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Circular dichroism and 13C nuclear magnetic resonance spectroscopy of pennisetin from pearl millet
Plant Science, 83 (1992) 15-22 Elsevier Scientific Publishers Ireland Ltd.
15
Circular dichroism and nuclear magnetic resonance spectroscopy of pennisetin from pearl millet Mohini N. Sainani a, Vinod K. Mishra b Vidya S. Gupta a and Prabhakar K. Ranjekar a aDivision of Biochemical Sciences, National Chemical Laboratory, Pune 411 008 and hMolecular Biophysics Unit. Indian Institute of Science, Bangalore, 560 012 (India)
(Received September 3rd, 1991; revision received December 3rd, 1991; accepted January 2nd, 1992) The conformation and stability of pearl millet prolamin (pennisetin) were examined by using circular dichroism and 13C nuclear magnetic resonance spectroscopy. The far UV spectrum of pennisetin in 70% (v/v) aqueous ethanol showed the presence of predominant a helical structure and its occurrence in the c~ + /3 class of protein. The far and near UV spectra of pennisetin in ethanol: trifluoroethanol also supported this observation. However pennisetin showed the presence of some helical structure in 8 M urea which is known to be a highly unordered structure forming solvent. A decrease in c~ helical content of native pennisetin was observed with rise in temperature from 5-75°C and this effect of temperature was found to be reversible. A 13C NMR spectrum of pennisetin in 70% ethanol suggested a high degree of molecular mobility in ethanol, Comparison of the cross polarization spectrum with the single pulse excitation spectrum suggested pennisetin to be a heterogeneous protein. Key words." pearl millet; pennisetin; circular dichroism; nuclear magnetic resonance spectroscopy
Introduction
Material and Methods
Pearl millet p r o l a m i n (pennisetin) constitutes a b o u t 40% o f the total seed protein. A l t h o u g h partial biochemical c h a r a c t e r i z a t i o n o f this p r o t e i n has been r e p o r t e d [1], no d a t a are available with respect to its structure a n d c o n f o r m a t i o n . In this direction, we have earlier r e p o r t e d the h y d r o dynamic properties and molecular dimensions of pennisetin in the native as well as d e n a t u r e d state by m e a s u r i n g its intrinsic viscosity [2]. Recently, we have also investigated the e n v i r o n m e n t o f tyrosine a n d t r y p t o p h a n residues using intrinsic fluorescence s p e c t r o s c o p y [3]. In the present work, we r e p o r t structural a n d c o n f o r m a t i o n a l changes o f pennisetin using circular d i c h r o i s m ( C D ) a n d nuclear magnetic r e s o n a n c e ( N M R ) spectroscopy. Such d a t a are expected to p r o v i d e a better basis for u n d e r s t a n d i n g the functional p r o p e r t i e s o f p r o lamins in pearl millet.
Preparation o f pennisetin Pearl millet (Pennisetum americanum, varA w a s a r i ) seeds were p o w d e r e d in a R e m i m a k e blender and the fine p o w d e r was d e f a t t e d by giving several changes with n-hexane until it was free from fats. Pennisetin was then p r e p a r e d from defatted meal, after the r e m o v a l o f water and salt soluble fractions, using 70% e t h a n o l at 60°C. The p r o l a m i n c o n t a i n i n g s u p e r n a t a n t was mixed with an equal v o l u m e o f 1.5 M N a C I a n d kept overnight at 4°C. The p r e c i p i t a t e d p r o l a m i n was recovered by c e n t r i f u g a t i o n and lyophilised. It was then purified on an Octyl S e p h a r o s e c o l u m n as described by M a w a l et al. [4]. The c o n c e n t r a t i o n o f protein was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y
Correspondence to: Prabhakar K. Ranjekar, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.
15]. CD measurements C D m e a s u r e m e n t s were m a d e using a Jasco J-500 A s p e c t r o p o l a r i m e t e r with DP-501 N d a t a processor. Variable t e m p e r a t u r e spectra were obtained using a heat cell h o l d e r a t t a c h e d to a w a t e r
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 bath mgw Lauda RC-3. Quartz cells of different pathlengths (0.1-10.0 mm) were used to take the CD spectra in far and near UV regions. The molar ellipticity was calculated from the following equation. [0]m = (0)ob~ X 100 d e g . c m 2 drool_ l
Single-pulse excitation experiments were carried out with a 90 ° pulse (duration 5 #s) on 13C, followed immediately by a decoupling pulse on the protons during which the spectra were acquired. A repetition delay of 2 s was employed for the data collection. This sequence will tend to enhance the signal from a more mobile region [6].
CxL Where (0)obs is observed ellipticity in degrees, C is concentration in moles per litre and L is the pathlength in cm. For making measurements at different temperatures, the solution was heated to the desired temperature by circulating water through a double w',dled cell holder from a preheated water bath and then allowed to remain at that temperature for 15-20 rain for attainment of thermal equilibrium before taking a spectrum reading.
13C NMR spectrum of pennisetin in solution state A Bruker MSL 300 N M R spectrometer was used to carry out 75.47 MHz 13C measurements at ambient temperature, For solution spectra, sample tubes of 10-ram diameter containing 3 ml aqueous ethanolic solution (70% v/v) of pennisetin (60 mg/ml) were used. The spectra were recorded at ambient temperature (30°C) with broad band proton decoupling. An external capillary of D20 was used for field frequency lock and chemical shifts were referred to dioxane in D20 taken as 67.8 ppm. Data were collected with 16 384 data points and a spectral width of 20 kHz. ~r/2 pulses of 9 ~s duration were used with a repetition rate of 4 s. Cross polarization (CP)/single pulse excitation ( SPE) MAS spectrum of pennisetin The solid state spectra were obtained under conditions of dipolar decoupling and magic angle spinning. About 300 mg dry powdered pennisetin was packed into a 7-ram Zirconia type rotor and spun at 4 kHz. The H a r t m a n n - H a h n match condition was achieved using adamentane. For cross polarization, a contact time of I ms and recycle time of 4 s were used. One thousand transients were collected with 4 K data point and processed with a 10 Hz line broadening. The chemical shifts were with respect to the methine carbon of adamentane taken as 37.8 ppm.
Results and Discussion
Circular dichroism is a technique which is sensitive to secondary structure and hence can be applied to proteins and polypeptides in solution. Assignment of c~-helical and t3-sheet features in proteins from C D data are, however, based on calculations using known structures such as myoglobin (c~ proteins), lysozyme (c~ + /3 proteins), lactate dehydrogenase (c~/~3 proteins), and c~-Cobratoxin (/3 protein). The structural data obtained from CD spectra are, therefore, considered as predictions for the secondary structure of proteins. The conformation of pennisetin was studied by using CD spectroscopy in the far (190-260 nm) and near UV (250-320 nm) regions. The spectrum in the far UV region is indicative of the backbone structure, whereas the spectrum in the near UV region arises from aromatic and disulphide chromophores.
CD spectra in 70% ethanol The far UV CD spectrum of pennisetin in 70'7,, ethanol is shown in Fig. 1A. The curve shows two negative minima at 206 nm and 218 nm with a positive band at 192 rim. The molar ellipticity (0)m values at 206 nm and 218 nm are -195.8 × 10 -4 and -189.2 × 10 -4 deg. cm 2 dmol -I, respectively. The negative CD bands at 206 nm and 218 nm, crossover at 199 nm and a positive CD band at 192 nm indicate that a substantial part of the polypeptide chain is a right handed c~ helical [7] and that pennisetin is a c~ + t3 type of protein [8]. The near UV CD spectrum of pennisetin in 70% ethanol is depicted in Fig. lB. It shows shoulders and peaks at 256, 260, 261, 265, 267, 275 and 295 nm. The peaks at 275 nm and 295 nm are due to tyrosine and tryptophan residues, respectively. The peaks/shoulders at 256, 260, 261,265 and 267 nm
17
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205 nm and 218 nm having molar ellipticities of -268.4 x 10 -4 and -255.2 x 10 -4 d e g . c m 2 dmo1-1, respectively. The spectrum is comparable to that of 70% ethanol except for a little increase in the percent of c~-helix as judged by molar ellipticity value. The near UV CD spectrum of pennisetin in T F E (Fig. 2B) shows distinct bands of phenylalanine at 259 nm and 265 nm and tyrosine and tryptophan bands at 274 nm and 295 nm, respectively. Although pennisetin has 10% proline residues, it has a high level of c~-helix indicating the presence of long segments of c~-helix free of proline. This high level of helical structure of pennisetin is comparable to that reported for zein of maize, [10,11] a, {3, and r gliadins of wheat [12] and kafirin in sorghum [13].
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WAVELENGTH (nm) Fig. 1. CD spectra ofpennisetin in 70% (v/v) aqueous ethanol. The protein concentration was 1 mg/ml and molar ellipticity was expressed as [0]m x 10 -4 deg ' cm 2 dmol I. (A) Far UV spectrum. (B) Near UV spectrum.
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are due to the aromatic groups of phenylalanine [9] which indicate that these groups are present in fixed conformation in pennisetin.
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CD spectra in trifiuoroethanol ( TFE) The structure of pennisetin was next studied in ethanol: trifluoroethanol (7:3 v/v) mixture, since this solvent promotes a regular secondary structure by eliminating competition by water for hydrogen bonding. Figure 2A represents the far UV CD spectrum of pennisetin in the above solvent. The spectrum exhibits two negative peaks at
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WAVELENGTH Fig. 2. C D spectra of pennisetin in ethanol: t r i f l u o r o e t h a n o l solvent. Protein c o n c e n t r a t i o n was 1 mg/ml and m o l a r ellipticity was expressed as [0]m x 10 -4 deg • cm 2 d m o l - < (A) F a r UV spectrum. (B) Near UV spectrum.
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WAVELENGTH Fig. 3. F a r UV C D spectrum of pennisetin in 8 M urea. Protein concentration was l mg/ml and molar ellipticity was expressed as [0]m x 10 -4 deg " cm 2 dmol I.
CD spectra in 8 M urea
The spectra of pennisetin were then studied in 8 M urea, a denaturant of protein molecules, to check the conformation of pennisetin in the denatured state. The far UV spectrum was not obtained below 210 nm because of the strong absorbance of urea. From Fig. 3, it can be seen that pennisetin in 8 M urea shows a weak negative ellipticity at 222 nm. According to the data of Holzwarth and Doty [7], poly-L-lysine in randomly disordered conformation shows a positive dichroism near 220 nm and a strong negative dichroism below 210 nm. The negative ellipticity
of pennisetin at 222 nm suggests the presence of a stable portion of the protein molecule that resists unfolding in 8 M urea solution and that contains some helical structure, or it may result from restrictions on the conformation of the unfolded chain [14]. Alternatively, the negative ellipticity may be a result of the residual optical activity of aromatic side chains of the unfolded polypeptides as suggested in the case of ribonuclease [15]. The near UV spectrum of pennisetin shows the presence of some fine structures of phenylalanine, tyrosine and tryptophan thus supporting the fact that denaturation of pennisetin was incomplete in 8 M urea (data not shown). CD spectra at variable temperatures
C D spectra of pennisetin at different temperatures were recorded and (0)222 values were plotted against temperature (Fig. 4A). When the temperature of pennisetin solution is increased from 5-75°C, a decrease in the c~-helix content is observed in the far UV region. The near UV C D spectrum (Fig. 4B) shows a decrease in the intensity of the CD bands with rise in temperature. At 75°C, the fine structure of pennisetin is destroyed to a considerable extent. This suggests a change in
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Fig. 4. Effect of temperature on molar ellipticity of pennisetin (A) Molar ellipticity (0)222 as a function of temperature. (B) Near UV spectrum at 25°C and 75°C.
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Fig. 5. Solution-state ]3C NMR spectra of pennisetin. (A) In 70% (v/v) aqueous ethanol• Peak positions 1-15, 16-20 and 21-24 are from aliphatic, aromatic and carbonyl regions, respectively. (B) In 7 M guanidine hydrochloride.
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Solid-state 13C N M R spectra of pennisetin. (A) Cross polarization spectrum. Peak positions l - 7 are from aliphatic, 8 and 9 from aromatic and 10 from carbonyl regions. (B) Single pulse excitation (SPE) spectrum. Peak positions 1-9 are from aliphatic, 10 and 11 from aromatic and 12 from carbonyl regions. Peaks identified by + sign indicate spinning side bands from aromatic and carbonyl regions.
21 the conformation of aromatic residues with increase in temperature. These conformational changes with rise in temperature were found to be reversible (data not shown).
NMR spectroscopy After getting some insight into its secondary structure, ~3C N M R spectroscopy was used to get information about molecular properties of pennisetin. From solid-state NMR, the chemical environment of the protein within the system is understood, while the solution state 13C NMR approach reveals interactional and conformational changes of the protein with the solvent. Both these approaches are used in the present study. Solution state 13C N M R spectra of pennisetin Figure 5A shows 13C NMR spectrum of pennisetin at ambient temperature in 70% (v/v) aqueous ethanol. Aliphatic, aromatic and carbonyl regions in pennisetin show fifteen, five and four well resolved 13C resonances, respectively. As such the assignment of chemical shift to 13C resonances is difficult for proteins and is more complicated for proteins like pennisetin because of the overlap of peaks from the organic solvent. It can be readily seen from the figure that the regions from 50-60 ppm and 15-20 ppm are masked by the strong solvent. Spectral assignment of these resonances were made by reference to chemical shift tables of Howarth and Lilly [16]. Since the chemical shift values reported by them are in D20 and our measurements were made in aqueous ethanol, the comparison is approximate. In the aliphatic region, peak no. 8 and 9 of proline and glutamine residues respectively are intense because of the predominance of these amino acids in pennisetin. The resolved peaks present in the aromatic region can be readily assigned to phenylalanine and tyrosine residues which suggests the occurrence of a good interaction of the protein molecule with the solvent. Although 13C NMR spectrum of pennisetin is comparable with 13C NMR spectrum o f ' C ' hordein in 0.1 M acetic acid [17], it is more resolved than 13C NMR of zein in 70°/,, ethanol which shows only seven 13C NMR resonances [18]. The well resolved 13C resonances of pennisetin thus suggest a high
degree of molecular mobility of pennisetin in 70% (v/v) ethanol. The ~3C N M R spectrum of pennisetin in 7 M guanidine-hydrochloride (Gdn.HCI) is depicted in Fig. 5B. Due to the unfolding of pennisetin in 7 M Gdn.HCk the residues in pennisetin behave like free amino acids and are exposed to the solvent resulting in sharp 13C resonances in all the three regions indicating denaturation of pennisetin in 7 M Gdn.HCI. This observation is in contrast with the reported spectrum of ~C' hordein in 6 M Gdn.HCI where there was a complete loss of spectral activity [17].
CP/SPE M A S 13C-NMR spectrum o/ penn&et& Proteins of the kind which we are looking at are known to exhibit a dynamic heterogeneity that is the protein consists of different domains having different molecular mobilities. Solid state 13C NMR technique can roughly differentiate the regimes having a solid-like or rigid-lattice nature, and a liquid like or mobile regime. CP-MAS NMR spectroscopy selectively picks up the 13C signals arising from the former category while SPE technique prefers the signals from the latter category [6]. These two techniques were employed for pennisetin and the spectra obtained are given in Fig. 6. CP-MAS 13C NMR spectrum (Fig. 6A) of pennisetin shows seven ~3C resonances in the aliphatic region and two ~3C resonances in both the aromatic and carbonyl regions whereas in the SPE spectrum, aliphatic, aromatic and carbonyl regions show 9, 2 and 1 13C resonances, respectively. 13C NMR resonances of the aliphatic region are more resolved while aromatic and carbonyl regions are less resolved in the SPE spectrum as compared to the CP-MAS spectrum. These differences in CP and SPE-MAS spectra suggest that in pennisetin there exists a mobile liquid environment along with a rigid environment. However, in case of ~C' hordein, being a homogenous protein, neither change in peak width nor new resonances were observed in the SPE spectrum [17]. These structural and conformational details are important for an ongoing work of protein structure and function.
22
Acknowledgements The authors are grateful to Professor P. Balaram, Indian Institute of Science, Bangalore, for his invaluable help in evaluation of the CD data. Thanks are due to Dr. Rajmohan, Dr. S. Ganpathy and Dr. V.K. Bhalerao from the National Chemical Laboratory, Pune for their assistance in carrying out NMR experiments and in the interpretation of the results.
8
9
10
11
References 12 1
2
3
4
5
6
7
P.N. Okoh, C.C. Nwasike and C.O. Ikediobi, Studies of seed protein of pearl millet: amino acid composition of protein fractions of early and late maturing varieties. J. Agric. Food Chem., 33 (1985) 55-57. M.N. Sainani, A.H. Lachke, N.A. Sahasrabudhe and P.K. Ranjekar, Viscometric characterization of pennisetin from pearl millet. Biochem. Biophys. Res. Commun., 165 (1989) 334-341. M.N. Sainani, V.S. Gupta and P.K. Ranjekar, Conformational changes in pennisetin using intrinsic fluorescence spectroscopy. Phytochemistry, (in press). Y.R. Mawal, M.R. Mawal and P.K. Ranjekar, A novel method for iodination of a hydrophobic protein: rice prolamin. Biotechnol. Tech., 3 (1989) 167-172. V. Jagannathan, K. Singh and M. Damodaran, Carbohydrate metabolism in citric acid fermentation. Biochem. J., 63 (1956) 94-105. P.S. Belton, P.R. Shewry and A.S. Tatham, t3c solid state nuclear magnetic resonance study of wheat gluten. J. Cereal Sci., 3 (1985) 305-317. G. Holzwarth and P. Dory, The ultraviolet circular
13
14
15
16
17 18
dichroism of polypeptides. J. Am. Chem, Soc., 87 (1965) 218-228. P. Manavalan and W,C. Johnson, Sensitivity of circular dichroism to protein tertiary structure class. Nature, 305 (1983) 831-832. E. Strickland, Aromatic contribution to circular dichroism spectra ~;f proteins. CRC Crit. Rev. Biochem., 2 (1974) 113-175. P. Argos, K. Pederson, M.D. Marks and B.A. Larkins, A structure model for maize zein protein. J. Biol. Chem., 257 (1982) 9984-9990. Y.V. Wu, J.W. Paulis, K.R. Sexson and J.S. Wall, Conformation of corn zein and glutelin fractions with unusual amino acid sequence. Cereal Chem., 60 (1983) 342-344. A.S. Tatham, P.R. Shewry and B.J. Miflin, Wheat gluten elasticity: a similar molecular basis to elastin?. FEBS Lett., 177 (1984) 205-208. Y,V. Wu, J.E. Cluskey and R.W. Jones, Sorghum prolamins: their opti,,al rotatory dispersion, circular dichroism and infra-red spectroscopy. J. Agric. Food Chem., 19 (1971) 1139-1143. D.D. Kasarda, J.E. Bernardin and W. Gaffield, Circular dichroism and optical rotatory dispersion of ~-gliadin. Biochemistry, 7 (1968) 3950-3957. H, Hashizume, M. Shiraki and K. lmahori, Study of circular dichroism of proteins and polypeptides in relation to their backbone and side chain conformation. J. Biochem., 62 (1967) 543-551. O.D. Howarth and D.M. Lilley, Carbon-13 NMR of peptides and proteins. Prog. Nucl. Magn. Reson. Spectrosc., 12 (1978) 1-40. A.S. Tatham, P.R. Shewry and P.S. Belton, 13C NMR study of C hordein. Biochem. J., 232 (1985) 617-620. M.E. Augustine and I.C. Baianu, High resolution carbon-13 nuclear magnetic resonance studies of maize proteins. J. Cereal Sci,, 4 (1986) 371-378.
15
Circular dichroism and nuclear magnetic resonance spectroscopy of pennisetin from pearl millet Mohini N. Sainani a, Vinod K. Mishra b Vidya S. Gupta a and Prabhakar K. Ranjekar a aDivision of Biochemical Sciences, National Chemical Laboratory, Pune 411 008 and hMolecular Biophysics Unit. Indian Institute of Science, Bangalore, 560 012 (India)
(Received September 3rd, 1991; revision received December 3rd, 1991; accepted January 2nd, 1992) The conformation and stability of pearl millet prolamin (pennisetin) were examined by using circular dichroism and 13C nuclear magnetic resonance spectroscopy. The far UV spectrum of pennisetin in 70% (v/v) aqueous ethanol showed the presence of predominant a helical structure and its occurrence in the c~ + /3 class of protein. The far and near UV spectra of pennisetin in ethanol: trifluoroethanol also supported this observation. However pennisetin showed the presence of some helical structure in 8 M urea which is known to be a highly unordered structure forming solvent. A decrease in c~ helical content of native pennisetin was observed with rise in temperature from 5-75°C and this effect of temperature was found to be reversible. A 13C NMR spectrum of pennisetin in 70% ethanol suggested a high degree of molecular mobility in ethanol, Comparison of the cross polarization spectrum with the single pulse excitation spectrum suggested pennisetin to be a heterogeneous protein. Key words." pearl millet; pennisetin; circular dichroism; nuclear magnetic resonance spectroscopy
Introduction
Material and Methods
Pearl millet p r o l a m i n (pennisetin) constitutes a b o u t 40% o f the total seed protein. A l t h o u g h partial biochemical c h a r a c t e r i z a t i o n o f this p r o t e i n has been r e p o r t e d [1], no d a t a are available with respect to its structure a n d c o n f o r m a t i o n . In this direction, we have earlier r e p o r t e d the h y d r o dynamic properties and molecular dimensions of pennisetin in the native as well as d e n a t u r e d state by m e a s u r i n g its intrinsic viscosity [2]. Recently, we have also investigated the e n v i r o n m e n t o f tyrosine a n d t r y p t o p h a n residues using intrinsic fluorescence s p e c t r o s c o p y [3]. In the present work, we r e p o r t structural a n d c o n f o r m a t i o n a l changes o f pennisetin using circular d i c h r o i s m ( C D ) a n d nuclear magnetic r e s o n a n c e ( N M R ) spectroscopy. Such d a t a are expected to p r o v i d e a better basis for u n d e r s t a n d i n g the functional p r o p e r t i e s o f p r o lamins in pearl millet.
Preparation o f pennisetin Pearl millet (Pennisetum americanum, varA w a s a r i ) seeds were p o w d e r e d in a R e m i m a k e blender and the fine p o w d e r was d e f a t t e d by giving several changes with n-hexane until it was free from fats. Pennisetin was then p r e p a r e d from defatted meal, after the r e m o v a l o f water and salt soluble fractions, using 70% e t h a n o l at 60°C. The p r o l a m i n c o n t a i n i n g s u p e r n a t a n t was mixed with an equal v o l u m e o f 1.5 M N a C I a n d kept overnight at 4°C. The p r e c i p i t a t e d p r o l a m i n was recovered by c e n t r i f u g a t i o n and lyophilised. It was then purified on an Octyl S e p h a r o s e c o l u m n as described by M a w a l et al. [4]. The c o n c e n t r a t i o n o f protein was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y
Correspondence to: Prabhakar K. Ranjekar, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.
15]. CD measurements C D m e a s u r e m e n t s were m a d e using a Jasco J-500 A s p e c t r o p o l a r i m e t e r with DP-501 N d a t a processor. Variable t e m p e r a t u r e spectra were obtained using a heat cell h o l d e r a t t a c h e d to a w a t e r
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 bath mgw Lauda RC-3. Quartz cells of different pathlengths (0.1-10.0 mm) were used to take the CD spectra in far and near UV regions. The molar ellipticity was calculated from the following equation. [0]m = (0)ob~ X 100 d e g . c m 2 drool_ l
Single-pulse excitation experiments were carried out with a 90 ° pulse (duration 5 #s) on 13C, followed immediately by a decoupling pulse on the protons during which the spectra were acquired. A repetition delay of 2 s was employed for the data collection. This sequence will tend to enhance the signal from a more mobile region [6].
CxL Where (0)obs is observed ellipticity in degrees, C is concentration in moles per litre and L is the pathlength in cm. For making measurements at different temperatures, the solution was heated to the desired temperature by circulating water through a double w',dled cell holder from a preheated water bath and then allowed to remain at that temperature for 15-20 rain for attainment of thermal equilibrium before taking a spectrum reading.
13C NMR spectrum of pennisetin in solution state A Bruker MSL 300 N M R spectrometer was used to carry out 75.47 MHz 13C measurements at ambient temperature, For solution spectra, sample tubes of 10-ram diameter containing 3 ml aqueous ethanolic solution (70% v/v) of pennisetin (60 mg/ml) were used. The spectra were recorded at ambient temperature (30°C) with broad band proton decoupling. An external capillary of D20 was used for field frequency lock and chemical shifts were referred to dioxane in D20 taken as 67.8 ppm. Data were collected with 16 384 data points and a spectral width of 20 kHz. ~r/2 pulses of 9 ~s duration were used with a repetition rate of 4 s. Cross polarization (CP)/single pulse excitation ( SPE) MAS spectrum of pennisetin The solid state spectra were obtained under conditions of dipolar decoupling and magic angle spinning. About 300 mg dry powdered pennisetin was packed into a 7-ram Zirconia type rotor and spun at 4 kHz. The H a r t m a n n - H a h n match condition was achieved using adamentane. For cross polarization, a contact time of I ms and recycle time of 4 s were used. One thousand transients were collected with 4 K data point and processed with a 10 Hz line broadening. The chemical shifts were with respect to the methine carbon of adamentane taken as 37.8 ppm.
Results and Discussion
Circular dichroism is a technique which is sensitive to secondary structure and hence can be applied to proteins and polypeptides in solution. Assignment of c~-helical and t3-sheet features in proteins from C D data are, however, based on calculations using known structures such as myoglobin (c~ proteins), lysozyme (c~ + /3 proteins), lactate dehydrogenase (c~/~3 proteins), and c~-Cobratoxin (/3 protein). The structural data obtained from CD spectra are, therefore, considered as predictions for the secondary structure of proteins. The conformation of pennisetin was studied by using CD spectroscopy in the far (190-260 nm) and near UV (250-320 nm) regions. The spectrum in the far UV region is indicative of the backbone structure, whereas the spectrum in the near UV region arises from aromatic and disulphide chromophores.
CD spectra in 70% ethanol The far UV CD spectrum of pennisetin in 70'7,, ethanol is shown in Fig. 1A. The curve shows two negative minima at 206 nm and 218 nm with a positive band at 192 rim. The molar ellipticity (0)m values at 206 nm and 218 nm are -195.8 × 10 -4 and -189.2 × 10 -4 deg. cm 2 dmol -I, respectively. The negative CD bands at 206 nm and 218 nm, crossover at 199 nm and a positive CD band at 192 nm indicate that a substantial part of the polypeptide chain is a right handed c~ helical [7] and that pennisetin is a c~ + t3 type of protein [8]. The near UV CD spectrum of pennisetin in 70% ethanol is depicted in Fig. lB. It shows shoulders and peaks at 256, 260, 261, 265, 267, 275 and 295 nm. The peaks at 275 nm and 295 nm are due to tyrosine and tryptophan residues, respectively. The peaks/shoulders at 256, 260, 261,265 and 267 nm
17
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205 nm and 218 nm having molar ellipticities of -268.4 x 10 -4 and -255.2 x 10 -4 d e g . c m 2 dmo1-1, respectively. The spectrum is comparable to that of 70% ethanol except for a little increase in the percent of c~-helix as judged by molar ellipticity value. The near UV CD spectrum of pennisetin in T F E (Fig. 2B) shows distinct bands of phenylalanine at 259 nm and 265 nm and tyrosine and tryptophan bands at 274 nm and 295 nm, respectively. Although pennisetin has 10% proline residues, it has a high level of c~-helix indicating the presence of long segments of c~-helix free of proline. This high level of helical structure of pennisetin is comparable to that reported for zein of maize, [10,11] a, {3, and r gliadins of wheat [12] and kafirin in sorghum [13].
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WAVELENGTH (nm) Fig. 1. CD spectra ofpennisetin in 70% (v/v) aqueous ethanol. The protein concentration was 1 mg/ml and molar ellipticity was expressed as [0]m x 10 -4 deg ' cm 2 dmol I. (A) Far UV spectrum. (B) Near UV spectrum.
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are due to the aromatic groups of phenylalanine [9] which indicate that these groups are present in fixed conformation in pennisetin.
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CD spectra in trifiuoroethanol ( TFE) The structure of pennisetin was next studied in ethanol: trifluoroethanol (7:3 v/v) mixture, since this solvent promotes a regular secondary structure by eliminating competition by water for hydrogen bonding. Figure 2A represents the far UV CD spectrum of pennisetin in the above solvent. The spectrum exhibits two negative peaks at
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WAVELENGTH Fig. 2. C D spectra of pennisetin in ethanol: t r i f l u o r o e t h a n o l solvent. Protein c o n c e n t r a t i o n was 1 mg/ml and m o l a r ellipticity was expressed as [0]m x 10 -4 deg • cm 2 d m o l - < (A) F a r UV spectrum. (B) Near UV spectrum.
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WAVELENGTH Fig. 3. F a r UV C D spectrum of pennisetin in 8 M urea. Protein concentration was l mg/ml and molar ellipticity was expressed as [0]m x 10 -4 deg " cm 2 dmol I.
CD spectra in 8 M urea
The spectra of pennisetin were then studied in 8 M urea, a denaturant of protein molecules, to check the conformation of pennisetin in the denatured state. The far UV spectrum was not obtained below 210 nm because of the strong absorbance of urea. From Fig. 3, it can be seen that pennisetin in 8 M urea shows a weak negative ellipticity at 222 nm. According to the data of Holzwarth and Doty [7], poly-L-lysine in randomly disordered conformation shows a positive dichroism near 220 nm and a strong negative dichroism below 210 nm. The negative ellipticity
of pennisetin at 222 nm suggests the presence of a stable portion of the protein molecule that resists unfolding in 8 M urea solution and that contains some helical structure, or it may result from restrictions on the conformation of the unfolded chain [14]. Alternatively, the negative ellipticity may be a result of the residual optical activity of aromatic side chains of the unfolded polypeptides as suggested in the case of ribonuclease [15]. The near UV spectrum of pennisetin shows the presence of some fine structures of phenylalanine, tyrosine and tryptophan thus supporting the fact that denaturation of pennisetin was incomplete in 8 M urea (data not shown). CD spectra at variable temperatures
C D spectra of pennisetin at different temperatures were recorded and (0)222 values were plotted against temperature (Fig. 4A). When the temperature of pennisetin solution is increased from 5-75°C, a decrease in the c~-helix content is observed in the far UV region. The near UV C D spectrum (Fig. 4B) shows a decrease in the intensity of the CD bands with rise in temperature. At 75°C, the fine structure of pennisetin is destroyed to a considerable extent. This suggests a change in
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Fig. 4. Effect of temperature on molar ellipticity of pennisetin (A) Molar ellipticity (0)222 as a function of temperature. (B) Near UV spectrum at 25°C and 75°C.
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Fig. 5. Solution-state ]3C NMR spectra of pennisetin. (A) In 70% (v/v) aqueous ethanol• Peak positions 1-15, 16-20 and 21-24 are from aliphatic, aromatic and carbonyl regions, respectively. (B) In 7 M guanidine hydrochloride.
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Solid-state 13C N M R spectra of pennisetin. (A) Cross polarization spectrum. Peak positions l - 7 are from aliphatic, 8 and 9 from aromatic and 10 from carbonyl regions. (B) Single pulse excitation (SPE) spectrum. Peak positions 1-9 are from aliphatic, 10 and 11 from aromatic and 12 from carbonyl regions. Peaks identified by + sign indicate spinning side bands from aromatic and carbonyl regions.
21 the conformation of aromatic residues with increase in temperature. These conformational changes with rise in temperature were found to be reversible (data not shown).
NMR spectroscopy After getting some insight into its secondary structure, ~3C N M R spectroscopy was used to get information about molecular properties of pennisetin. From solid-state NMR, the chemical environment of the protein within the system is understood, while the solution state 13C NMR approach reveals interactional and conformational changes of the protein with the solvent. Both these approaches are used in the present study. Solution state 13C N M R spectra of pennisetin Figure 5A shows 13C NMR spectrum of pennisetin at ambient temperature in 70% (v/v) aqueous ethanol. Aliphatic, aromatic and carbonyl regions in pennisetin show fifteen, five and four well resolved 13C resonances, respectively. As such the assignment of chemical shift to 13C resonances is difficult for proteins and is more complicated for proteins like pennisetin because of the overlap of peaks from the organic solvent. It can be readily seen from the figure that the regions from 50-60 ppm and 15-20 ppm are masked by the strong solvent. Spectral assignment of these resonances were made by reference to chemical shift tables of Howarth and Lilly [16]. Since the chemical shift values reported by them are in D20 and our measurements were made in aqueous ethanol, the comparison is approximate. In the aliphatic region, peak no. 8 and 9 of proline and glutamine residues respectively are intense because of the predominance of these amino acids in pennisetin. The resolved peaks present in the aromatic region can be readily assigned to phenylalanine and tyrosine residues which suggests the occurrence of a good interaction of the protein molecule with the solvent. Although 13C NMR spectrum of pennisetin is comparable with 13C NMR spectrum o f ' C ' hordein in 0.1 M acetic acid [17], it is more resolved than 13C NMR of zein in 70°/,, ethanol which shows only seven 13C NMR resonances [18]. The well resolved 13C resonances of pennisetin thus suggest a high
degree of molecular mobility of pennisetin in 70% (v/v) ethanol. The ~3C N M R spectrum of pennisetin in 7 M guanidine-hydrochloride (Gdn.HCI) is depicted in Fig. 5B. Due to the unfolding of pennisetin in 7 M Gdn.HCk the residues in pennisetin behave like free amino acids and are exposed to the solvent resulting in sharp 13C resonances in all the three regions indicating denaturation of pennisetin in 7 M Gdn.HCI. This observation is in contrast with the reported spectrum of ~C' hordein in 6 M Gdn.HCI where there was a complete loss of spectral activity [17].
CP/SPE M A S 13C-NMR spectrum o/ penn&et& Proteins of the kind which we are looking at are known to exhibit a dynamic heterogeneity that is the protein consists of different domains having different molecular mobilities. Solid state 13C NMR technique can roughly differentiate the regimes having a solid-like or rigid-lattice nature, and a liquid like or mobile regime. CP-MAS NMR spectroscopy selectively picks up the 13C signals arising from the former category while SPE technique prefers the signals from the latter category [6]. These two techniques were employed for pennisetin and the spectra obtained are given in Fig. 6. CP-MAS 13C NMR spectrum (Fig. 6A) of pennisetin shows seven ~3C resonances in the aliphatic region and two ~3C resonances in both the aromatic and carbonyl regions whereas in the SPE spectrum, aliphatic, aromatic and carbonyl regions show 9, 2 and 1 13C resonances, respectively. 13C NMR resonances of the aliphatic region are more resolved while aromatic and carbonyl regions are less resolved in the SPE spectrum as compared to the CP-MAS spectrum. These differences in CP and SPE-MAS spectra suggest that in pennisetin there exists a mobile liquid environment along with a rigid environment. However, in case of ~C' hordein, being a homogenous protein, neither change in peak width nor new resonances were observed in the SPE spectrum [17]. These structural and conformational details are important for an ongoing work of protein structure and function.
22
Acknowledgements The authors are grateful to Professor P. Balaram, Indian Institute of Science, Bangalore, for his invaluable help in evaluation of the CD data. Thanks are due to Dr. Rajmohan, Dr. S. Ganpathy and Dr. V.K. Bhalerao from the National Chemical Laboratory, Pune for their assistance in carrying out NMR experiments and in the interpretation of the results.
8
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References 12 1
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