(4) Medek, A.; Olejniczak, E. T.; Meadows, R. P.; Fesik, S. W. J. Biomol.
NMR 2000, 18, 229-238.
(5) Yamazaki, T.; Yoshida, M.; Nagayama, K. Biochemistry 1993, 32, 5656-
5669.
2
and sensitive, we developed a method to introduce novel H,
13C labeling patterns to Phe and Tyr and assignment strategies
for the amino acids. As shown in Figure 1, one of the patterns
is that all of the nonexchangeable hydrogens in the aromatic
rings, except for the ꢀ position, were replaced with 2H, and the
carbons at the γ and ꢀ positions were labeled with 13C, denoted
(6) Seavey, B. R.; Farr, E. A.; Westler, W. M.; Markley, J. L. J. Biomol. NMR
1991, 1, 217-236.
(7) Yamazaki, T.; Forman-Kay, J. D.; Kay, L. E. J. Am. Chem. Soc. 1993,
115, 11054-11055.
(8) Grzesiek, S.; Bax, A. J. Am. Chem. Soc. 1995, 117, 6527-6531.
(9) Carlomagno, T.; Maurer, M.; Sattler, M.; Schwendinger, M. G.; Glaser, S.
J.; Griesinger, C. J. Biomol. NMR 1996, 8, 161-170.
(10) Lohr, F.; Ruterjans, H. J. Magn. Reson. B 1996, 112, 259-268.
(11) Prompers, J. J.; Groenewegen, A.; Hilbers, C. W.; Pepermans, H. A. M. J.
Magn. Reson. 1998, 130, 68-75.
(12) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York, 1986.
9
12620
J. AM. CHEM. SOC. 2005, 127, 12620-12626
10.1021/ja051386m CCC: $30.25 © 2005 American Chemical Society

Aromatic Ring Resonances of Phe and Tyr in Proteins
A R T I C L E S
Scheme 1 a
a Reagents and conditions: (i) (1) (EtOCO)2, NaOEt, (2) conc DCl, (3) Cu; (ii) (1) [13C3]-ethyl malonate, t-BuOK/BuOD; (iii) (1) MeI, acetone,
(2) LiAlD4, (3) PDC; (iv) (1) [13C2;15N]-NAc-glycine, Ac2O, AcONa, (2) MeOH, Et3N; (v) (1) (S,S)-Et-DuPhos-Rh, H2, (2) 1N-HCl, (3) HBr-AcOH.
Scheme 2 a
a Reagents and conditions: (i) (1) HCl, ∆, (2) Tet-Cl, K2CO3, (3) D2, Pd/C; (ii) (1) LiAlD4, (2) PDC; (iii) (1) [13C2;15N]-NAc-glycine, Ac2O, AcONa,
(2) MeOH, Et3N; (iv) (1) (S,S)-Et-DuPhos-Rh, H2, (2) 1N-HCl.
as [13Cγ-13Cꢀ-1Hꢀ]-Tyr (1a)/Phe (1b). The other is that the
hydrogens at the δ and ꢀ positions of Phe were replaced with
2H, and the carbons at the γ and ú positions were labeled with
13C, denoted as [13Cγ-13Cú-1Hú]-Phe (2), as shown in Figure
1. In both cases, the aliphatic moieties were uniformly labeled
with 13C and 15N, and the pro-R proton of the â-methylene was
stereospecifically labeled with 2H, thus enabling the unambigu-
ous chiral assignment for the prochiral methylene signals.
Therefore, to use this novel isotope labeling strategy for NMR
structure determinations of proteins, we chemically synthesized
the labeled Phe and Tyr and then successfully incorporated these
labeled amino acids into calmodulin, with a molecular weight
of 17 kDa, by an E. coli cell-free system.13-15 With the help of
the extremely high protein yield of this cell-free system, as
compared to in vivo expression systems,15 only a few milligrams
of the labeled amino acids were required to prepare the NMR
samples. Here, the effectiveness of the samples is evaluated from
the viewpoints of sensitivities and spectral overlap, and the
assignment schemes are introduced.
We previously reported the stereoselective syntheses of Phe
labeled with deuterium on the Hδ, Hꢀ, and Hú positions.16
However, these synthetic routes were not suitable for the
synthesis of these amino acids because of the difficulty in the
regioselective incorporation of 13C in the aromatic ring. In this
work, the labeled amino acids described above were synthesized
(15) Torizawa, T.; Shimizu, M.; Taoka, M.; Miyano, H.; Kainosho, M. J. Biomol.
NMR 2004, 30, 311-325.
(16) Nishiyama, K.; Oba, M.; Ueno, R.; Morita, A.; Nakamura, Y.; Kainosho,
M. J. Labelled Compds. 1994, 9, 831-837.
(13) Torizawa, T.; Terauchi, T.; Kainosho, M. Seikagaku 2002, 74, 1279-1284.
(14) Torizawa, T.; Terauchi, T.; Kainosho, M. Seibutsu Butsuri 2004, 44, 200-
205.
9
J. AM. CHEM. SOC. VOL. 127, NO. 36, 2005 12621

A R T I C L E S
Scheme 3 a
Torizawa et al.
a Reagents and conditions: (i) (1) (EtOCO)2, NaOEt, (2) conc DCl, (3) Cu; (ii) (1) [13C3]-ethyl malonate, t-BuOK/BuOD, (2) Tet-Cl, K2CO3, (3) H2,
Pd/C; (iii) (1) LiAlD4, (2) PDC; (iv) (1)
[
13C2;15N]-NAc-glycine, Ac2O, AcONa, (2) MeOH, Et3N; (v) (1) (S,S)-Et-DuPhos-Rh, H2,
(2) 1N-HCl.
Phe and [ul-13C,15N]-Tyr were purchased from Cambridge Isotope
efficiently, using a hydroxyl benzoic acid derivative as key
intermediate, starting from various types of 13C-labeled acetone
and ethyl malonate.
Laboratories. Each unlabeled amino acid was added to the system to a
final concentration of 1 mM. All of the NMR samples were prepared
according to the previously published method.15,26,27 Each NMR sample
contained 0.5 mM calmodulin in 5 mM MES-d13 (Cambridge Isotope
Laboratories), ca. 10 mM bis-Tris-d19 (Cambridge Isotope Laboratories)
to adjust the pH to 6.5,28 5 mM CaCl2, 0.1 mM NaN3, and 10% D2O
or 100% D2O. The NMR measurements were performed on a Bruker
DRX600 spectrometer equipped with a CryoProbe at 37 °C.
From 3, 4H-pyran-4-one 4 was obtained by a modification
of reported procedure.17,18 Then, 4H-pyran-4-one 4 was sub-
jected to a condensation/aromatization reaction to give the key
intermediate 5.19 For the synthesis of Tyr 1a (Scheme 1), the
hydroxyl group of 5 was methylated, and the ester was reduced
by LiAlD4. The following oxidation by PDC yielded 6.20 Then,
6 was converted into the dehydrotyrosine derivative 7 by a
condensation with labeled Gly derivatives.21,22 Compound 7 was
subjected to hydrogenation in the presence of (S,S)-Et-DuPhos-
Rh, as a catalyst in MeOH,23 and then was deprotected to give
Tyr 1a.24
For the preparation of Phe 1b, it is possible to synthesize it
only by the deoxygenation of the hydroxyl group of the key
intermediate 5. Thus, a reductive deuteration of the hydroxyl
group of 5 was carried out, using our recently reported methods
to place a deuterium at the Hú position.16,25 Then, as in the
method for the synthesis of Tyr, 9 was converted into Phe 1b.
For the synthesis of Phe 2 labeled simultaneously with 13C
in the ú position and with deuterium in the δ and ꢀ positions,
[2-13C]acetone was used instead of [1,3-13C2]acetone.
1
The H-13C HSQC spectra29 and the constant time (ct) version30
for the aromatic resonances shown in Figure 2 were obtained using
the samples in 100% D2O. The other spectra were measured using the
samples in 10% D2O. The 1H-13C (ct) HSQC spectra shown in Figures
4 and 5 were acquired using coherence selection with sensitivity
enhancement.31-33 All of the HSQC measurements for aromatic
resonances, except for the ct version, were performed under the
following conditions: data size, 128 (t1) × 1024 (t2) complex points;
13
t
( C) ) 30.3 ms and t2max(1H) ) 128 ms; and spectral widths in
1max
ω1(13C) and ω2(1H) of 4200 and 8000 Hz, respectively. The carrier
frequencies of 13C and 1H were 126 and 4.7 ppm, respectively. In each
t1 increment, eight transients were accumulated, and the repetition delay
was 1.2 s. The data set was zero-filled to 256 (t1) × 2048 (t2) complex
points. The ct-HSQC spectrum for the aromatic resonances was obtained
under the same conditions, except that the t1 complex points were 70
and t1max(13C) ) 16.6 ms, as limited by the constant evolution period
of 17 ms. Compared to the ct-HSQC measurements for the aromatic
resonances, the altered parameters in those for the aliphatic resonances
were as follows: 256 (t1) complex points, t1max(13C) ) 20.7 ms, ct
evolution ) 26.6 ms, spectral width in 13C ) 12 000 Hz, carrier
frequency of 13C ) 48 ppm, and zero-filling to 512 (t1) complex points
Experimental Section
Calmodulin was synthesized by the E. coli cell-free system, which
we developed.13-15 For the synthesis, the amounts of the labeled Phe
and Tyr added were 1.32 and 0.37 mg, respectively. The [ul-13C,15N]-
(17) Riegel, E.; Zwilgmeyer, F. Organic Syntheses, Coll. Vol. II; Wiley: New
York, 1943; p 126.
(26) Ikura, M.; Marion, D.; Kay, L. E.; Shih, H.; Krinks, M.; Klee, C. B.; Bax,
A. Biochem. Pharmacol. 1990, 40, 153-160.
(18) Organic Syntheses with Isotopes, Part II; Interscience Publishers, Inc.: New
York, 1958; p 1388.
(27) Ikura, M.; Kay, L. E.; Bax, A. Biochemistry 1990, 29, 4659-4667.
(28) Kelly, A. E.; Ou, H. D.; Withers, R.; Dotsch, V. J. Am. Chem. Soc. 2002,
124, 12013-12019.
(19) Lang, M.; Lang-Fugmann, S.; Steglich, W. Organic Syntheses; Wiley: New
York, 2002; Coll. Vol. 78, pp 113-122.
(29) Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185-189.
(30) Vuister, G. W.; Bax, A. J. Magn. Reson. 1998, 98, 428-435.
(31) Palmer, A. G., III; Cavanagh, J.; Wright, P. E.; Rance, M. J. Magn. Reson.
1991, 93, 151-170.
(32) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114, 10663-
10665.
(33) Schleucher, J.; Schwendinger, M.; Sattler, M.; Schmidt, P.; Schedletzky,
O.; Glaser, S. J.; Sorensen, O. W.; Griesinger, C. J. Biomol. NMR 1994, 4,
301-306.
(20) Beyer, J.; Lang-Fugmann, S.; Mu¨hlbauer, A.; Steglich, W. Synthesis 1998,
1047-1051.
(21) Erlenmeyer, E. Justus Liebigs. Ann. Chem. 1893, 275, 1-20.
(22) Ojima, I.; Kato, K.; Fujita, M. J. Org. Chem. 1989, 54, 4511-4522.
(23) Burk, M.; Feaster, J.; Nugent, W.; Harlow, R. J. Am. Chem. Soc. 1993,
115, 10125-10138.
(24) Li, G.; Patel, D.; Hurby, V. Tetrahedron Lett. 1993, 34, 5393-5396.
(25) Viswanatha, V.; Hruby, V. J. J. Org. Chem. 1980, 45, 2010-2012.
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Aromatic Ring Resonances of Phe and Tyr in Proteins
A R T I C L E S
scans per increment ) 16 and zero-filling to 16 (t2) complex points
prior to transformation. The total measurement time of the 3D spectrum
was 3.4 h. Prior to Fourier transformation, the data matrices were
multiplied with a 60° -shifted sine bell window in the indirect
dimensions and a 45°-shifted sine bell window in the acquisition
dimension.34 Data processing was performed with the program XWIN-
NMR, version 3.5 (Bruker), and for the data analysis, the program
SPARKY, version 3.106 (Goddard and Kneller, University of Califor-
nia, San Francisco), was used. The 1H chemical shifts were referenced
to external DSS, and the 13C and 15N shifts were indirectly refer-
enced.35,36
Results and Discussion
We compared the 1H-13C HSQC spectra of calmodulin
samples labeled according to the patterns in Figure 1 (Figure
2b) using a constant time (ct) version of the measurement of
the protein, in which the Phe and Tyr residues were uniformly
13C-labeled (Figure 2a). As a result, the signal overlapping was
alleviated in Figure 2b, due to the disappearance of some
resonances and the sharpening of the residual resonances.
Sensitivity improvements were also observed (averaged ratios
of the signal intensities to those of the uniformly 13C-labeled
rings: Phe ꢀ: 13, ú: 26, Tyr: 4). One of the reasons for the
improvement is the attenuation of the dipolar interactions, by
replacing the hydrogens adjacent to the detecting nuclei with
2H. In the case of the uniformly 13C-labeled aromatic residues,
a long ct (∼17 or 34 ms) evolution period, which causes a
1
reduction of the magnetization, must be used in the H-13C
HSQC measurement so that the 13C-13C coupling can be
refocused. On the other hand, the novel labeling patterns do
not require the constant time evolution in the measurement. This
is another reason the spectra had improved sensitivity. TROSY
in a ct evolution manner has been developed for the sensitive
detection of aromatic resonances in uniformly 13C-labeled
proteins.37 By altering the evolution period to remove the
constant time, this measurement technique would allow more
sensitive detection of the aromatic resonances of the novel
labeling patterns in larger proteins.
The schemes for the assignments of the aromatic resonances
are shown in Figure 3. The pathways of magnetization are as
follows:
Figure 2. 1H-13C correlated spectra of calmodulin. (a) ct-HSQC spectrum
of [ul-13C,15N]-Phe and -Tyr selectively labeled calmodulin. (b) HSQC
spectrum of [13Cγ-13Cꢀ-1Hꢀ]-Tyr (1a) and Phe (1b) selectively labeled
calmodulin. (c) HSQC spectrum of [13Cγ-13Cú-1Hú]-Phe (2) selectively
labeled calmodulin. In (a), the 13C region between 130 and 135 ppm contains
the resonances of the δ, ꢀ, and ú of Phe, and the δ of Tyr. The corresponding
regions in (b) and (c) contain only the resonances of ꢀ and ú of Phe,
respectively. In another region near 118 ppm in (a) and (b), the ꢀ resonances
of Tyr are detected. The small chemical shift differences between (a) and
(b) or (a) and (c) are caused by isotope shifts.
(a) HBCB(CG)HE (Hâ (t1) f Câ (t2) f Cγ f Hꢀ (t3))
(b) HBCB(CGCZ)HZ (Hâ (t1) f Câ (t2) f Cγ f
Cú f Hú (t3))
These pulse sequences were composed of previously sum-
marized building blocks.38 The most similar pulse sequence is
either (Hâ)Câ(CγCδ) Hδ or (Hâ)Câ(CγCδCꢀ)Hꢀ, previously
reported by Yamazaki et al.7 One of the major differences
between the previously published sequences and our sequences
is the incorporation of Hâ evolution. The overlap of the Hâ
resonances is alleviated, and the relaxation time of the evolving
Hâ is prolonged, due to the stereoselective deuteration in the
2
prior to transformation. During the ct evolution, H continuous wave
decoupling was applied with a field strength of 0.72 kHz. For the 3D
HBCB(CG)HE measurement, the acquisition parameters were set as
follows: data size, 16 (t1) × 16 (t2) × 512 (t3) complex points;
t
1max(1Hâ) ) 13.3 ms, t2max(13Câ) ) 8.8 ms and t3max(1Hꢀ) ) 64 ms; and
spectral widths in ω1 (1Hâ), ω2 (13Câ), and ω3 (1Hꢀ) of 1200, 1800, and
8000 Hz, respectively. The eight scans per increment and the 1 s
repetition delay gave a measuring time of 3.4 h. The data set was zero-
filled to 32 (t1) × 32 (t2) × 1024 (t3) complex points. For the 2D
(HB)CB(CG)HE measurement, the parameters for the 3D measurement
were used by reducing the dimension. The 32 scans per increment
resulted in a measuring time of 25 min. The 3D HBCB(CGCZ)HZ
spectrum was obtained under the same conditions as the 3D HBCB-
(CG)HE measurement, except for the differences of t2 complex points
) 8, t2max(13Câ) ) 8.8 ms, spectral width in 13C ) 9000 Hz, number of
(34) DeMacro, A.; Wuthrich, K. J. Magn. Reson. 1976, 24, 201-204.
(35) Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard, F.; Dyson, H. J.; Oldfield,
E.; Markley, J. L.; Sykes, B. D. J. Biomol. NMR 1995, 6, 135-140.
(36) Markley, J. L.; Bax, A.; Arata, Y.; Hilbers, C. W.; Kaptein, R.; Sykes, B.
D.; Wright, P. E.; Wuthrich, K. Eur. J. Biochem. 1998, 256, 1-15.
(37) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. J. Am. Chem. Soc. 1998,
120, 6394-6400.
(38) Edison, A. S.; Abildgaard, F.; Westler, W. M.; Mooberry, E. S.; Markley,
J. L. Methods Enzymol. 1994, 239, 3-79.
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A R T I C L E S
Torizawa et al.
Figure 3. Pulse sequences of the (a) HBCB(CG)HE and (b) HBCB(CGCZ)HZ measurements. In both pulse sequences, the radio frequency (rf) pulses on
1H between points a and b, 1H between b and acquisition, and 2H were applied at 3, 4.7, and 2.5 ppm, respectively. In (a), the carrier frequency of 13Câ was
set to 39 ppm, and that of 13Cγ was set to 137 ppm for Phe or to 129 ppm for Tyr. In (b), the rf pulses on 13Câ and 13Cγ/ú were irradiated at 38 and 134 ppm.
Narrow and wide black bars indicate nonselective 90 and 180°, respectively. Sine bell shapes on the lines marked with 13Cγ and 13Cγ/ú indicate selective 180°
pulses at the corresponding frequencies with a g3 shape.40 The pulses were applied along x if no phase is marked above the pulses. All 1H pulses were
1
applied with a 20 kHz field, while the H DIPSI-2 decoupling41 employed a 5 kHz field. All of the rectangular 13C 90 and 180° pulses were applied with
37 and 18.5 kHz fields, respectively. The first selective 13Câ 180° pulses were applied with a duration of 1.2 ms. Selective 180° pulses with a duration of
600 µs were used for the second and third pulses on 13Câ and for the first and second pulses on 13Cγ/13Cγ/ú. The third and fourth selective 180° pulses for
13Cγ/13Cγ/ú were applied with durations of 2.2 ms in (a) and 600 µs in (b). 13C decoupling during acquisition was achieved using 2 kHz WATLZ-16 decoupling.42
2
For H decoupling, continuous wave irradiation was accomplished with a field strength of 0.72 kHz during the blocks between points b and c (denoted as
CW in the sequences). Pulsed-field gradients were applied along the z-axis with a sine bell shape and with a duration of 1 ms. Amplitudes of the gradients
are as follows: g1 ) 5 G/cm, g2 ) 40 G/cm, g3 ) 15 G/cm, g4 ) 40 G/cm, g5 ) 10.1 G/cm. The first gradient (g1) was applied to ensure that the
magnetization giving rise to the signal originates on 1H, and not 13C.43 The second and third gradients (g2 and g3) were used for a heteronuclear zz-filter.44
The fourth and fifth gradients (g4 an g5) were applied for coherence pathway selection.33 The delays commonly used in (a) and (b) were τ1 ) 1.8 ms,
τ2 ) 3.7 ms, τ3 ) 5.2 ms, and T ) 5.2 ms. The delays in (a) were τ4 ) 7.2 ms and τ5 ) 12 ms, and in (b), τ4 ) 6 ms, τ5 ) 6 ms, τ6 ) 3.1 ms, τ7 ) 1.5
ms, and τ8 ) 1.2 ms. The phases were cycled as: φ1 ) {x, -x}; φ2 ) {x}; φ3 ) {4x, -4x}; φ4 ) {2x, -2x}; φ5 ) {-2y, 2y}; φrec ) {x, -2x, x, -x,
2x, -x}. Quadrature detections in the t1 and t2 dimensions were obtained by altering the phases φ1 and φ2 according to the States-TPPI manner,39 respectively.
â-methylene group. These are the reasons why our pulse
sequences include the Hâ evolution. In addition, the slower
relaxation of 13Câ, due to the deuteration, also enhances the
measurement sensitivity. In the meantime, 2H decoupling must
be applied during the 13C evolution time to avoid splitting by
the 13C-2H coupling. In the case of HBCB(CG)HE, the
magnetization is transferred from Cγ to detect Hꢀ directly via
3JC H (7.9 Hz for Phe, 7.1 Hz for Tyr), after the transfer to Cγ.
the HBCB(CG)HE correlations of Phe and Tyr could be detected
individually by setting the frequencies to the individual averaged
shifts, as shown in Figure 4b,c,f. Calmodulin has eight Phe (12,
16, 19, 65, 68, 89, 92, and 141) and two Tyr (99 and 138)
residues. In Figure 4, all of the correlations of the Phe and Tyr
residues could be detected. For the assignment of the Hꢀ/Cꢀ of
Phe, the 3D spectrum was measured (∼3.4 h). However, the
reduced dimensional 2D spectrum, like the spectrum shown in
Figure 4f for Tyr, would be sufficient even for the assignment
of Phe. Yamazaki et al. measured the 2D (Hâ)Câ(CγCδCꢀ)Hꢀ
spectrum of a 104 residue protein complexed with a 12 residue
peptide at 30 °C on a 500 MHz spectrometer, resulting in a
measurement time of 20 h.7 Compared to this previous method,
our labeling pattern and the 2D measurement (with the assistance
of a cryogenic probe) could reduce the time needed to detect
the correlations between â and ꢀ to 25 min.
γ
ꢀ
On the other hand, HBCB(CGCZ)HZ transfers the magnetiza-
3
tion from Cγ to Cú via JCγCú (9.3 Hz), before the last Cú-Hú
one-bond INEPT transfer. The site-specific 2H- and 13C-labeling
patterns made it possible to transfer the magnetizations through
the long-range couplings. One should optimize the delays for
magnetization transfer using such small J couplings by consid-
ering the relaxation times. Here, we determined the values so
that the intensities of the correlations could be maximized in
the calmodulin spectra. Figures 4 and 5 illustrate the spectra
for the Hꢀ/Cꢀ assignments of Phe and Tyr, and for the Hú/Cú
assignments of Phe, respectively. The averaged Cγ chemical
shifts of Phe and Tyr are separated well (136.78 and 128.97
ppm, respectively, obtained from BioMagResBank). Therefore,
Figure 5 also shows all of the correlations of HBCB(CGCZ)-
HZ of Phe. The 3D measurement had a sensitivity similar to
that of 3D HBCB(CG)HE (∼3.4 h). The Hú/Cú assignments of
Phe would also be performed easily, even if the 2D version is
measured, as inferred from the 2D projections shown in Figure
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Aromatic Ring Resonances of Phe and Tyr in Proteins
A R T I C L E S
Figure 4. Connection between Hꢀ/Cꢀ and Hâ/Câ of Phe and Tyr using the 3D or 2D HBCB(CG)HE spectrum of [13Cγ-13Cꢀ-1Hꢀ]-Phe and -Tyr selectively
1
labeled calmodulin. (a) H-13C HSQC spectrum for aromatic resonances (Phe-Hꢀ/Cꢀ region). (b) 2D Câ-Hꢀ projection of the 3D HBCB(CG)HE spectrum
for Phe. (c) 2D Hâ-Hꢀ projection of the 3D HBCB(CG)HE spectrum for Phe. (d) 1H-13C ct-HSQC spectrum for aliphatic resonances (Phe and Tyr-Hâ/Câ
region). (e) 1H-13C HSQC spectrum for aromatic resonances (Tyr-Hꢀ/Cꢀ region). (f) 2D (HB)CB(CG)HE spectrum for Tyr. Assignments are indicated
alongside the corresponding signals with the one-letter amino acid code and the residue number. Aromatic and aliphatic resonances are connected with
dotted lines through the HBCB(CG)HE spectra.
Figure 5. Connection between Hú/Cú and Hâ/Câ of Phe using the 3D HBCB(CGCZ)HZ spectrum of [13Cγ-13Cú-1Hú]-Phe, selectively labeled calmodulin.
(a) 1H-13C HSQC spectrum for aromatic resonances. (b) 2D Câ-Hú projection of the 3D HBCB(CGCZ)HZ spectrum. (c) 2D Hâ-Hú projection of the 3D
1
HBCB(CGCZ)HZ spectrum. (d) H-13C ct-HSQC spectrum for aliphatic resonances (Hâ/Câ region).
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A R T I C L E S
Torizawa et al.
Conclusions
5. In the measurements of both HBCB(CG)HE and HBCB-
(CGCZ)HZ, we utilized the relatively well separated Hâ, Câ,
and Hꢀ/Hú chemical shifts. However, if necessary, Cγ evolution
in HBCBCGHE can easily be introduced to the pulse scheme,
by modifying the building block between points c and d, to
contain the evolution time, and by altering φ3, the receiver
phases, and g4 to exploit the echo/anti-echo manner for
quadrature detection.31 In the same way, the building block
between points c and d, which includes an evolution period,
and the alteration of φ3 for the States-TPPI quadrature detection
manner39 are necessary for Cγ evolution in HBCBCGCZHZ,
and the block between points d and e for evolution and the
alteration of φ5 and g4 for the echo/anti-echo manner are
required for Cú evolution.
We have proposed novel stable isotope labeling patterns for
Phe and Tyr in proteins. The NMR samples of calmodulin, in
which the amino acids were introduced, were successfully
prepared by chemical synthesis of the amino acids and protein
production with the E. coli cell-free system. The labeling
patterns diminished the spectral overlap and allowed sensitive
detection. The straightforward pulse schemes developed for the
labeling patterns also afforded sensitive spectra. The unambigu-
ously assigned aromatic resonances are expected to be useful
for the structural determinations of proteins, as will be described
elsewhere.
Acknowledgment. We thank Prof. Mitsuhiko Ikura of Tor-
onto University for providing the calmodulin gene. This work
was supported by CREST/JST.
(39) Marion, D.; Ikura, M.; Tschudin, R.; Bax, A. J. Magn. Reson. 1989, 85,
393.
(40) Emsley, L.; Bodenhausen, G. Chem. Phys. Lett. 1987, 165, 469.
(41) Shaka, A. J.; Lee, C. J.; Pines, A. J. Magn. Reson. 1988, 77, 274-293.
(42) Shaka, A. J.; Keeler, J.; Frenkiel, T.; Freeman, R. J. Magn. Reson. 1993,
52, 335.
JA051386M
(44) Montelione, G. T.; Lyons, B. A.; Emerson, S. D.; Tashiro, M. J. Am. Chem.
Soc. 1992, 114, 10974-10975.
(43) Kay, L. E. J. Am. Chem. Soc. 1993, 115, 2055-2057.
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