Molecular aggregation and conformational change of wasp venom mastoparan as induced by salt in aqueous solution

Biochimica et Biophysica Acta, 802 (1984) 157-161

157

Elsevier BBA 21870

MOLECULAR AGGREGATION AND CONFORMATIONAL CHANGE OF WASP VENOM MASTOPARAN AS INDUCED BY SALT IN AQUEOUS SOLUTION TSUTOMU HIGASHIJIMA a, KAORI WAKAMATSU a, KAZUKI SAITO a MASAHIKO FUJINO b, TERUMI N A K A J I M A c and TATSUO MIYAZAWA a.,

a Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, b Chemistry Laboratories, Central Research Division, Takeda Chemical Industries, Yodogawa-ku, Osaka 532, and c Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Chiyoda.ku, Tokyo 101 (Japan) (Received April 17th, 1984)

Key words: Wasp venom; Mastoparan; Conformational change," Salt effect

The effect of salt on molecular aggregation and conformation has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270 MHz 1H.NMR and CD spectra of mastuparan in solution have been analyzed. On addition of salt at high concentration in aqueous solution, the monomeric form of mastoparan in largely unordered conformation is converted to the molecular aggregate (probably tetramer) with a largely a-helical conformation. The molecular aggregation of mastoparan depends on pH and ionic strength, but not on the kind of counter anion, in contrast to the case with melittin from bee venom.

Introduction Mastoparan is the major component of wasp venom (Vespula lewisii) and causes the degranulation of mast cells [1,2]. The primary structure of mastoparan is Ile-Asn-Leu-Lys-Ala-Leu-Ala-AlaLeu-Ala-Lys-Lys-Ile-Leu-NH2, with a molecular weight of 1480 [1,2]. As a first step for elucidating the conformational basis of the physiological action, the molecular conformations of mastoparan in solution have been analyzed [3]. The conformation of mastoparan in methanol solution is largely a-helical, while the conformation of mastoparan at a low concentration (50 #M) in aqueous solution is predominantly unordered in the presence of salt at a low concentration, such as in 0.1 M sodium phosphate buffer [3]. However, the conformation of mastoparan in aqueous solution is converted to a helix-rich form upon binding with phospholipid

* To whom correspondence should be addressed. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

membrane [3,4], probably because of the interaction of the hydrophobic moiety of mastoparan with the hydrophobic interior of phospholipid membrane [3,5]. The conformation and action of mastoparan may be compared with those of melittin, the major component of bee venom. Melittin is also a peptide (with 26 residues) with mast cell degranulating activity [6]. In clear contrast to mastoparan, the conformation of melittin in aqueous solution is significantly affected by the presence of 0.1 M phosphate buffer. Furthermore, in aqueous solution, melittin is known to be in an equilibrium of the monomeric form (largely unordered) and tetrameric form (largely a-helical), depending on the peptide concentration, pH, kind of counter ion and ionic strength [7-10]. Interestingly, the molecular weight of mastoparan has been obtained to be as high as 6310 from the size-exclusion chromatography at an ionic strength of about 1.5 [11]. This suggests that mastoparan also forms a tetramer in solution at high ionic strength. The present paper

158

reports on the molecular association and concomitant a-helix formation of mastoparan in aqueous solution in the presence of salt at high concentration. Materials and Methods Mastoparan was synthesized by the solution method [12]. N-Acetylglycine methylamide was also synthesized. The 270-MHz 1H-NMR spectra were recorded on a Bruker WH-270 spectrometer. Chemical shifts were measured from the internal standard of sodium 2,2-dimethyl-2-silapentane-5sulfonate. Nuclear Overhauser enhancement experiments were performed with the gated irradiation of a proton for 10 s, and negative nuclear Overhauser enhancements of other proton resonances were extracted by the difference method. Spin-lattice relaxation times (T1) were measured by the 180°-r-90°-t pulse sequence method (t = 10 s). All the N M R measurements were carried out at 23°C. CD spectra were recorded on a Jasco J-40S spectrometer. The value of [0] was expressed as the molar ellipticity as divided by 14, the number of main-chain C O N H groups. Results Circular dichroism The concentration dependences of [0] at 222 nm ([0]222) of mastoparan were observed, as shown in Fig. 1. As the concentration of peptide is raised from 0.01 m M to 5 mM, [0]222 increases gradually

from - 3 0 0 0 to - 7 0 0 0 in salt-free aqueous solution and from - 4 0 0 0 to - 1 2 0 0 0 in 0.1 M TrisHC1 buffer (pH 7.2). However, a remarkable concentration dependence of [0]222 is observed in 0.5 M sodium phosphate buffer (pH 7.2); [0]222 increases from - 3500 at 0.01 mM to - 20 500 at 5 mM. Such a concentration dependence of [0]222 is due to the molecular association (and concomitant conformation change) of mastoparan in aqueous solution. The C D spectra of mastoparan at a constant concentration (0.2 mM) in aqueous solution were observed, as shown in Fig. 2, in the presence of phosphate buffer (pH 7.2). At a low concentration (5 mM) of phosphate anion, the CD spectrum of mastoparan exhibits a negative peak at around 204 nm. However, as the phosphate concentration is raised to 500 mM, the CD band at 204 nm is shifted to 208 nm with intensity enhancement. The negative band at around 220 nm is also enhanced in intensity, and, at the highest concentration (500 mM) of phosphate anion, the CD spectrum of mastoparan with the negative bands at 208 and 220 nm is typical of the a-helical conformation. Furthermore, the CD curves of mastoparan in the presence of phosphate anion over the concentration range 5-500 mM exhibit an isodichroic point at 203 nm. These observations indicate that the conformational change from a largely unordered form to a helix-rich form of mastoparan is induced by the interaction with the phosphate anion. The C D spectra of mastoparan (0.2 mM) in 0.1 M Tris-HC1 buffer (pH 7.2) were also observed in the

T" -6

"6 ,4 20 E ?

,

WAVELENGTH

!,200210220230240250 "o

× ?

o:° 5

0 I

i,,1

o

I

o.ol

o.~;~ ,;.1

I

o.~

CONCENTRATION

I

1

I

I

lo

( mM )

Fig. 1. The dependences of [0]222 on the concentration of mastoparan in salt-free aqueous solution (A), in 0.1 M Tris-HCl buffer (pH 7.2) (m), and in 0.5 M sodium phosphate buffer (pH 7.2) (O) a t 23°C.

-2o I Fig. 2. The C D spectra of mastoparan (0.2 m M ) in aqueous solution (pH 7.2, 23°C) in the presence of phosphate buffer at 5 m M ( . . . . . . ), 115 m M ( . . . . ), 250 m M ( - - - - - ) and 500 mM( ).

159 I

~

I

(b')--~

~-.

(b)

A

I

I

~.

x4

lO WAVELENGTH (nm)

o1:0 2io 2!o

22o 23o 2,4o 250

,", -10 X

-2ot Fig. 3. The CD spectra of mastoparan (0.2 mM) in 0.1 M Tris-HCl buffer (pH 7.2, 23°C) in the presence of NaCI at 1.00 M ( . . . . . . ), 1.25 M ( - - - - - ) , and 1.50 M ( ).

(a)"]~-~ ~-~ ~ x 1 (a')..... ,.~x4 --

i

l

i

I

4

3

2

1

CHEMICAL SHIFT (PPM)

presence of NaC1, As the concentration of NaC1 is raised from 1.0 M to 1.5 M (Fig. 3), the negative band at 206 nm is shifted to 208 nm (with intensity enhancement) and the negative band at around 220 nm is enhanced in intensity, and the isodichroic point is observed at 203 nm. Thus, the conformational change from an unordered form to an a-helical form of mastoparan is also induced by the addition of NaC1. Furthermore, the pH dependences of CD spectra of mastoparan (0.2 mM) were observed in aqueous solution in the presence of 0.01 M NaC1 (23°C). The [0]222 value is increased from -5900 at pH 6.0 to -8900 at pH 8.3, with the isodichroic point again at 203 nm. Nuclear magnetic resonance

The 270 MHz 1H-NMR spectrum (in the aliphatic region) of mastoparan (2 mM) in salt-free 2H20 solution is shown in Fig. 4a. Major resonance peaks are readily assigned as, 4.23 ppm to a-CH, 2.99 ppm to Lys e-CH2, 1.69 ppm to fl-CH, 1.42 ppm to Ala-CH3, and 0.90 ppm to lie- and Leu-CH 3. In the presence of 0.5 M phosphate buffer (Fig. 4b), the proton resonances become broad and the a-CH resonances are shifted upfield, as compared with the proton resonances in salt-free solution. Such changes in 1H-NMR spectrum are due to the molecular association and concomitant conformational change of mastoparan upon addition of 0.5 M sodium phosphate. For mastoparan in aqueous solution, nuclear Overhauser enhancement difference spectra at 270 MHz were recorded on irradiation of Ala-CH 3

Fig. 4. The 270 MHz ] H - N M R spectra (in the aliphatic region) of mastoparan (2 mM) in salt-free 2H20 solution (a) and in 0.5 M deuterated phosphate buffer (b). (a') and (b') are nuclear Overhauser enhancement difference spectra on the irradiation of AIa-CH 3 protons at 1.42 ppm.

protons. Significant negative nuclear Overhauser enhancements were observed in 0.5 M phosphate buffer (Fig. 4b'), while nuclear Overhauser enhancements were negligible in salt-free solution (Fig. 4a'). In order to elucidate the effect of phosphate buffer on the correlation times of solute molecules, the spin-lattice relaxation rates (1//'1) of N-acetylglycine methylamide were measured. By the addition of 0.5 M phosphate buffer, the 1 / T 1 value of the Gly-a-CH 2 protons is increased from 0.60 to 0.72 s-1. Discussion The 270 MHz 1H-NMR spectrum and the CD spectrum of mastoparan in salt-free aqueous solution have been reported previously [3]. The CD spectrum of mastoparan is typical of unordered form, with a negative band around 200 nm. This is consistent with the large coupling constants (3JNH.C~H) and the large temperature coefficients of chemical shifts as obtained for most amide NH protons. These observations indicate that the mastoparan molecule largely takes unordered form in salt-free aqueous solution [3]. In the present study, and nuclear Overhauser enhancement experiment has been made of mastoparan in salt-free 2HzO solution. On irradiation of the CH 3 protons

160 of alanine residues (1.42 ppm), however, nuclear Overhauser enhancements are are hardly observed at the resonance frequency (u) of 270 MHz (Fig. 4a'). This indicates that the correlation time of mastoparan in salt-free aqueous solution (at 23°C) is nearly equal to (5/4)]/2/2~v = 0.66 ns [13]. The correlation time for the dipole interaction is known to be nearly proportional to the solution viscosity. In order to elucidate the viscosity effect on correlation times, the longitudinal relaxation rates (1//'1) have been measured of the a - C H 2 proton resonance of N-acetylglycine methylamide. The 1 / T 1 value of this small molecule in the 0.5 M phosphate buffer has been found to be 1.2-times as large as that in salt-free aqueous solution. This suggests that the correlation time of free mastoparan molecule is increased from 0.66 ns to nearly 0.79 ns on addition of 0.5 M phosphate, and then the maximum negative nuclear Overhauser enhancement is expected to be about - 10% at the resonance frequency of 270 M H z [13]. Actually, however, for mastoparan (2 mM) in 0.5 M phosphate buffer at 23°C, the negative nuclear Overhauser enhancement of the a - C H proton resonance has been found to be as large as - 30% on irradiation of the C H 3 proton resonance of alanine residues (Fig. 4b'). This indicates that the effective correlation time of mastoparan in 0.5 M phosphate buffer is longer than 1.1 ns, and the averaged molecular weight of mastoparan is much increased on addition of 0.5 M phosphate ion. In fact, in the size-exclusion chromatography at the ionic strength of about 1.5, the effective molecular weight of mastoparan has been obtained as high as 6310 [11]. This suggests that the mastoparan molecules primarily form the tetramer in aqueous solution at high ionic strength. The CD curves of mastoparan in aqueous solution with various concentrations of phosphate buffer exhibit an isodichroic point at 203 nm (Fig. 2), indicating the chemical equilibrium between the monomer and the aggregate (tetramer). In order to elucidate the effects of salt on such tetramer formation, the CD curves of mastoparan have also been studied in aqueous solution with various concentrations of Na2SO 4 (Fig. 5). Again, an isodichroic point is observed at 203 nm, and the effect of 0.5 M NaRSO 4 (ionic strength of 1.5) on the tetramer formation is slightly stronger than the

effect of 0.5 M phosphate buffer (ionic strength of 1.3 at p H 7.2). Furthermore, the effect of 0.5 M NazSOn on the tetramer formation is nearly equal to the effect of 1.5 M NaC1 (ionic strength of 1.5) (Fig. 3). Such salt effects on mastoparan are in clear contrast to the case with melittin, where the effect of phosphate ion is about three-times as strong as the effect of sulfate ion and is about twenty-times as strong as the effect of chloride ion [10]. Such a specific effect of phosphate ion on the tetramer formation of melittin has been ascribed to the interaction of the phosphate ion with the guanidinium group of arginine residues in melittin [10]. By contrast, mastoparan does not have arginine residues, and the salt effect on the tetramer formation may be explained largely in relation with the ionic strength of the aqueous solution. The tetramer formation of mastoparan at high ionic strength is accompanied by the conformational change from an unordered form to an ahelix-rich form, as is evident from the observation of C D spectra (Figs. 2, 3 and 5). In the a-helical form of mastoparan, the positively charged groups of three lysine residues are all directed to one side of the helix, while the other side is occupied by hydrophobic amino acid residues [5]. Previously, for mastoparan as bound to phospholipid micelles, the positively charged groups of lysine residues have been found to be exposed to the aqueous phase [5]. Probably in the tetramer of mastoparan, the hydrophobic sides of the four monomeric units

0

E

"0

U

o

,'-, - 1 0 × i

~

-20

Fig. 5. The CD spectra of mastoparan (0.2 mM) in 0.01 M Tris-HC1 buffer (pH 7.2, 23°C) in the presence of Na2SO4 at 5 mM (. . . . . . ), 115 mM (. . . . ), 250 mM ( - - - - - ) and 500 mM ( ).

161 form the aggregate, while the positively charged groups of lysine residues (and N-terminal a - N H ~ groups) are exposed to the aqueous phase. In fact, for m a s t o p a r a n (2 m M ) in aqueous solution ( p H 7.2) with 0.5 M N a 2 S O 4, the [Cr(CN)6]3--induced relaxation rate of the e-CH 2 p r o t o n resonance (2.99 ppm) of lysine residues is higher than that of the C H 3 p r o t o n resonance (approx. 1.5 ppm) of alanine residues (data not shown). Such a tetramer of m a s t o p a r a n will be stabilized b y the presence of salt at high concentration, which effectively reduces the electrostatic repulsion a m o n g the charged groups as exposed outside. This is consistent with the observation that the a-helix content of m a s t o p a r a n is increased by raising the p H from 6.0 to 8.3, and appreciable aggregation occurs at p H higher than 8.3.

Acknowledgement This work was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References 1 Hirai, Y., Yasuhara, T., Yoshida, H. and Nakajima, T. (1978) in Peptide Chemistry 1977 (Shiba, T., ed.), pp. 155-160, Protein Research Foundation, Osaka

2 Hirai, Y., Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M. and Kitada, C. (1979) Chem. Pharm. Bull. 27, 1942-1944 3 Higashijima, T., Wakamatsu, K., Takemitsu, M., Fujino, M., Nakajima, T. and Miyazawa, T. (1983) FEBS Lett. 152, 227-230 4 Wakamatsu, K., Higashijima, T., Fujino, M., Nakajima, T. and Miyazawa, T. (1983) FEBS Lett. 162, 123-126 5 Wakamatsu, K., Higashijima, T., Nakajima, T., Fujino, M. and Miyazawa, T. (1983) in Peptide Chemistry 1982 (Sakakibara, S., ed.), pp. 323-326, Protein Research Foundation, Osaka 6 Habermann, E. (1972) Science 177, 314-322 7 Talbot, J.C., Dufourcq, J., de Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191-193 8 Lauterwein, J., Brown, L.R. and Wiithrich, K. (1980) Biochim. Biophys. Acta 622, 219-230 9 Brown, L.R., Lauterwein, J. and Wiithrich, K. (1980) Biochim. Biophys. Acta 622, 231-244 10 Tatham, A.S., Hider, R.C. and Drake, A.F. (1983) Biochem. J. 211, 683-686 11 Shioya, Y., Yoshida, H. and Nakajima, T. (1982) J. Chromatogr. 240, 341-348 12 Saito, K., Higashijima, T., Miyazawa, T., Wakimasu, M. and Fujino, M. (1984) Chem. Pharm. Bull., Vol. 32, 2187-2193 13 Noggle, J.H. and Schirmer, R.E. (1971) The Nuclear Overhauser Effect: Chemical Applications, Academic Press, New York