- Email: [email protected]
The structure and mobility of l -alanine residues of tropomyosin in the solid state as studied by high resolution solid state 13C NMR spectroscopy
Journal of Molecular Structure, 221 (1990) 289-297 Elsevier Science Publishers B.V., Amsterdam - Printed
289
in The Netherlands
THE STRUCTURE AND MOBILITY OF L-ALANINE RESIDUES OF TROPOMYOSIN IN THE SOLID STATE AS STUDIED BY HIGH RESOLUTION SOLID STATE 13CNMR SPECTROSCOPY
SATORU TUZI, SATOSHI
SAKAMAKI
and ISA0 AND0
Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo (Japan) (Received
10 July 1989)
ABSTRACT High resolution 13C NMR spectra of tropomyosin, one of the muscle proteins, in the solid state were recorded by the cross polarization-magic angle spinning method and the inversion-recovery method, in order to investigate the higher order structure of the protein through the observation of the 13C chemical shifts of the amino acid residues and their mobility. From these results, the two peaks at 15.8 and 16.7 ppm were assigned to the L-Ala C, carbons in the external and internal sites of the coiled-coil structure, respectively, and the molecular motion of the latter carbons is found to be more restricted than that of the former carbons.
INTRODUCTION
It has been demonstrated by recent 13CNMR studies of polypeptides in the solid state that 13CNMR chemical shifts of the C,, C, and carbonyl carbons as determined by the W cross polarization-magic angle spinning (CP-MAS) method are remarkably displaced, depending on their characteristic conformations as defined by the torsional angles (# and v) of the skeletal bonds such as the a-helix, P-sheet, 3,-helix, o-helix form, etc. [l-15]. Furthermore, it seems that the 13Cchemical shift values for the amino acid residues in proteins are influenced not only by the secondary structures, but also by the higher order structure composed of the secondary structures. The assignment of these chemical shift displacements caused by the higher order structure is necessary for interpreting the relationship between the protein structure and its function. In previous work, we observed the 13C chemical shifts of the L-alanine (LAla) residues of tropomyosin in the solid state by means of the 13CCP-MAS method, in order to elucidate the chemical shift values corresponding to one of the simplest higher order structures, coiled-coil structure [ 161. The higher order structure of tropomyosin is a right-handed a-helix coiled-coil structure, and contains two different sites, which are characterized as the internal hy-
0022-2860/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
290 External
Internal
hydrophilic site
hydrophobic site
Fig. 1. A schematic picture of the cross-section of the coiled-coil structure in tropomyosin.
drophobic sites and the external hydrophilic sites (Fig. 1) [ 171. In aqueous solution, the internal site which is located between the two a-helices is isolated from the water molecules, and the external site which is located on the surface of the molecule is exposed to them. In the present work we have extended the previous study in an attempt to make more clear the assignment of the 13C chemical shifts of the L-Ala C, carbons at the two sites of the coiled-coil structure. Furthermore, we discuss the mobility of the L-Ala C, carbons in the coiledcoil structure in order to assist the assignment of the chemical shift data. EXPERIMENTAL
Materials Tropomyosin was prepared from chicken-breast muscle according to the procedure reported by Edward and Sykes [ 181. It is known that the breast muscle of adult chicken contains only cx-tropomyosin and no isofroms. Characterization of the prepared protein was made on the basis of the ‘H NMR spectra [ 191. NMR measurement 13CNMR spectra in the solid state were recorded on a JNM GSX-270 NMR spectrometer operating at 67.8 MHz with a CP-MAS accessory and a variabletemperature accessory. The CP-MAS method and the inversion-recovery method with MAS were used to record the spectra. The inversion-recovery method was carried out using the pulse sequence shown in Fig. 2 [ 201. The recovery time, r, was changed arbitrarily in the measurements. The CP contact time was usually 2 ms, but was altered in some experiments. The repetition time for the CP-MAS method was 2-5 s and for the inversion-recovery method was 10 s. Samples (ca. 150 mg) were contained in a cylindrical rotor made of zirconia, and spun at 4-5 kHz. The spectral width and data points were 27 kHz and 8 k, respectively. Spectra were usually accumulated 1000-3000 times in
291
Fig. 2. A pulse sequence of the inversion-recovery
method. r indicates the recovery time.
order to achieve a reasonable signal-to-noise ratio. The 13C chemical shifts were calibrated indirectly through external adamantane (29.5 ppm relative to tetramethylsilane [ (CH3)$i] ). The temperature of the sample was calibrated from the temperature of the NMR probe using the calibration curve. When the measurement was carried out at room temperature (25 “C), the temperature of the sample was about 44°C. When the 13C NMR peaks overlapped one another, a computer-fitting procedure was used to obtain the exact chemical shift values and the peak intensities.
RESULTS AND DISCUSSION
A typical 13C CP-MAS NMR spectrum for tropomyosin in the solid state is shown in Fig. 3. As shown in Fig. 3, the 13C signals for the carbonyl and the C, carbons of the constituent amino acid residues overlap one another. In all experiments the carbonyl carbons gave only one peak at 176 ppm. This suggests that the secondary structure of tropomyosin takes the right-handed a-helix form. However, the resolved 13C signals which arise from the side chain aliphatic carbons can be assigned using reference data of homopolypeptides reported previously (see Table 1) [ 1,2,16,21]. Among the many amino acid residues of which tropomyosin is comprised, L-Ala is one of the major components and the corresponding C, 13C signals which appear at 15-17 ppm in the aliphatic side chain region can be easily assigned to the L-Ala C, carbons. Only the L-Met C, and L-Ile C, carbons have 13C chemical shift values close to 1517 ppm (see Table 1). These two kinds of residue, however, contribute only extremely minor amounts to the amino acid composition of tropomyosin (LAla 13%, L-Ile 3%, and L-Met 2%) [22,23]. Therefore, the contribution of the L-Ile and L-Met residues to the spectrum is quite small, and we consider that the peak intensity at 15-17 ppm arises mainly from the C, carbon atoms of the L-Ala residue. Since the assignment of the other peaks in the aliphatic side
292
Side chain allphalic c-o
I
carbons 1
Ca
L-Ala CP
\ j,~:I:I:_:
200
150
0
50
100
6/PPM
Fig. 3. The typical i3C CP-MAS spectrum of tropomyosin at room temperature. TABLE 1 The observed i3C chemical shift values of tropomyosin and some homopolypeptides taking the right-handed a-helix form (in ppm from (CH3)$i) Sample Tropomyosin (L-Ala), (L-Ile), (L-Met), (L-Leu), CL-LYS),
Solid Solid Solid Solid Solid Solution
c=o
c,
c,
176 177.0 175.9 176.5 176.7 176.5
54-58 53.3 64.9 58.2 56.7 57.4
15.8/16.7(L-Ala) 15.7 35.8 31.6 40.5 29.9
CY
28.2/15.9 31.6 25.1 23.4
C6
11.9 23.5121.2 27.6
C,
Ref.
This work This work 1 16.3 15 2 40.4 20
chain region is difficult, we discuss here only the peaks of the L-Ala C!, carbon atoms. 13CNMR chemical shifts and mobility of the L-alunine C, carbon atoms in the coiled-coil structure The L-Ala residue has no long side chains whose conformational changes are expected to affect the 13C chemical shift values, and the residue is included both in the internal and the external sites of tropomyosin. Therefore, the LAla residue must be a good probe for estimating the 13Cchemical shift displacement corresponding to the higher order structure. We have reported previously that the L-Ala residues located in the internal sites (positions a and d in Fig. 1) and in the external sites (positions b, c, e, f and g in Fig. 1) give different chemical shift values for the C, carbon atoms. However, because of the limited resolution of the CP-MAS 13C spectra, the discussion of the relationship be-
293
tween the structure and the 13CNMR chemical shift was limited. In the present paper we give a more exact and deeper discussion of this aspect through the mobility of the L-Ala C, carbons. It is expected that the L-Ala C, carbons which occupy the different sites of the higher order structure may be in different mobile states. The expanded L-Ala C!, signal, measured using the inversion-recovery method, is shown in Fig. 4. When the recovery time is very short (10ms), an inverse peak appears at 15.8 ppm. The spectrum recorded at a recovery time of 500 ms has a peak at 16.7 ppm, and when the recovery time is long (3500 ms), two peaks appear at 15.9 and 16.8 ppm. This means that there are at least two kinds of L-Ala C, carbon atoms. The carbon atoms contributing to the peak at 15.8 ppm have a longer T1 value than those at 16.7 ppm. This shows that there are two kinds of site with different mobility in the coiled-coil structure. In order to compare the mobilities of the L-Ala C, carbon atoms in the two sites, we must know whether the correlation time (2,) for their molecular motions is in the extremely narrow region or not. According to the proton T1 data reported by Andrew et al. [ 24,251, the correlation times of the L-Ala CH3 protons are very short in polypeptides and proteins in the solid state, and are in the extremely narrow region at room temperature (z, = 10-l’ s and o = 67.8 MHz; i.e. w 7= << 1). Therefore, it is predicted that the L-Ala C, carbons in the L-AloCp
r,“~,.‘~‘I”‘,I”‘.I’~“I”“I”“I”~~I
21
20
19
16
17
16
15
1L
13 b/wM
Fig. 4. The expanded 13CNMR spectra of the L-Ala C, carbon atoms of tropomyosin in the solid state, measured using the inversion-recovery method. Recovery time, r: (a) 10 ms; (b) 500 ms; (c) 3500 ms.
294
a-helix coiled-coil structure may also have the correlation times in the extremely narrow region for 67.8 MHz i3C NMR measurement at room temperature. To ascertain whether or not this is true, inversion-recovery measurements were made at various temperatures (Fig. 5). As mentioned below, the a-helix form in the coiled-coil structure is found to be retained even at 77°C. Obviously, the recovery of the peaks at 15.8 and 16.7 ppm at 44’ C is faster than that at 77 oC. This means that the decrease in the correlation times for the L-Ala C, carbons in tropomyosin leads to an increase in their T1 values. Therefore, both the L-Ala C, carbons contributing to the peaks at 15.8 and 16.7 ppm have correlation times in the extremely narrow region, and the former carbons with a longer T1 value are more mobile than the latter carbons. The expanded CP-MAS spectra of L-Ala C, carbons as a function of CP contact time is shown in Fig. 6. It is expected that the mobility affects the CP efficiency. Therefore, the CP efficiency depends on the cross-relaxation time, Ten, and the relaxation time of the proton, T1 ,,. When the molecular motion is in the extremely narrow region, Ten and T, p decrease as the mobility increases. Thus, the optimum contact time for obtaining the maximum magnetization decreases as the mobility increases. The optimum contact times for the peaks at 15.8 and 16.7 ppm are found to be about 2 and 1 ms, respectively. Under the above condition, this shows that the mobility of the carbon atoms contributing to the peak at 15.8 ppm is greater than that at 16.7 ppm. This is consistent with the results obtained from the inversion-recovery measurements. As mentioned above, there are two sites in the coiled-coil structure whose environments are quite different from one another. It seems to be natural to assign the two peaks (15.8 and 16.7 ppm) for the L-Ala CB carbons to these L-Ala 550
Cp
ms
\
Tl7 1Oms
77 “C
I
I
I
I
I
,
I
19
16
17
16
15
14
13
6/PPM
1
I
I
I
I
1
1
19
16
17
16
15
14
13
%PM
Fig. 5. The expanded 13CNMR spectra of the L-Ala C, carbons of tropomyosin in the solid state at 44 and 77”C, measured using the inversion-recovery method. Top spectra, r=550 ms; bottom spectra, 7= 10 ms.
295 L-Ala Cg 9
a
,,..,,....,....,....,....,...‘,.”‘I‘.,~
2,
20
19
18
17
16
15
14
13
--?
\
\
\_ I‘\,
%PM
Fig. 6. The expanded 13CCP-MAS NMR spectra of the L-Ala C, carbons of tropomyosin in the solid state, measured as a function of contact time. Contact time: (a) 0.5 ms; (b) 1 ms; (c) 2 ms; (d) 4 ms. Fig. 7. The expanded 13CCP-MAS NMR spectra of the L-Ala C, carbons of tropomyosin in the solidstate, measuredasafunctionoftemperature. (a) 44°C; (b) 57°C; (c) 77°C; (d) 116°C; (e) 135°C; (f) 155°C; (g) 44°C measured after the high temperature experiments (a-f).
two sites. The positions of the L-Ala residues in the primary structure do not affect the chemical shift values; it has been reported that the influence on the 13Cchemical shift values of the amino acid sequence is negligible in the solid state [5]. X-Ray studies on paramyosin which has almost the same higher order structure as tropomyosin show that the distance between the two a-helical axes in the coiled-coil structure is about lo-11 A and the closest distance between the a-helical axes in the other coiled-coil structure is at least about 12-13 A where the coiled-coil helices are packed in parallel in native muscle [ 261. Therefore, the mobility of the L-Ala C, carbons in the internal site is expected to be more restricted than that in the external site. From such a situation we can assign the peak at 15.8 ppm to the more mobile L-Ala C, carbons in the external site and the peak at 16.7 ppm to the less mobile L-Ala C, carbons in the internal site. Higher order structure of tropomyosin at high temperature In order to obtain information on a change in the higher order structure at high temperature, the 13C CP-MAS NMR spectra for tropomyosin was mea-
296
sured as a function of temperature. The chemical shift values of the carbonyl carbons (about 176 ppm) were found to be independent of temperature in going from 44°C to 155°C. This indicates that the a-helix structures in tropomyosin are maintained within this temperature range. The expanded 13C CP-MAS spectra of the L-Ala C, carbons at various temperatures are shown in Fig. 7. Since the two peaks corresponding to the two sites in the coiled-coil structure overlap one another, the peak fitting procedure was used to determine the chemical shift value and intensity of each peak (Table 2). There are two peaks at all temperatures, and their determined chemical shift values, which are close to 15.8 and 16.7 ppm, are almost independent of temperature. However, the relative intensity of the two peaks changes as the temperature is increased. The intensity of the downfield peak increases as the temperature is increased, but that of the upfield peak decreases. This seems to be caused by a change in the mobility of the L-Ala C, carbons in the two sites and a change in the optimum CP efficiency associated with a change of mobility. As mentioned above, the optimum contact time for the L-Ala C, carbons in the internal and the external sites at 44°C are about 1 and 2 ms, respectively. On the other hand, the optimum contact times increase as the temperature is increased. Therefore, this leads to the experimental fact that the optimum contact time of the carbons in the internal site approaches 2 ms, and that of the carbons in the external site becomes more than 2 ms. This description is consistent with the behaviour of the peak intensity corresponding to the two sites at high temperatures. Fig. 7 (g ) shows the 13CCP-MAS spectrum for tropomyosin at 44 oC measured again after the high temperature experiments. Since the line shape in this spectrum resembles that recorded at 155’ C, it seems that the mobility of the carbons at 44°C is similar to that at 155 “C due to any change in the coiled-coil structure by heating. However, it is necessary to obtain further NMR data in order to elucidate details of changes in the higher order structure. TABLE
2
The observed Y! chemical shift values and peak intensities for the L-Ala C, carbons in tropomyosin at various temperatures (ppm from (CHs)$i)
Temperature (“C) 44 57 77 116 135 155
13Cchemical shift Downfield peak
Upfield peak
16.9 (35)” 17.0 (36)
15.6 (65) 15.8 (64)
16.9 17.1 17.0 16.8
15.7 15.9 15.8 15.7
(43) (44) (45) (60)
(57) (56) (55) (40)
“The values in parenthese are relative peak heights (in per cent).
297
CONCLUSIONS
The characteristic 13C chemical shift values and mobility of the L-Ala CR carbons corresponding to the coiled-coil structure were obtained. These dad give information about the spatial positions of the residues in the higher order structure, and can be related to the higher order structures in other proteins which are similar to the coiled-coil structure of tropomyosin. The collection of such fundamental 13CNMR data corresponding to the higher order structures is essential in interpreting the structure of globular proteins.
REFERENCES 1 2 3 4
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
T. Taki, S. Yamashita, M. Satoh, A. Shibata, T. Yamashita, R. Tabeta and H. SaitB, Chem. Lett., (1981) 1803. H. SaitB, R. Tabeta, A. Shoji, T. Ozaki and I. Ando, Macromolecules, 16 (1983) 1050. H. Saito, R. Tabeta, I. Ando, T. Ozaki and A. Shoji, Chem. Lett., (1983) 1437. H. Saito, R. Tabeta, T. Asakura, Y. Iwanaga, A. Shoji, T. Ozaki and I. Ando, Macromolecules, 17 (1984) 1405. A. Shoji, T. Ozaki, H. Saito, T. Tabeta and I. Ando, Macromolecules, 17 (1984) 1472. H. Saito, R. Tabeta, A. Shoji, T. Ozaki, I. Ando and T. Miyata, Biopolymers, 23 (1984) 2279. H. Sait8 and I. Ando, Ann. Rep. NMR Spectrosc., 21 (1989) 209. H.R. Kricheldorf, D. Mutter, F. Mazer, D. Mtiller and D. Foster, Biopolymers, 22 (1983) 1357. H.R. Kricheldorf and D. Miiller, Macromolecules, 16 (1983) 615. H.R. Kricheldorf, D. Miiller and K. Ziegler, Polym. Bull., 9 (1983) 284. 1. Ando, H. SaitB, R. Tabeta, A. Shoji and T. Ozaki, Macromolecules, 17 (1984) 457. T. Yamanobe, I. Ando, H. Saitci, R. Tabeta, A. Shoji and T. Ozaki, Bull. Chem. Sot. Jpn., 58 (1985) 23. T. Yamanobe, I. Ando, H. Saite, R. Tabeta, A. Shoji and T. Ozaki, Chem. Phys., 99 (1985) 259. S. Ando, T. Yamanobe, I. Ando, A. Shoji, T. Ozaki, R. Tabeta and H. SaitB, J. Am. Chem. Sot., 107 (1985) 7648. S. Tuzi, T. Komoto, I. Ando, H. Sait& A. Shoji and T. Ozaki, Biopolymers, 26 (1987) 1983. S. Tuzi and I. Ando, J. Mol. Struct., 196 (1989) 317. P.L. Privalov, Adv. Protein Chem., 35 (1982) 1. F.P. Edward and B.D. Sykes, Biochemistry, 19 (1980) 2577. M. Stewart and G.C.K. Roberts, J. Mol. Biol., 166 (1983) 219. T. Fujito, K. Deguchi, M. Ohuchi, M. Imanari and M.J. Albright, 20th Meeting of NMR, Tokyo, 1981, p. 68. H. Saiti, T. Ohki, M. Kodama and C. Nagata, Biopolymers, 17 (1978) 2587. P. Cummins and S.V. Perry, Biochem. J., 141 (1974) 43. A.R. MacLeod, Eur. J. Biochem., 126 (1982) 293. E.R. Andrew, R. Gaspar, Jr., and W. Vennert, Biopolymers, 17 (1978) 1913. E.R. Andrew, D.N. Bone, D.J. Bryant, E.M. Cashell, R. Gaspar, Jr., and Q.A. Meng, Pure Appl. Chem., 54 (1982) 585. C. Cohn and KC. Holmes, J. Mol. Biol., 6 (1963) 423.
289
in The Netherlands
THE STRUCTURE AND MOBILITY OF L-ALANINE RESIDUES OF TROPOMYOSIN IN THE SOLID STATE AS STUDIED BY HIGH RESOLUTION SOLID STATE 13CNMR SPECTROSCOPY
SATORU TUZI, SATOSHI
SAKAMAKI
and ISA0 AND0
Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo (Japan) (Received
10 July 1989)
ABSTRACT High resolution 13C NMR spectra of tropomyosin, one of the muscle proteins, in the solid state were recorded by the cross polarization-magic angle spinning method and the inversion-recovery method, in order to investigate the higher order structure of the protein through the observation of the 13C chemical shifts of the amino acid residues and their mobility. From these results, the two peaks at 15.8 and 16.7 ppm were assigned to the L-Ala C, carbons in the external and internal sites of the coiled-coil structure, respectively, and the molecular motion of the latter carbons is found to be more restricted than that of the former carbons.
INTRODUCTION
It has been demonstrated by recent 13CNMR studies of polypeptides in the solid state that 13CNMR chemical shifts of the C,, C, and carbonyl carbons as determined by the W cross polarization-magic angle spinning (CP-MAS) method are remarkably displaced, depending on their characteristic conformations as defined by the torsional angles (# and v) of the skeletal bonds such as the a-helix, P-sheet, 3,-helix, o-helix form, etc. [l-15]. Furthermore, it seems that the 13Cchemical shift values for the amino acid residues in proteins are influenced not only by the secondary structures, but also by the higher order structure composed of the secondary structures. The assignment of these chemical shift displacements caused by the higher order structure is necessary for interpreting the relationship between the protein structure and its function. In previous work, we observed the 13C chemical shifts of the L-alanine (LAla) residues of tropomyosin in the solid state by means of the 13CCP-MAS method, in order to elucidate the chemical shift values corresponding to one of the simplest higher order structures, coiled-coil structure [ 161. The higher order structure of tropomyosin is a right-handed a-helix coiled-coil structure, and contains two different sites, which are characterized as the internal hy-
0022-2860/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
290 External
Internal
hydrophilic site
hydrophobic site
Fig. 1. A schematic picture of the cross-section of the coiled-coil structure in tropomyosin.
drophobic sites and the external hydrophilic sites (Fig. 1) [ 171. In aqueous solution, the internal site which is located between the two a-helices is isolated from the water molecules, and the external site which is located on the surface of the molecule is exposed to them. In the present work we have extended the previous study in an attempt to make more clear the assignment of the 13C chemical shifts of the L-Ala C, carbons at the two sites of the coiled-coil structure. Furthermore, we discuss the mobility of the L-Ala C, carbons in the coiledcoil structure in order to assist the assignment of the chemical shift data. EXPERIMENTAL
Materials Tropomyosin was prepared from chicken-breast muscle according to the procedure reported by Edward and Sykes [ 181. It is known that the breast muscle of adult chicken contains only cx-tropomyosin and no isofroms. Characterization of the prepared protein was made on the basis of the ‘H NMR spectra [ 191. NMR measurement 13CNMR spectra in the solid state were recorded on a JNM GSX-270 NMR spectrometer operating at 67.8 MHz with a CP-MAS accessory and a variabletemperature accessory. The CP-MAS method and the inversion-recovery method with MAS were used to record the spectra. The inversion-recovery method was carried out using the pulse sequence shown in Fig. 2 [ 201. The recovery time, r, was changed arbitrarily in the measurements. The CP contact time was usually 2 ms, but was altered in some experiments. The repetition time for the CP-MAS method was 2-5 s and for the inversion-recovery method was 10 s. Samples (ca. 150 mg) were contained in a cylindrical rotor made of zirconia, and spun at 4-5 kHz. The spectral width and data points were 27 kHz and 8 k, respectively. Spectra were usually accumulated 1000-3000 times in
291
Fig. 2. A pulse sequence of the inversion-recovery
method. r indicates the recovery time.
order to achieve a reasonable signal-to-noise ratio. The 13C chemical shifts were calibrated indirectly through external adamantane (29.5 ppm relative to tetramethylsilane [ (CH3)$i] ). The temperature of the sample was calibrated from the temperature of the NMR probe using the calibration curve. When the measurement was carried out at room temperature (25 “C), the temperature of the sample was about 44°C. When the 13C NMR peaks overlapped one another, a computer-fitting procedure was used to obtain the exact chemical shift values and the peak intensities.
RESULTS AND DISCUSSION
A typical 13C CP-MAS NMR spectrum for tropomyosin in the solid state is shown in Fig. 3. As shown in Fig. 3, the 13C signals for the carbonyl and the C, carbons of the constituent amino acid residues overlap one another. In all experiments the carbonyl carbons gave only one peak at 176 ppm. This suggests that the secondary structure of tropomyosin takes the right-handed a-helix form. However, the resolved 13C signals which arise from the side chain aliphatic carbons can be assigned using reference data of homopolypeptides reported previously (see Table 1) [ 1,2,16,21]. Among the many amino acid residues of which tropomyosin is comprised, L-Ala is one of the major components and the corresponding C, 13C signals which appear at 15-17 ppm in the aliphatic side chain region can be easily assigned to the L-Ala C, carbons. Only the L-Met C, and L-Ile C, carbons have 13C chemical shift values close to 1517 ppm (see Table 1). These two kinds of residue, however, contribute only extremely minor amounts to the amino acid composition of tropomyosin (LAla 13%, L-Ile 3%, and L-Met 2%) [22,23]. Therefore, the contribution of the L-Ile and L-Met residues to the spectrum is quite small, and we consider that the peak intensity at 15-17 ppm arises mainly from the C, carbon atoms of the L-Ala residue. Since the assignment of the other peaks in the aliphatic side
292
Side chain allphalic c-o
I
carbons 1
Ca
L-Ala CP
\ j,~:I:I:_:
200
150
0
50
100
6/PPM
Fig. 3. The typical i3C CP-MAS spectrum of tropomyosin at room temperature. TABLE 1 The observed i3C chemical shift values of tropomyosin and some homopolypeptides taking the right-handed a-helix form (in ppm from (CH3)$i) Sample Tropomyosin (L-Ala), (L-Ile), (L-Met), (L-Leu), CL-LYS),
Solid Solid Solid Solid Solid Solution
c=o
c,
c,
176 177.0 175.9 176.5 176.7 176.5
54-58 53.3 64.9 58.2 56.7 57.4
15.8/16.7(L-Ala) 15.7 35.8 31.6 40.5 29.9
CY
28.2/15.9 31.6 25.1 23.4
C6
11.9 23.5121.2 27.6
C,
Ref.
This work This work 1 16.3 15 2 40.4 20
chain region is difficult, we discuss here only the peaks of the L-Ala C!, carbon atoms. 13CNMR chemical shifts and mobility of the L-alunine C, carbon atoms in the coiled-coil structure The L-Ala residue has no long side chains whose conformational changes are expected to affect the 13C chemical shift values, and the residue is included both in the internal and the external sites of tropomyosin. Therefore, the LAla residue must be a good probe for estimating the 13Cchemical shift displacement corresponding to the higher order structure. We have reported previously that the L-Ala residues located in the internal sites (positions a and d in Fig. 1) and in the external sites (positions b, c, e, f and g in Fig. 1) give different chemical shift values for the C, carbon atoms. However, because of the limited resolution of the CP-MAS 13C spectra, the discussion of the relationship be-
293
tween the structure and the 13CNMR chemical shift was limited. In the present paper we give a more exact and deeper discussion of this aspect through the mobility of the L-Ala C, carbons. It is expected that the L-Ala C, carbons which occupy the different sites of the higher order structure may be in different mobile states. The expanded L-Ala C!, signal, measured using the inversion-recovery method, is shown in Fig. 4. When the recovery time is very short (10ms), an inverse peak appears at 15.8 ppm. The spectrum recorded at a recovery time of 500 ms has a peak at 16.7 ppm, and when the recovery time is long (3500 ms), two peaks appear at 15.9 and 16.8 ppm. This means that there are at least two kinds of L-Ala C, carbon atoms. The carbon atoms contributing to the peak at 15.8 ppm have a longer T1 value than those at 16.7 ppm. This shows that there are two kinds of site with different mobility in the coiled-coil structure. In order to compare the mobilities of the L-Ala C, carbon atoms in the two sites, we must know whether the correlation time (2,) for their molecular motions is in the extremely narrow region or not. According to the proton T1 data reported by Andrew et al. [ 24,251, the correlation times of the L-Ala CH3 protons are very short in polypeptides and proteins in the solid state, and are in the extremely narrow region at room temperature (z, = 10-l’ s and o = 67.8 MHz; i.e. w 7= << 1). Therefore, it is predicted that the L-Ala C, carbons in the L-AloCp
r,“~,.‘~‘I”‘,I”‘.I’~“I”“I”“I”~~I
21
20
19
16
17
16
15
1L
13 b/wM
Fig. 4. The expanded 13CNMR spectra of the L-Ala C, carbon atoms of tropomyosin in the solid state, measured using the inversion-recovery method. Recovery time, r: (a) 10 ms; (b) 500 ms; (c) 3500 ms.
294
a-helix coiled-coil structure may also have the correlation times in the extremely narrow region for 67.8 MHz i3C NMR measurement at room temperature. To ascertain whether or not this is true, inversion-recovery measurements were made at various temperatures (Fig. 5). As mentioned below, the a-helix form in the coiled-coil structure is found to be retained even at 77°C. Obviously, the recovery of the peaks at 15.8 and 16.7 ppm at 44’ C is faster than that at 77 oC. This means that the decrease in the correlation times for the L-Ala C, carbons in tropomyosin leads to an increase in their T1 values. Therefore, both the L-Ala C, carbons contributing to the peaks at 15.8 and 16.7 ppm have correlation times in the extremely narrow region, and the former carbons with a longer T1 value are more mobile than the latter carbons. The expanded CP-MAS spectra of L-Ala C, carbons as a function of CP contact time is shown in Fig. 6. It is expected that the mobility affects the CP efficiency. Therefore, the CP efficiency depends on the cross-relaxation time, Ten, and the relaxation time of the proton, T1 ,,. When the molecular motion is in the extremely narrow region, Ten and T, p decrease as the mobility increases. Thus, the optimum contact time for obtaining the maximum magnetization decreases as the mobility increases. The optimum contact times for the peaks at 15.8 and 16.7 ppm are found to be about 2 and 1 ms, respectively. Under the above condition, this shows that the mobility of the carbon atoms contributing to the peak at 15.8 ppm is greater than that at 16.7 ppm. This is consistent with the results obtained from the inversion-recovery measurements. As mentioned above, there are two sites in the coiled-coil structure whose environments are quite different from one another. It seems to be natural to assign the two peaks (15.8 and 16.7 ppm) for the L-Ala CB carbons to these L-Ala 550
Cp
ms
\
Tl7 1Oms
77 “C
I
I
I
I
I
,
I
19
16
17
16
15
14
13
6/PPM
1
I
I
I
I
1
1
19
16
17
16
15
14
13
%PM
Fig. 5. The expanded 13CNMR spectra of the L-Ala C, carbons of tropomyosin in the solid state at 44 and 77”C, measured using the inversion-recovery method. Top spectra, r=550 ms; bottom spectra, 7= 10 ms.
295 L-Ala Cg 9
a
,,..,,....,....,....,....,...‘,.”‘I‘.,~
2,
20
19
18
17
16
15
14
13
--?
\
\
\_ I‘\,
%PM
Fig. 6. The expanded 13CCP-MAS NMR spectra of the L-Ala C, carbons of tropomyosin in the solid state, measured as a function of contact time. Contact time: (a) 0.5 ms; (b) 1 ms; (c) 2 ms; (d) 4 ms. Fig. 7. The expanded 13CCP-MAS NMR spectra of the L-Ala C, carbons of tropomyosin in the solidstate, measuredasafunctionoftemperature. (a) 44°C; (b) 57°C; (c) 77°C; (d) 116°C; (e) 135°C; (f) 155°C; (g) 44°C measured after the high temperature experiments (a-f).
two sites. The positions of the L-Ala residues in the primary structure do not affect the chemical shift values; it has been reported that the influence on the 13Cchemical shift values of the amino acid sequence is negligible in the solid state [5]. X-Ray studies on paramyosin which has almost the same higher order structure as tropomyosin show that the distance between the two a-helical axes in the coiled-coil structure is about lo-11 A and the closest distance between the a-helical axes in the other coiled-coil structure is at least about 12-13 A where the coiled-coil helices are packed in parallel in native muscle [ 261. Therefore, the mobility of the L-Ala C, carbons in the internal site is expected to be more restricted than that in the external site. From such a situation we can assign the peak at 15.8 ppm to the more mobile L-Ala C, carbons in the external site and the peak at 16.7 ppm to the less mobile L-Ala C, carbons in the internal site. Higher order structure of tropomyosin at high temperature In order to obtain information on a change in the higher order structure at high temperature, the 13C CP-MAS NMR spectra for tropomyosin was mea-
296
sured as a function of temperature. The chemical shift values of the carbonyl carbons (about 176 ppm) were found to be independent of temperature in going from 44°C to 155°C. This indicates that the a-helix structures in tropomyosin are maintained within this temperature range. The expanded 13C CP-MAS spectra of the L-Ala C, carbons at various temperatures are shown in Fig. 7. Since the two peaks corresponding to the two sites in the coiled-coil structure overlap one another, the peak fitting procedure was used to determine the chemical shift value and intensity of each peak (Table 2). There are two peaks at all temperatures, and their determined chemical shift values, which are close to 15.8 and 16.7 ppm, are almost independent of temperature. However, the relative intensity of the two peaks changes as the temperature is increased. The intensity of the downfield peak increases as the temperature is increased, but that of the upfield peak decreases. This seems to be caused by a change in the mobility of the L-Ala C, carbons in the two sites and a change in the optimum CP efficiency associated with a change of mobility. As mentioned above, the optimum contact time for the L-Ala C, carbons in the internal and the external sites at 44°C are about 1 and 2 ms, respectively. On the other hand, the optimum contact times increase as the temperature is increased. Therefore, this leads to the experimental fact that the optimum contact time of the carbons in the internal site approaches 2 ms, and that of the carbons in the external site becomes more than 2 ms. This description is consistent with the behaviour of the peak intensity corresponding to the two sites at high temperatures. Fig. 7 (g ) shows the 13CCP-MAS spectrum for tropomyosin at 44 oC measured again after the high temperature experiments. Since the line shape in this spectrum resembles that recorded at 155’ C, it seems that the mobility of the carbons at 44°C is similar to that at 155 “C due to any change in the coiled-coil structure by heating. However, it is necessary to obtain further NMR data in order to elucidate details of changes in the higher order structure. TABLE
2
The observed Y! chemical shift values and peak intensities for the L-Ala C, carbons in tropomyosin at various temperatures (ppm from (CHs)$i)
Temperature (“C) 44 57 77 116 135 155
13Cchemical shift Downfield peak
Upfield peak
16.9 (35)” 17.0 (36)
15.6 (65) 15.8 (64)
16.9 17.1 17.0 16.8
15.7 15.9 15.8 15.7
(43) (44) (45) (60)
(57) (56) (55) (40)
“The values in parenthese are relative peak heights (in per cent).
297
CONCLUSIONS
The characteristic 13C chemical shift values and mobility of the L-Ala CR carbons corresponding to the coiled-coil structure were obtained. These dad give information about the spatial positions of the residues in the higher order structure, and can be related to the higher order structures in other proteins which are similar to the coiled-coil structure of tropomyosin. The collection of such fundamental 13CNMR data corresponding to the higher order structures is essential in interpreting the structure of globular proteins.
REFERENCES 1 2 3 4
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
T. Taki, S. Yamashita, M. Satoh, A. Shibata, T. Yamashita, R. Tabeta and H. SaitB, Chem. Lett., (1981) 1803. H. SaitB, R. Tabeta, A. Shoji, T. Ozaki and I. Ando, Macromolecules, 16 (1983) 1050. H. Saito, R. Tabeta, I. Ando, T. Ozaki and A. Shoji, Chem. Lett., (1983) 1437. H. Saito, R. Tabeta, T. Asakura, Y. Iwanaga, A. Shoji, T. Ozaki and I. Ando, Macromolecules, 17 (1984) 1405. A. Shoji, T. Ozaki, H. Saito, T. Tabeta and I. Ando, Macromolecules, 17 (1984) 1472. H. Saito, R. Tabeta, A. Shoji, T. Ozaki, I. Ando and T. Miyata, Biopolymers, 23 (1984) 2279. H. Sait8 and I. Ando, Ann. Rep. NMR Spectrosc., 21 (1989) 209. H.R. Kricheldorf, D. Mutter, F. Mazer, D. Mtiller and D. Foster, Biopolymers, 22 (1983) 1357. H.R. Kricheldorf and D. Miiller, Macromolecules, 16 (1983) 615. H.R. Kricheldorf, D. Miiller and K. Ziegler, Polym. Bull., 9 (1983) 284. 1. Ando, H. SaitB, R. Tabeta, A. Shoji and T. Ozaki, Macromolecules, 17 (1984) 457. T. Yamanobe, I. Ando, H. Saitci, R. Tabeta, A. Shoji and T. Ozaki, Bull. Chem. Sot. Jpn., 58 (1985) 23. T. Yamanobe, I. Ando, H. Saite, R. Tabeta, A. Shoji and T. Ozaki, Chem. Phys., 99 (1985) 259. S. Ando, T. Yamanobe, I. Ando, A. Shoji, T. Ozaki, R. Tabeta and H. SaitB, J. Am. Chem. Sot., 107 (1985) 7648. S. Tuzi, T. Komoto, I. Ando, H. Sait& A. Shoji and T. Ozaki, Biopolymers, 26 (1987) 1983. S. Tuzi and I. Ando, J. Mol. Struct., 196 (1989) 317. P.L. Privalov, Adv. Protein Chem., 35 (1982) 1. F.P. Edward and B.D. Sykes, Biochemistry, 19 (1980) 2577. M. Stewart and G.C.K. Roberts, J. Mol. Biol., 166 (1983) 219. T. Fujito, K. Deguchi, M. Ohuchi, M. Imanari and M.J. Albright, 20th Meeting of NMR, Tokyo, 1981, p. 68. H. Saiti, T. Ohki, M. Kodama and C. Nagata, Biopolymers, 17 (1978) 2587. P. Cummins and S.V. Perry, Biochem. J., 141 (1974) 43. A.R. MacLeod, Eur. J. Biochem., 126 (1982) 293. E.R. Andrew, R. Gaspar, Jr., and W. Vennert, Biopolymers, 17 (1978) 1913. E.R. Andrew, D.N. Bone, D.J. Bryant, E.M. Cashell, R. Gaspar, Jr., and Q.A. Meng, Pure Appl. Chem., 54 (1982) 585. C. Cohn and KC. Holmes, J. Mol. Biol., 6 (1963) 423.