H-MAS および 15 N 交差分極 -MAS-NMR による研究：反平行および平行βシート構造と2つの結晶学的に独立な分子の峻別

Yu Suzuki

^a

, Kazuo Yamauchi

^a

, Tetsuo Asakura

^a

, Masataka Tansho

^b

, Tadashi Shimizu

^b 鈴木優^a、山内一夫^a、朝倉哲夫^a、丹所正孝^b、清水禎^b

aDepartment of Biotechnology, Tokyo UniVersity of Agriculture and Technology, 東京農工大学 b National Institute for Material Science

、

物質・材料研究機構

β-Strand peptides are known to assemble into either antiparallel (AP) or parallel (P) β-sheet forms which

are very important motifs for protein folding and fibril formations occurring in silk fibroin or amyloid proteins. Well-resolved

¹

H NMR signals including NH protons were observed for alanine tripeptides (Ala)3 with the AP and P structures as well as (Ala)n (n ) 4-6) by high-field/fast magic-angle spinning NMR.

Amide NH and amino NH3 +

¹

H signals of (Ala)3 with the P structure were well resonated at 7.5 and 8.9 ppm, respectively, whereas they were not resolved for the AP structure. Notably, NH

¹

H signals of (Ala)3 and (Ala)4 taking the P structure are resonated at higher field than those of the AP structure by 1.0 and 1.1 ppm, respectively. Further, NH

¹⁵

N signals of (Ala)3 with the AP structure were resonated at lower field by 2 to 5 ppm than those of (Ala)3 with the P structure. These relative

¹

H and

¹⁵

N hydrogen bond shifts of the P structure with respect to those of the AP structure are consistent with the relative hydrogen bond lengths of the interstrand N-H…O=C bonds. Distinction between the two crystallographically independent chains present in the AP and P structures was feasible by

¹⁵

N chemical shifts but not by

¹

H chemical shifts because of insufficient spectral resolution in the latter. Calculated

¹

H and

¹⁵

N shielding constants by density functional theory are generally consistent with the experimental data, although some discrepancies remain depending upon the models used.

βストランドペプチドが結集して生じる反平行または平行βシート構造は、絹素やアミロイド蛋 白質における蛋白質のフォールディングやフィブリルの形成などにおける重要なモチーフとなっ ている。我々は、平行／反平行構造を持つアラニントリペプチドの¹Hと¹⁵Nの強磁場／高速マジ ック角回転NMRを測定した。その結果、反平行と平行構造内の結晶学的に非等価な2つの鎖の区

別は¹Hでは困難で、¹⁵Nの化学シフトにより初めて可能であることがわかった。また、¹Hと¹⁵Nの

遮蔽係数は、密度汎関数理論計算の結果とほぼ一致していることが判明した。

Results and Conclusion

Figure 1 shows eight and six peptide chains in the crystalline AP and P (Ala)3 used for the theoretical calculation of NMR shielding constants. Figure 2A illustrates the 1H NMR spectra of the AP (solid line) and the P (gray line) (Ala)3 at 60 °C. At least three peaks are resolved for the amide NH proton and the HR and Hβ protons from the lower to upper field. Note that the narrow peak resonates at 0.12 ppm arising from the peak from silicon rubber used for the chemical shift reference. A less intense 1H signal of the crystalline water molecules from the AP (Ala)3 may be superimposed upon a peak at 4

ppm. It is noteworthy that the lowermost peaks of

the P (Ala)3 (7.5-8.9 ppm) were split into the two

components and ascribed to NH and NH3 + protons,

respectively, as judged from their relative peak

intensities (4:6). These peaks in the AP tripeptide

were resonated at 8.5 and 9.3 ppm as shown in

Figure 2B. It is noted that some HR and Hβ signals

were split into several peaks after this procedure. To

clarify this further, the calculated 1H spectra, based

on the shielding constants obtained by DFT theory,

were represented by the stick diagram for NH3 +,

NH, HR, and Hβ protons of the individual

molecular chains (see Figure 1): they are arranged

so as to adjust the shielding constants with the highest Hβ signals (calculated 1H chemical shift of the higher most peak of P (Ala)3 (the shielding constant 30.7 ppm was set to 0.9 ppm)) (Figure 2C).

Here, it is anticipated that three separated theoretical peaks are available from the three NH3 + protons for A an B molecules, respectively:

N(1A)H…O(3B), N(1A)H…O(4B), and (1A)H…O(w2), and N(1B)H…O(3A), N(1B)H…O(4A), and N(1B)H…O(w1)3 (see Figure 1). Their electronic structures may significantly be different from each other,depending upon the manner of hydrogen-bonding nteractions.

Nevertheless, it appears that a time-averaged singlet peak is observable for the NH3 + 1H peak recorded at 60 °C, as a consequence of the presence of its C3 rotation. Otherwise, the observed NH3 + 1H signal should spread as large as 6 ppm on the basis of the calculated shielding constants (data not shown). For this reason, the calculated 1H signal for NH3+ was presented in Figure 2C after time-averaging of these three peaks for A and B molecules, respectively. Several calculated peak-splittings arising from the crystallographically independent molecules A and B cannot be distinguished as compared with the experimental spectra. Therefore, distinction of the A and B molecules is not feasible by 1H NMR spectra because the spectral resolution is not enough for this purpose.

Figure 1. Eight and six peptide chains in the crystalline AP and P(Ala)3 used for the theoretical calculation of NMR shielding constants. Two crystallographically independent molecules are indicated by A and B.

Figure 2. 1H MAS NMR spectra of AP (solid trace) and P structures (gray trace) of crystalline (Ala)3 (A).

Resolution-enhanced spectra are also shown (B). Stick diagram for 1H chemical shifts of N-H, HR, and Hâ protons obtained by the calculated shielding constants by DFT theory using molecular arrangements of the individual chains illustrated in Figure 1. Spectra for the A and B molecules are expressed by the solid and dotted peaks, respectively. Chemical shift scale for the calculated spectra is the same as that of the experimental ones (A and B). Stick diagram for NH3 + protons was shown after 1H chemical shifts from three different protons (see text) were averaged to be compared with the experimental data, taking into account the C3 rotation (C).

共同研究型支援 強

2004-73

A Solid-state

¹⁷

O NMR Study in Biological Compounds 生体分子の固体酸素 17 核 NMR 研究

Kazuhiko Yamada,

^a,

Toshio Yamazaki,

^a

Tadashi Shimizu

^b

and Shinobu Ohki

^b 山田和彦^a、山崎俊夫^a、清水禎^b、大木忍^b

a

Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN

b

National Institute for Materials Science

a理研ゲノム科学総合研究センター

b

物質・材料研究機構

Solid-state

¹⁷

O NMR is expected to be a useful tool for investigating biological systems such as metalloproteins and protein-protein interactions. This is because oxygen generally plays important roles in such biological activities and

¹⁷

O NMR tensors exhibit large ranges depending upon a local molecular environment. In this presentation, we will present a systematic experimental and theoretical investigation of oxygen-17 EFG and CS tensors in biological solids including amino acids, peptides, and proteins. In addition, we will report a solid-state

¹⁷

O NMR study of metal-organic compounds in which oxygen atoms are directly involved with metal ions. It is expected that they serve as model compounds for the future investigations of metalloproteins.

酸素は広範な分野で重要な原子である．例えば、生体分子の研究分野では、酸素が多くの生 理活性や水素結合を主とした構造維持に深く関与するため、酸素原子周辺の分子・電子情報は極 めて有益であるが、¹H や¹³C や¹⁵NNMR と比べると酸素 NMR の実験報告例は極めて少ない．酸素安 定同位体中で、原子量 17（以下¹⁷O）が唯一の NMR 測定可能核（

I

= 5/2）として存在するが、こ の天然存在比が 0.037%と極端に低いこと、また、その四極子モーメント（

Q

= -2.6 fm²）が比較 的大きいことから、観測が難しく、これまで研究者に敬遠されてきた．これまでの¹⁷ONMR の研究 において、無機化合物に対しては早い時期から実験が行われている一方で、有機化合物において は、通常、核四極相互作用の影響が大きく、多くの場合、線幅が広くなり測定自体が困難である．

また、しばしば有機化合物中に、標識として ¹⁷O を導入する必要があるために、主に有機化学者 を中心に溶液 ¹⁷O NMR 法を使った実験報告が、僅かではあるが、発表されてきた．しかしながら、

近年の固体 NMR 装置の進歩や測定技術と安定同位体標識技術の向上に伴って、有機化合物を対象 にした固体 ¹⁷O NMR の実験報告は着実に増えている．本章では固体 NMR 法を利用した有機化合物 における¹⁷O NMR 法について述べる

Introduction

Solid-state

¹⁷

O Nuclear Magnetic Resonance (NMR) is expected to be a useful tool for investigating biological systems such as metalloproteins and protein-protein interactions.

This is because oxygen generally plays important roles in such biological activities and

¹⁷

O NMR parameters such as electric-field-gradient (EFG) and chemical shielding (CS) tensors exhibit large ranges depending upon a functional group and a local molecular environment. For example, the ranges of

¹⁷

O CS tensors are distributed around approximately 1500 ppm from urea to aldehyde.

Recently, by using

¹⁷

O stationary, MAS, and MQMAS experiments at multiple magnetic fields combined with MO calculations, our group has presented experimental

¹⁷

O NMR tensors for a series of biological compounds including amino acids, peptides, and proteins.

Experiments

In this report, we will present a systematic experimental and theoretical investigation of oxygen-17 EFG and CS tensors in biological solids.

In particular, it is possible to discuss the trends and

the nature for the

¹⁷

O NMR tensors in comparison

of the obtained NMR parameters with the

already-known molecular structures. In addition, we will report a solid-state

¹⁷

O NMR study of metal-organic compounds in which oxygen atoms in amino acids are directly involved with metal ions.

They are very attractive small molecules since they may serve as model compounds for the future investigations of metalloproteins.

This work is part of a systematic investigation of amino acids, peptides, and proteins by solid-state

17

O NMR performed at RIKEN Genomic Sciences Center and NIMS.

Figure 1 shows experimental

¹⁷

O MAS spectra for (a) [

¹⁷

O]--glycine, (b) Li

₂

SO

₄[¹⁷

O]-glycine, and (c) NaNO

₃ [¹⁷

O]-glycine, observed at 11.7 T with sample spinning frequencies of 12-15 kHz.

The analysis of these MAS spectra can yield C

Q,

_Q, and _iso

for each oxygen atom in the carboxylate group of glycine molecules.

Compared to the

¹⁷

O NMR tensors for -glycine, previously reported by our group, it can be found that

¹⁷

O NMR parameters are highly sensitive to hydrogen bonding environments. For instance, there are differences of approximately 5-8 ppm in

iso

between -glycine and -glycine This clearly demonstrates that solid-state

¹⁷

O NMR is useful for the investigations of polymorphs.

In Li

₂

SO

₄glycine, the two carboxlate oxygen

atoms of a glycine molecule are directly linked to Li ions. At a glance, the

¹⁷

O MAS NMR line shape for the Li

₂

SO

₄[¹⁷

O]-glycine is different from that of [

¹⁷

O]--glycine, but the line widths are roughly similar to each other.

In fact, the value of 

_iso

is reduced to approximately 20 ppm, while the value of C

_Q

exhibits relatively similar value of -glycine.

Although it is well known that hydrogen bonds make it change the values of

¹⁷

O

iso

and C

_Q

, the present data suggest that the effects of ion-binds on the

¹⁷

O NMR tensors may be different from that of hydrogen bonds. According to the molecular structure of NaNO

₃glycine, the glycine molecules

are sandwiched between the NaNO

₃

layers, and the oxygen atoms of the glycine molecule are bonded to Na ions. Interestingly, the

¹⁷

O MAS spectrum of NaNO

3 [¹⁷

O]-glycine exhibits a shapeless one.

We carried out the

¹⁷

O NMR experiments from 210 K to room temperature, and it can be found that a dynamical process exists in this system since the line shapes are varied with each temperature. It is deduced that glycine molecules have a local motion between the NaNO

₃

layers.

Conclusion

In summary, we have presented the first experimental determination of the

¹⁷

O NMR tensors for

-glycine, which are different from those of

-glycine. The magnitudes of

¹⁷

O CS and EFG tensors

are sensitive to the hydrogen bond environments. It is expected that the present results for carboxylate functional groups in amino acids are also relevant to the situations in other functional groups, which will establish a foundation for future solid-state

¹⁷

O NMR studies for polymorph studies.

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

C

O O

H N

N H H N

H N

C

O O

Li Li

C

O O

Na Na

(a)

(b)

(c)

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

Chemical shift / ppm

-300 0

300 600

C

O O

H N

N H H N

H N

C

O O

Li Li

C

O O

Na Na C

O O

H N

N H H N

H N

C

O O

H N

N H H N

H N

C

O O

Li Li

C

O O

Li Li

C

O O

Na Na

C

O O

Na Na

(a)

(b)

(c)

Figure 1. Experimental ¹⁷O MAS spectra for (a) [¹⁷

O]-glycine, (b) Li2SO4[¹⁷O]-glycine, and (c) NaNO₃[¹⁷O]-glycine. Schematic representations of the corresponding intermolecular interactions around the carboxylate oxygen of glycine molecules are also given at the rights sides of the spectra.

共同研究型支援 強

2005-4

Analysis of

¹¹

B NMR Powder Lineshape of MgB

₂

in the Normal Phase

Toshiki Yamaji

^a

, Kiyonori Takegoshi

^a

, and Tadashi Shimizu

^b 山路俊樹^a 、竹腰清乃理^a、清水禎^b

a

Department of Chemistry, Graduate School of Natural Science and Technology, Kyoto University

b

National Institute for Materials Science,

a京都大学、^b物質・材料研究機構

11

B NMR measurements have been performed on MgB

₂

powder sample at several magnetic fields ranging from 6.3 to 21.9 T. The isotropic Knight-shift, as well as quadrupole coupling frequency, was accurately determined from magic-angle spinning (MAS) spectra by analyzing its magnetic-field dependence. These NMR parameters were then used to interpret its static powderpattern spectra observed at various magnetic fields, which reflect both the anisotropic Knight-shift tensor and the second-order quadrupole coupling tensor. We found that the Knight-shift tensor and the quadrupole coupling are both axially symmetric and their relative orientation to the molecular frame was also determined. This is the first experimental determination of the anisotropy of the Knight-shift of MgB

2

in a powder form from an exact analysis of the powder lineshape of the

¹¹

B NMR spectra. The anisotropy was found to be very small (~4 ppm), being in consistent with previous observations indicating that anisotropy of electronic conductivity in MgB

₂

is small.

超伝導MgB₂の電子状態を調べるために¹¹

BのNMR測定を行った。

¹¹

BのMAS-NMRスペクトルおよ

びStatic-NMRスペクトルの磁場依存性から、ナイトシフトと四極子核パラメーターなどの正確な 値を得ることが出来た。いずれのパラメーターも非常に小さい一軸性の異方性を持っているが、

異方性成分も含めて実験的にパラメーターを決めることが出来た。

Systematic investigation of the static (Fig.1) and MAS (Fig.2)

¹¹

B NMR spectra of powder MgB

₂

at variable magnetic fields up to the super-high magnetic field 21.9 T were performed, and the quadrupole and Knight-shift tensors were successfully determined. It is shown that the use of various magnetic fields is useful to extract reliable NMR parameters from a NMR lineshape affected by several interactions. This is also the first experimental determination of the anisotropy of the Knight-shift of MgB

₂

by the exact analysis of the lineshape of the

¹¹

B NMR

powder-pattern spectra The results of the

Knight-shift suggest that MgB

₂

has an isotropic

electronic conductivity, which is consistent with

the previous studies. This property indicates that

MgB

₂

should be versatilely suitable as a line

material such as a superconductive coil. This

report thus showed the very significant potential

of the combination of a super high-magnetic field

NMR approach and an analysis of a

magnetic-field dependence.

Fig. 1.: Experimental (left) and simulated (right) MAS-NMR spectra of ¹¹B. The spinning sidebands are also marked by _.

The vertical dotted line is drawn for eye-guidance. The zero shift corresponds to the value of the ¹¹B resonance frequency in H₃BO₃-saturated aqueous solution.

Fig.2: Experimental (left) and simulated (right) powder-pattern static spectra of the central +1/21/2 transition. The zero shift corresponds to the value of the ¹¹B resonance frequency in H3BO3-saturated aqueous solution.

共同研究型支援 強

2006-14

強磁場 NMR による YbAl

3

C

3

の研究 NMR Study of YbAl

₃

C

₃

in High Magnetic Field

冨澤 智^a、水戸 毅^a、和田 信二^a、端 健二郎^b、清水 禎^b、後藤 敦^b、大木 忍^b、 加藤 慶顕^c、小阪 昌史^c

Satoru Tomisawa^a, Takeshi Mito^a, Shinji Wada^a, Kenjiro Hashi^b, Tadashi Shimizu^b, Atsushi Goto^b, Shinobu Ohki^b, Yoshiaki Kato^c and Masashi Kosaka^c

a神戸大学、^b物質・材料研究機構、^c埼玉大学

a

Kobe Univ.,

^b

NIMS,

^c

Saitama Univ.

希土類化合物

YbAl

₃

C

₃は六方晶の層状結晶構造を持ち、帯磁率、比熱、超音波、中性子回折など の測定から８０Ｋ付近で相転移を起こし、その起源として高温四極子秩序の可能性が指摘させて きた。相転移温度が高いため磁気効果を観測するためには強磁場が必要となる。そこで本研究で は微視的な観点からこの転移の本質を明らかにするために物質・材料研究機構が所有するハイブ リッド磁石を用いた強磁場下でのアルミニウム核をプローブとした

NMR

測定を行なった。

Rare-earth compound YbAl

₃

C

₃

with a hexagonal crystal structure exhibits a phase transition around 80 K.

The properties of the phase transition have been investigated by magnetic susceptibility, specific heat, elastic constant and neutron diffraction measurements. In order to clarify the origin of the phase transition from microscopic point of view, high field NMR measurements with a hybrid magnet at NIMS were performed.

Introduction:

The elusive phase transition of the ytterbium (Yb)-based compound YbAl

₃

C

₃

has attracted much attention. The transition temperature T*~80K is much higher than ordinary magnetic ordering temperatures in Yb-based strongly correlated electron systems. Although the susceptibility of YbAl

₃

C

₃

at high temperatures is well described by the Curie Weiss law with a large absolute value of the Weiss temperature and the effective moment close to the magnetic Yb

³⁺

ion value, the magnetic properties at low temperatures are rather nonmagnetic. In the early stage of the investigation, it was supposed that the transition of this compound is associated with

‘‘high-temperature quadrupolar ordering’’.1) Moreover, neither magnetic nor structural anomalies had been detected by the X-ray or neutron diffraction measurement of powder samples.

In this paper, we report the results of

²⁷

Al nuclear magnetic resonance (NMR) experiments on YbAl

₃

C

₃

. The purpose of this study is to clarify whether or not the transition mechanism involves the properties of Yb-4f electrons. If the transition is associated with the quadrupole degrees of freedom, NMR will be useful in detecting it: The secondary magnetic moments possibly induced by magnetic field give rise to the splitting or broadening of resonance lines as indicated by earlier works.

In order to investigate the field dependence of the transition, the present measurements were required to use a substantially high magnetic field, because the transition temperature of YbAl

₃

C

₃

is fairly high. We therefore used a hybrid magnet installed at the

National Institute for Materials Science for the measurement at the highest field, which allows us to perform measurements up to 30 T.2)

Results and discussion：

We have carried out the spectrum measurements up to a field of 30 T. Figure 1 shows the temperature dependence of full width at a half of the maximum (FWHM) at fields of 1.0, 14.3, and 30.1 T. At each field, the rapid increase in FWHM is observed. We plot IT vs T in the insets to determine T*(H), which demonstrates the anomaly at the transition more explicitly. Here, IT is the maximum intensity of the central line multiplied by temperature. The decrease in IT implies the broadening of the resonance line, since the whole integrated signal intensity is generally proportional to T. The estimated T*(H) is indicated by the arrows in Fig. 1.

Obviously, T*(H=30T) is increased by ~10K

compared to T*(H=1T). This finding provides

microscopic evidence that confirms the previous

report on the field dependence of T*. The H−T

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