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High-resolution 1H-NMR studies of self-aggregation of melittin in aqueous solution
231
Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 2 3 1 - - 2 4 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38405
H I G H - R E S O L U T I O N IH-NMR STUDIES OF S E L F - A G G R E G A T I O N OF MELITTIN IN A Q U E O U S SOLUTION
L A R R Y R. B R O W N , J U R G E N L A U T E R W E I N * a n d K U R T W O T H R I C H
Institut fiir Molekularbiologie und Biophysik, EidgeniSssische Technische Hochschule, 8093 Ziirich-H6nggerberg (Switzerland) ( R e c e i v e d A u g u s t 31st, 1 9 7 9 )
Key words: Melittin; Polypeptide conformation; Aggregation; 1H-NMR; (.4. mellifera)
Summary 4 mM melittin solution in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C was shown by ultracentrifugation to contain tetrameric melittin. Using the spectra of this species and the previously characterized monomeric melittin as references, high-resolution ~H-NMR at 360 MHz was used to investigate selfaggregation of melittin at variable temperatures, pH and ionic strength. The NMR parameters show that the spatial structure of aggregated melittin is different from monomeric melittin in aqueous solution but resembles closely the conformation adopted by melittin bound to detergent micelles. Comparison of melittin b o u n d to different detergent miceUes and self-aggregated melittin in different aqueous media indicates that the melittin monomers adopt similar conformations in all these systems. The present data suggest that melittin assumes an amphiphilic spatial structure which is stabilized both b y the formation of mixed micelles with detergents or b y self-aggregation.
Introduction
We have used high-resolution 'H-NMR to study conformational features of melittin b o u n d to a variety of detergent and phospholipid micelles [2,3]. To provide reference data for interpreting the influence of peptide-lipid interactions on conformational features of micelle-bound melittin, the accompanying paper [ 1] presents a ~H-NMR study of monomeric melittin in aqueous solution. With the present paper we attempt to further clarify the relationships between the conformations of monomeric, self-aggregated and micelle-bound melittin * Present address: Institut de Chimie Organique, Universitd de Lausanne, 2, Rue de la Barze, Lausanne, Switzerland.
232
[ 1--6]. In addition, the influence of a variety of solution conditions on aggregation of melittin and on conformational features of fully aggregated melittin have been studied in order to compare the nature of the peptide-peptide interactions in tetrameric melittin with the nature of the peptide-lipid interactions in mixed detergent-melittin micelles. Materials and Methods The preparation of melittin and the techniques used to record 360 MHz 1H-NMR spectra have been described in the accompanying paper [1] and elsewhere [8--12]. Ultracentrifugation experiments were performed on a Beckman LB-70 ultracentrifuge using Schlieren optics. Quasi-elastic light-scattering measurements utilized an argon laser (Spectra Physics) at 514.5 nm and a 96 channel correlator (Malvon). The autocorrelation function was fit with a single exponential function [ 7 ]. Results
Solution conditions which lead to self-association o f melittin Equilibrium ultracentrifugation experiments with 3 mM melittin * in 0.05 M sodium phosphate buffer at pH 7.0 and 20°C yielded an apparent molecular weight of 11 800 +- 600. Comparison with the molecular weight of 2840 calculated from the amino acid sequence [4] indicates that a tetrameric aggregate is most consistent with the observed molecular weight. This agrees with previous ultracentrifugation [ 5 ] and gel filtration [ 4,6 ] experiments indicating that at neutral or basic pH in the presence of various buffer salts, melittin probably forms tetrameric aggregates although pentamers could not be rigorously excluded [4--6]. In the accompanying paper we have shown that at acidic pH, low ionic strengths and melittiu concentrations up to 4 mM, melittin exists as a m o n o m e r [1]. Fig. l a shows a 360 MHz 1H-NMR spectrum of 4 mM melittin in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C. Under these conditions, the melittin IH-NMR spectrum was identical for melittin concentrations between 0.8 mM and 4.0 mM, indicating that melittin forms a tetrameric aggregate over this concentration range. In the accompanying paper [1], we have characterized the 1H-NMR spectrum of monomeric mehttin in considerable detail and have shown that monomeric melittin has a predominantly flexible, extended conformation. The 'H-NMR spectrum of tetrameric melittin (Fig. 1) is very different from the spectrum observed for monomeric melittin [1], which indicates that major conformational changes accompany self-association of mehttin. In the following, the differences between the 1H-NMR spectra of monomeric and tetrameric mehttin are used to investigate the influence of pH, ionic strength, melittin concentration and temperature upon the self-association of melittin and upon conformational features of aggregated melittin. As
* I n all t h e m e l i t t i n s a m p l e s u s e d in t h i s p a p e r t h e m i n o r f o r m o f m e l i t t i n a m i n o t e r m i n u s f o r m y l a t e d , h a d b e e n r e m o v e d (see Ref. 1).
found in bee venom,
with the
233
II
a
_ . . _
I 9
I 8
_
I 7
_
.
_
t ,5
I 4
I 3
I 2
I 1
I 0
PPM Fig. 1. 3 6 0 M H z 1 H - N M R s p e c t r a o f s e l f - a g g r e g a t e d m e l i t t i n . (a) T e t r a m e r i c m e l i t t i n in 4 m M m e l i t t i n s o l u t i o n in 2 H 2 0 c o n t a i n i n g 5 0 m M s o d i u m p h o s p h a t e b u f f e r a t p 2 H 7 . 0 a n d 3 0 ° C . All e x c h a n g e a b l e p r o t o n s w e r e p r e - e x c h a n g e d w i t h s o l v e n t d e u t e r o n s . (b) S p e c t r a l r e g i o n f r o m 6 t o 9 p p m o f a g g r e g a t e d m e l i t t i n in 2 H 2 0 r e c o r d e d in a 4 m M m e l i t t i n s o l u t i o n c o n t a i n i n g 1 . 5 M N a C l a t p 2 H 3 . 8 a n d 3 0 ° C 3 0 r a i n a f t e r d i s s o l v i n g t h e p e p t i d e in 2 H 2 0 . In a d d i t i o n t o t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f T r p - 1 9 several s l o w l y e x c h a n g i n g a m i d e p r o t o n s a r e o b s e r v e d . (c) S p e c t r a l r e g i o n f~om 6 t o 9 p p m o f a g g r e g a t e d m e l i t t i n in H 2 0 r e c o r d e d w i t h 6 m M m e l i t t i n in 9 0 % H 2 0 / 1 0 % 2 H 2 0 c o n t a i n i n g 1 . 5 M N a C l a t p H 3 . 8 a n d 3 0 ° C . T h e s p e c t r u m s h o w s t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f "£rp-19 a n d t h e labile p r o t o n s . T h e c o n d i t i o n s f o r e x p e r i m e n t s (b) a n d (c), i.e. 1 . 5 M NaC1, p H 3 . 8 , a n d 3 0 ° C , w e r e c h o s e n i n o r d e r t o o b t a i n slow exchange of amide NH protons [14]. The number of melittin molecules per aggregate has not been d e t e r m i n e d u n d e r t h e s e c o n d i t i o n s , b u t it a p p e a r s t h a t t h e p r e v a i l i n g s p e c i e s is t e t r a m e r i c m e l i t t i n (see text).
will be described in more detail below, the ~H-NMR spectra indicated that melittin is fully aggregated for the following conditions: (i) 4 mM melittin in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C; (ii) 4 mM melittin in 1.5 M NaC1 solution at p2H 3.0 and 30°C; (iii) 6 mM melittin in salt-free solution at p2H 9.0 and 30°C; (iv) 40 mM melittin in salt-free solution at p2H 3.4 and 30°C, and (v) 20 mM melittin in 0.15 M sodium phosphate buffer at p2H 7.0 between 30 and 90°C. Resonance assignments in the ~H-NMR spectrum o f tetrameric melittin Chemical shifts and spin-spin coupling constants for tetrameric melittin under the solution conditions of Fig. l a are shown in Table I. In the following the experiments used for the assignment of the resonances listed in Table I are described. These assignments were considerably simplified by the observation that each m o n o m e r contained in the tetrameric species appeared to give the same 1H-NMR spectrum. In Fig. l a this spectroscopic equivalence is most clearly seen for the aromatic region at 7--8 ppm, which contains a single set of resonances for the indole ring o f Trp-19. For melittin concentrations b e t w e e n 0.4 and 4.0 mM, the 1H-NMR spectrum of melittin in 50 mM sodium phosphate buffer at p2H 7.0 and at 60 or 80°C indicated that melittin dissociated at higher temperatures. This allowed correlations between monomeric and tetrameric melittin to be obtained by the following procedure. Resonances from tetrameric melittin in 50 mM sodium
234 TABLE I RESONANCE ASSIGNMENTS AND 1H-NMR PARAMETERS
FOR TETRAMERIC
MELITTIN
M e a s u r e d w i t h 4 m M m e l i t t i n in 5 0 m M s o d i u m p h o s p h a t e b u f f e r in 2 H 2 0 a t p 2 H 7 . 0 a n d 3 0 ° C . C h e m ical shifts (5) are relative t o s o d i u m 3 - t r i m e t h y l s i l y l - [ 2 , 2 , 3 , 3 , - 2 H ] p r o p i o n a t e a t p 2 H 7.0 [ 8 ] . V a l u e s in parenthesis correspond to chemical shifts where irradiation caused decoupling of resolved multiplets. A m i n o acid residue
Spin-spin coupling constants (+0.3 Hz)
6 (ppm) aCH
flCH
A l a I (4 o r 1 5 ) A l a II (4 o r 1 5 ) V a l I (5 o r 8)
4.146 4,306 3.636
1.487 1.576 (2.19)
V a l II (5 o r 8)
3.813
(2.25)
3.515 3.505
(2.02) (1.90) 4.261 4.351 3.450 3.679
ne-2 Ile I (17 Ile II ( 1 7 Thr I (10 T h r II ( 1 0 Trp-19
or or or or
20) 20) 11) 11)
L y s I ( 7 , 21 o r 2 3 ) L y s II (7, 21 o r 2 3 ) L y s III ( 7 , 21 o r 2 3 ) ArgI (22 or 24) Arg n (22 or 24) Gln-25 and -26
Others
~'CH 3
1.020 0.875 ~/CH 3 1 . 1 3 7 0.971 7CH 3 0.54 5CH 3 0.30 7CH 3 0.950 ~CH 3 0.827 7CH 3 1.298 7CH 3 1.316 C2H 7.422 C4H 7.541 C5H 7.046 C6H 7.000 C7H 7.455 NH 10.318 eCH2 a 2.624 2.37 b eCH 2 2.894 eCH 2 2.957 5CH 3 3.197 6CH 2 2.94 6CH2 2.43 b
3 Jc~fl
Others
7.3 7.3 9.3
3jfl,y 6 . 4
8.8
3J~37 6 . 4
3J87r 6.1 3Jf17 6.1
a For Lys I the two e-methylene protons were not equivalent. b At 30°C the resonance of one eCH 2 proteon of Lys I and the 6CH 2 resonances of the two glutamines give rise t o a g r o u p o f o v e r l a p p e d lines (Fig. 1). T h e a p p r o x i m a t e c h e m i c a l s h i f t s given w e r e o b t a i n e d f r o m Fig. 1 a n d f r o m o b s e r v a t i o n s a t h i g h e r t e m p e r a t u r e .
phosphate buffer at p2H 7.0 and 30°C were followed first at variable temperatures up to 85°C and then at variable phosphate concentration between 50 and 0 mM at 85°C and p2H 7.0 (Fig. 2). At p2H 7.0 and 85°C in the absence of phosphate, 4 mM melittin was monomeric [1]. By using these conditions of p2H, temperature and phosphate concentration, conditions were obtained where exchange between monomeric and aggregated melittin was rapid on the NMR time scale over the entire range of melittin association. Fig. 2 shows plots of the chemical shift versus solution condition obtained for the alanine, threonine, valine and isoleucine residues. To obtain more reliable correlations for a larger number of lines, the complete spin systems for the individual residues were identified b y spin-decoupling difference spectroscopy [11] at the different solution conditions. While assignments of the spin systems to particular positions in the amino acid sequence were obtained for only two residues (Table I), correspondences between spin systems in monomeric and tetrameric
235
[P04]" m M
5o2s6
PPM
'
a
i
'
i
b
4.5f-
4.0
S
I
3.5
I I
50 25 0
50 25 0
50 25 0
e
C
,
I
'
•
i
,
.
i
d
i I
I J I
2.0 I I I i
1.5
i
I
I
I
1.O
i , hi , e l 25
55
85
I I
I
i
i
I I
0.5
I r
I , ii
Iill,ll
25
55
85
25
TEMPERATURE
55
r I,
i i I
25
85
,I,
55
,I
85
°C
Fig. 2. C o r r e l a t i o n o f c o r r e s p o n d i n g r e s o n a n c e s in m o n o m e r i c a n d t e t r a m e r i c m e l i t t i n . T h e c h e m i c a l shifts o f s e l e c t e d r e s o n a n c e s o f 4 raM r a e l i t t i n in 50 m M s o d i u m p h o s p h a t e b u f f e r at p 2 H 7.0 a n d 3 0 ° C ( t e t r a m e r i c m e l i t t i n ) w e r e f o l l o w e d as a f u n c t i o n o f t e m p e r a t u r e u p t o 8 5 ° C ( l o w e r scale) a n d t h e n as a f u n c t i o n o f p h o s p h a t e c o n c e n t r a t i o n at 85 ° C ( u p p e r scale). A t 85 ° C a n d p 2 H 7 . 0 in t h e a b s e n c e o f p h o s p h a t e , 4 raM r a e l i t t i n w a s fully m o n o r a e r i c [ 1 ] . T h e a s ~ g n r a e n t s o f t h e r e s o n a n c e s are: (a) 0, [] a n d m c~CH,~3CH a n d "),CH3 of Val I (5 or 8); A ~ a n d v , ~ C H , ~ 3 C H a n d ~ , C H 3 o f V a l I I ( 5 o r 8 ) . ( b ) s, [ ] a n d 0, ~CH, ~CH a n d 7 C H 3 o f U e - 2 ; A Lx a n d v ~ C H , ~3CH a n d 7 C H 3 o f I l e I ( 1 7 o r 2 0 ) ; o o a n d o a C H , J3CH a n d 9"CH3 o f Ile II (17 o r 20). (c) A a n d V ~xCH,~CH 3 of A l a I (4 o r 1 5 ) ; m O, 0~CH.~CH 3 o f A l a I I (4 o r 15). (d) m 0, ~3CH, 7 C H 3 o f T h r I ( 1 0 o r 11); A v ~3CH, 7 C H 3 o f T h r II ( 1 0 o r 11). T h e riCH r e s o n a n c e s o f t h e v a l i n e a n d i s o l e u c i n e r e s i d u e s w e r e o b s e r v e d i n d i r e c t l y as t h e f r e q u e n c y at w h i c h t h e ~ a n d T-protons were decoupled.
melittin could thus be established for all the resonances in Table I. Truncated~Iriven nuclear Overhauser enhancement difference spectra [12] obtained with preirradiation at 0.3 ppm indicated that the two methyl groups observed at 0.30 and 0.54 ppm in tetrameric melittin were located near each other and near the indole ring of Trp-19. Since proximity to Trp-19 may, depending on the location in the amino acid sequence of the residue in question, impose rather stringent constraints on possible spatial structures of the melttin tetramer, individual assignments for the two high-field methyl lines were of particular interest. At 65°C, where these lines were at 0.58 and 0.71 ppm, the multiplet structures resolved after resolution enhancement were a triplet and a doublet. At 65°C the doublet was decoupled by irradiation at 1.70 ppm, and at 85°C and low phosphate concentration it was found to be a component of a spin system identical to that assigned to Ile-2 in monomeric melittin [1]. Because of the above-mentioned evidence for close proximity of the two m e t h y l groups observed at high field and since the two high-field lines showed nearly identical temperature dependences, the triplet resonance was tentatively assigned to 5CH3 of Ile-2. The assignment of the spin system of Ile-2
236
was further supported by experiments with 6 mM melittin in 9 mM sodium phosphate buffer at p:H 7.0 and 15°C. Fig. 3a shows that under these conditions the exchange between monomeric and aggregated melittin is slow and the NMR spectrum is a superposition of the spectra of monomeric and tetrameric melittin, with approx. 30% of the observed intensity corresponding to tetrameric melittin. Saturation transfer experiments established correlations between the line at 0.58 ppm and a doublet at 0.96 ppm, and between the line at 0.30 ppm and a triplet at 0.89 ppm (Fig. 3b and c). In corresponding experiments with fully tetrameric melittin the doublet resonance at 0.96 ppm and the triplet resonance at 0.89 ppm were not observed, which confirms that these
i=
I
I
b
t cs
d PPM
2
1
0
Fig. 3. 3 6 0 M H z 1 H - N M R spectra showing double-resonance experiments used to correlate the resonances o f the m e t h y l groups o f isoteucine 2 in m o n o m e r i e and tetrameric m e l i t t i n . The arrows denote the frequencies at w h i c h double-resonance i r r a d i a t i o n was applied. S, i r r a d i a t i o n applied p r i o r to data acquisition in o r d e r to s a t u r a t e t h e i r r a d i a t e d r e s o n a n c e . D, d e c o u p l i n g i r r a d i a t i o n a p p l i e d d u r i n g a c q u i s i t i o n . (a) H i g h - f i e l d r e g i o n o f t h e 1 H . N M R s p e c t r u m o f 6 m M m e l i t t i n in 9 m M s o d i u m p h o s p h a t e b u f f e r at p 2 H 7.0 a n d 1 5 ° C . T h e o b s e r v e d s p e c t r u m is a s u p e r p o s i t i o n o f t h e s p e c t r a o f m o n o m e r i c a n d t e t r a m e r i c melittin, with approx. 30% of the total intensity c o r r e s p o n d i n g to tetrameric melittin. The inset shows t h e 0 - - 0 . 7 p p m r e g i o n w i t h a 5-fold i n c r e a s e d v e r t i c a l scale. ( b ) D i f f e r e n c e b e t w e e n s p e c t r a t a k e n w i t h a n d w i t h o u t p r e s a t u r a t i o n o f t h e b r o a d m e t h y l r e s o n a n c e o f t e t r a m e r i c m e l i t t i n at 0 . 3 0 p p m . (c) D i f f e r ence b e t w e e n spectra t a k e n with and w i t h o u t presaturation of the broad m e t h y l resonance from tetram e r i c m e l t t i n at 0 . 5 8 p p m . ( d ) D i f f e r e n c e b e t w e e n s p e c t r a t a k e n w i t h a n d w i t h o u t p r e s a t u r a t i o n o f t h e m e t h y l r e s o n a n c e f r o m t e t r a r n e r i c m e l i t t i n at 0 . 5 8 p p m , w i t h s p i n d e c o u p l i n g at 1 . 9 2 p p m .
237 resonances correspond to monomeric melittin. The doublet at 0.96 p p m coincides with the previously identified doublet of Ile-2 in monomeric melittin [1] and was decoupled b y irradiation at the chemical shift previously determined for riCH o f Ile-2 (Fig. 3d). The experiments of Fig. 4 further resulted in the tentative jassignment of the triplet resonance at 0.89 p p m to ~CH3 of Ile-2 in monomeric melittin.
Effects o f p H and salt concentration on self-association o f melittin Table II lists 1H-NMR chemical shifts for numerous amino acid residues of melittin in aggregates formed in different aqueous solvents. That similar line widths for the NMR resonances were observed under all conditions indicates that aggregates containing a small number of melittin molecules prevail in all the preparations in Table II. This was independently supported by gel filtration
2
c
2
65
I 8
I
I 7
PPM
Fig. 4. 3 6 0 M H z 1 H . N M R s p e c t r a s h o w i n g t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f T r p - 1 9 f o r 4 m M m e l i t t i n a t p 2 H 2 . 8 a n d 3 0 ° C as a f u n c t i o n o f NaC1 c o n c e n t r a t i o n . (a) s a l t free. (b) 0 . 0 8 M NaCI. (c) 0 . 2 M NaC1. (d) 1 . 2 M NaC1. T h e n u m b e r s i d e n t i f y i n d i v i d u a l i n d o l e r i n g p r o t o n lines. M e l i t t i n is m o n o m e r i c in (a) a n d f u l l y a g g r e g a t e d in (d)0 I n (b) a n d (c) m e l i t t i n is p a r t i a l l y a g g r e g a t e d a n d t h e o b s e r v e d s p e c t r a are s u p e r positions of resonances from monomeric and aggregated melittin.
C5H C6H C2H C7H C4H
eCH2 d
eCH 2 eCH 2 5CH 2 5CH 2 7CH~
T r p - 1 9 ring
L y s I (7, 21 or 23)
L y s II (7, 21 or 23) L y s I I I (7, 21 o r 23) Arg I (22 or 24) A r g II ( 2 2 or 24) G i n - 2 5 a n d -26
(2.62) (2.37) (2.89) (2.96) 3.197 (2.94) (2.43)
7.045 6.981 7.421 7.454 7.540
1.488 1.583 1.020 1.137 1.298 1.316 0.297
4 mM melittin, a 50 m M p h o s p h a t e p 2 H 7.0, 3 0 ° C
(2.43)
(2.90) 2.979 3.223
(2.62)
(7.02) (7.02) 7.438 7.458 7.533
1.507 1.572 1.017 1.128 1.307 1.307 0.334
4 mM melittin, b 1.5 M NaC1, p 2 H 3.0, 3 0 ° C
Self-aggregated melittin
6
(2.75) (2.64) (2.94) 2.999 3.200 (3.15) (2.45)
7.046 7.152 7.357 7.507 7.608
1.507 1.527 1.031 1.147 1.324 1.337 (0.40)
6 mM melittin, b n o salt, p 2 H 9.0, 3 0 ° C
(2.75) (2.60) (2.95) 3.005 3.216 3.070 (2.44)
7.048 7.086 7.420 7.491 7.585
1.522 1.588 1.040 1.162 1.334 1.334 0.421
40 mM melittin, b n o salt, p 2 H 3.4, 30OC
2.978 2.998 3.135 3.196 2.396
2.962
7.158 7.264 7.270 7.514 7.619
1.379 1.389 0.958 0.958 1.223 1.238 0.904
M o n o m e r i c melittin [1] 3 raM m e l i t t i n , n o salt, p 2 H 3.3, 3 0 ° C
0.13 0.27 0.06 0.05 0.03 0.21 0.02
0.03 0.17 0.08 0.05 0.08
0.03 0.06 0.02 0.03 0.04 0.03 0.12
ASA
--0.28 --0.43 --0.06 ---0.01 0.07 ---0.14 0.04
---0.12 --0.20 0.14 ---0.04 --0.05
0.13 0.18 0.07 0.19 0.09 0.09 --0.46
h A - - ~M
--0.13 --0.23 --0.04 --0.02 0.06 --0.04 0.02
--0.24 --O.18 0.09 --0.02 --0.03
0.18 0.18 0.18 0.23 0.12 0.12
~Mic
a F o r these c o n d i t i o n s m e ] i t t i n w a s s h o w n t o b e t e t r a m e r i c (see t e x t ) . b w h i l e c o r r e s p o n d e n c e s b e t w e e n i n d i v i d u a l spin s y s t e m s in m o n o m e r i c m e l i t t i n a n d in t h e species a w e r e e s t a b l i s h e d (see t e x t ) , o n l y c o r r e s p o n d e n c e s b e t w e e n t y p e s o f a m i n o acid residues w e r e d e t e r m i n e d f o r t h e o t h e r t h r e e s o l u t i o n c o n d i t i o n s . W h e n t h e r e w a s m o r e t h a n o n e a m i n o acid of a g i v e n t y p e t h e i n d i v i d u a l spin s y s t e m s w e r e c o r r e l a t e d so as to give g r e a t e s t s i m i l a r i t y in t h e s p e c t r a l p r o p e r t i e s for d i f f e r e n t s o l v e n t c o n d i t i o n s . c Only o n e 7 C H 3 of e a c h valine was r e s o l v e d in t h e s p e c t r a o f a g g r e g a t e d m e l i t t i n . d "rwn n n e - n r o t o n r e s o n a n c e s w e r e o b s e r v e d f o r t h e L y s I e C H 2 g r o u p .
~CH 3 ~CH 3 7CH3 c ~/CH3 c ~CH 3 "yCH3 5CH 3
Ala I (4 o r 15) Ala I I (4 or 15) Val I (5 o r 8) Val I I (5 or 8) T h r I ( 1 0 or 11) T h r II ( 1 0 or 11) ne-2
Resonance assignment
5 are t h e c h e m i c a l shifts relative to t h e r e f e r e n c e c o m p o u n d s o d i u m 3 - t r i m e t h y l s i l y l - [ 2 , 2 , 3 , 3 - 2 H ] p r o p i o n a t e at p 2 H 7.0 ( T o a c c o u n t f o r t h e t i t r a t i o n shift o f this r e f e r e n c e b e t w e e n p 2 H 3.0 a n d p 2 H 7.0 [ 8 ] , 0 . 0 1 9 p p m has b e e n a d d e d to t h e e x p e r i m e n t a l l y o b s e r v e d c h e m i c a l shifts f o r 4 m M m e l i t t i n in 1.5 M NaC1 a t p 2 H 3 . 0 a n d f o r 3 r a m m e l i t t i n at p 2 H 3.3 in t h e a b s e n c e of salt. C h e m i c a l shifts g i v e n in p a r e n t h e s i s are less a c c u r a t e , since t h e c o r r e s p o n d i n g r e s o n a n c e s w e r e b r o a d . ) A~ A is the m a x i m u m d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a of a g g r e g a t e d m e l i t t i n u n d e r t h e f o u r d i f f e r e n t s o l u t i o n c o n d i t i o n s c o n t a i n e d in this t a b l e . 6 A - 5 M is t h e a v e r a g e d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a o f a g g r e g a t e d m e l i t t i n u n d e r t h e f o u r d i f f e r e n t s o l u t i o n c o n d i t i o n s a n d m o n o m e r i c m e l i t t i n . 5M ic - - 5M is t h e a v e r a g e d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a o f m e l i t t i n b o u n d t o five d i f f e r e n t t y p e s o f d e t e r g e n t m i c e n e s [ 2 ] a n d m o n o m e r i c melittin.
I N F L U E N C E OF S O L U T I O N C O N D I T I O N S ON T H E 1H-NMR C H E M I C A L SHIFTS OF A G G R E G A T E D M E L I T T I N
T A B L E II b0 f~ 00
239 experiments for high NaC1 concentrations [13] and by light-scattering studies of salt-free solutions. Table III summarizes the melittin and/or salt concentrations which were necessary to induce aggregation of melittin. Several interesting facets of melittin aggregation were revealed by these experiments. Firstly, for salt-free solution conditions at both p2H 3.4 and p2H 9.0, greater than 90% aggregation was achieved for about a 5-fold change in melittin concentration. This is not consistent with an equilibrium between m o n o m e r and aggregates containing a small number of molecules, such as a tetramer, unless a high degree of cooperativity is involved in melittin association. Since a small number of melittin molecules are involved in the aggregates discussed in Table II, the concentration dependence gives clear indication that self-association of melittin in salt-free solution involves cooperativity between individual melittin molecules. Table III also shows that under salt-free conditions, much higher melittin concentrations were necessary to achieve aggregation at p~H 3.4 as compared to p~H 9.0. p2H titration of 6 mM melittin in salt-free solution at 30°C showed that in aggregated melittin, as previously shown for monomeric melittin [1], the a-amino group of Gly-1 was the only ionizable group which titrated between p2H 3.0 and p2H 9.0. The present results on aggregation of melittin at p2H 3.4 and at p2H 9.0 (Table III) thus show that removal of the positive charge on the a-amino group of Gly-1 by deprotonation greatly enhances the tendency to aggregate. This is consistent with our previous observation that at acidic pH the minar c o m p o n e n t of melittin, where the positive charge on the a-amino group of Gly-1 is replaced by an uncharged formyl group, shows a more pronounced tendency to aggregate than the major c o m p o n e n t of melittin
[11. When monomeric mehttin was titrated with either NaCI or sodium phosphate to induce aggregation, slow exchange on the NMR time scale between
TABLE
nI
INFLUENCE
OF SOLUTION
CONDITIONS
ON AGGREGATION
OF MELITTIN
D e p e n d i n g o n w h e t h e r fast o r s l o w e x c h a n g e o n t h e N M R t i m e s c a l e p r e v a i l e d , t h e d e g r e e o f a g g r e g a t i o n was determined either from the observed chemical shifts or from the relative intensities of the monomer and aggregate resonances, respectively [15]. Solution
conditions
p2H 9.0, 30°C, no salt p2H 3.4, 30°C, no salt
Melittin conSalt conceneentration (raM) tration (M) 0.7 2 6 3 10 35
Degree of aggregation
Exchange rate on the N M R t i m e scale
----
monomer ca. 50% aggregated ~90% aggregated
fast
----
monomer ca. 50% aggregated ~'90% aggregated
fast
p2H 2.8, 30°C NaCI
4
-0.06 ~1
monomer ca. 5 0 % a g g r e g a t e d fully aggregated
slow
p2H 7.0, 30°C, NaH2PO 4/Na2HPO4
4
-0.01 0.05
monomer ca. 50% aggregated fully aggregated
slow
240
monomeric and aggregated melittin was observed (Table III). Since a superposition of monomeric melittin resonances and aggregated melittin resonances could be observed for salt concentrations where melittin was partially aggregated (Fig. 4), these experiments led to the observation that the chemical shifts observed for monomeric melittin were independent of salt concentration whereas the chemical shifts observed for aggregated melittin varied at intermediate salt concentrations. This behaviour suggests that the conformation of monomeric melittin is essentially independent of salt concentration whereas for aggregated melittin either the aggregate conformation or the number of molecules per aggregate changes as a function of salt concentration.
Temperature dependence of the 1H-NMR parameters of aggregated melittin The temperature dependence of the ~H-NMR chemical shifts of a 20 mM melittin solution in 150 mM sodium phosphate buffer at p2H 7.0 was measured. Evidence that under these conditions no appreciable change in the degree of melittin aggregation occurred between 20 and 90°C was obtained from the observation that 20 mM and 28 mM melittin showed identical 1H-NMR spectra over this temperature range. Below approx. 40°C the spectrum was virtually identical to the spectrum of 4 mM melittin in 50 mM sodium phosphate buffer at p2H 7.0, i.e. the reference tetramer state, suggesting that melittin is probably also tetrameric at the higher concentrations of melittin and buffer. Above approx. 40°C large changes in chemical shift were observed (Fig. 5) for the resonances of one lysine, one arginine, Ile-2 and the protons of the indole ring of Trp-19. In addition, the two protons of the 7CH2 group of each of the two glutamines in melittin, which were inequivalent below approx.
PPM 74 7.o
3.3 29
C
~
=
-"
=
_
"-
~
°
D
~
E
* ~
F
G
25 17 1.3 0.9
L M
0.5 ]
20
i
i
~0
i
i
t
I
60
TEMPERATURE
=
80
°C
Fig. 5. P l o t s o f t h e c h e m i c a l s h i f t s o b s e r v e d f o r s e l e c t e d r e s o n a n c e s o f 2 0 m M p h o s p h a t e b u f f e r a t p 2 H 7.0 as a f u n c t i o n o f t e m p e r a t u r e . T h e a s s i g n m e n t s T r p - 1 9 i n d o l e ring; F, A r g - 2 2 a n d -24 6 C H 2 ; G, Lys-7, -21 a n d -23 e C H 2 ; Ala-4 a n d -15 ~ C H 3 ; J , T h r - 1 0 a n d -11 7 C H 3 ; K, Val-5 a n d -8 7 C H 3 ( o n l y e a c h r e s i d u e w a s i d e n t i f i e d ) ; L a n d M, I l e - 2 7 C H 3 a n d 6 C H 3.
melittin in 150 mm sodium o f t h e r e s o n a n c e s are: A - - E ,
H, G l n - 2 5 a n d -26 ~/CH2; I, one methyl
resonance
from
241 40°C, indicating slow rotation about the C~--C~ bond, became equivalent above 40°C. At 90°C the NMR parameters of the lysine, arginine and glutamine residues were very similar to the parameters observed for monomeric melittin, whereas the NMR parameters of the alanine, threonine and valine residues were similar to the parameters observed for tetrameric melittin at 30°C. Thus qualitatively different behaviour is observed for residues located in the hydrophobic C-terminal or the hydrophilic N-terminal region of the melittin sequence. The chemical shifts of the protons of the indole ring of Trp-19 showed a different behaviour yet, i.e. at 90°C t h e y did not coincide with shifts of either the m o n o m e r or tetramer. Discussion The 1H-NMR spectrum of aggregated melittin in H20 solution (Fig. 1) revealed that a number of the labile protons have chemical shifts which differ appreciably from the chemical shifts of the labile protons in both monomeric melittin [1] and the model peptides H-Gly-Gly-X-Ala-OH [14]. Furthermore, in aggregated melittin approx. 12 labile protons per m o n o m e r were found to be stabilized against exchange with solvent deuterons (Fig. 1). Under the conditions of Fig. l b and c, half-times with respect to exchange of several hours were measured for these protons implying that, in contrast to monomeric melittin [1], aggregated melittin assumes a globular conformation where a sizeable portion of the backbone amide protons are shielded from solvent contact [15]. Since, in addition, a variety of 1H-NMR resonances from amino acid residues distributed t h r o u g h o u t the amino acid sequence were found to have the same chemical shifts for all four polypeptide chains in tetrameric melittin (Table I), it seems that the apparent s y m m e t r y of tetrameric melittin is based on the propensity of the melittin polypeptide chain to assume a particular nonrandom conformation. This is extremely interesting in view of our previous results indicating that melittin also assumes a well-defined, non-random conformation when bound to various types of detergent and phospholipid micelles [2]. In the following the ~H-NMR data on tetrameric and micelle-bound [2,3] melittin are used to compare conformational features of melittin in these two environments and to investigate the nature of melittin-melittin interactions in the aggregates and melittin
242 caused variations of the chemical shifts comparable to those observed upon aggregation (Table II). For the resonances of the indole ring of Trp-19, one of lysines, 7 21 or 23 and one of arginines 22 and 24, a third type of behaviour was observed. These resonances showed both appreciable changes in chemical shift to either high or low field upon aggregation of melittin and appreciable differences in the chemical shifts observed for aggregated melittin under various solution conditions (Table II). Trp-19, and possibly all three of these residues, are near the boundary between the hydrophobic and hydrophilic regions of the melittin amino acid sequence. It seems likely that the NMR parameters observed for these residues reflect changes in population of a limited number of conformation states in tetrameric melittin. The following qualitative picture of aggregated melittin seems to emerge from these observations. The hydrophobic N-terminal region of melittin assumes a definite conformation which includes intra- and/or interchain hydrogen bonding and which, by association, forms a hydrophobic core of the aggregate. It is presumably the spatial folding of this part of the polypeptide chain which gives rise to circular dichroism reminiscent of u-helical structure [2,16]. The hydrophilic C-terminal region of the melittin amino acid sequence lies at the surface of the aggregate and its spatial arrangement is highly sensitive to solvent interactions. These conclusions are supported by the temperature dependence studies of aggregated melittin. In the hydrophilic C-terminal region of melittin, changes in chemical shifts as a function of temperature were comparable to the chemical shift changes upon aggregation, whereas in the hydrophobic N-terminal region, the changes in chemical shift as a function of temperature were small compared to the chemical shift changes upon aggregation. Overall this picture suggests that the melittin monomer-aggregate equilibrium represents a balance between folding of the polypeptide chain into a spatial structure which is stabilized by self-aggregation, attractive interactions between the hydrophobic regions of this structure and electrostatic repulsion between the highly positively charged hydrophilic regions. This is supported by the observations that either reducing the charge on melittin by deprotonation of Gly-1 or shielding of the charges by addition of salt favours aggregation. Even though ultracentrifugation and quasi-elastic light-scattering experiments indicated that melittin binds as a m o n o m e r to dodecylphosphocholine miceUes and NMR experiments suggested that melittin is also monomeric when bound to a variety of other types of detergent and phospholipid micelles [2], the circular dichroism spectra of tetrameric and miceUe-bound melittin were very similar and would be compatible with an a-helical conformation [2]. On the other hand, the IH-NMR spectra of tetrameric and micelle-bound melittin showed appreciable differences [2]. Despite the differences in the 1H-NMR spectra, detailed comparison of the present NMR results on aggregated melittin under variety of solution conditions with our previous NMR results on melittin bound to a variety of different types of micelles [2] suggests a considerable degree of conformational similarity. Thus, for all of the resonances in Table II except Ile-2, which was not assigned in micelle-bound melittin, the average change in chemical shift when monomeric melittin aggregates under a variety of solution conditions (6A - - ~ M in Table II) are in the same direction as the aver-
243 age changes in chemical shift when monomeric melittin binds to a variety of different micelles (~Mic- ~M in Table II). Furthermore, while the chemical shifts observed for micelle-bound melittin show some variation with the type of micelle, the pattern of the variations for different amino acid residues was very similar to the pattern noted above for the chemical shift variations observed for aggregated melittin under various solution conditions [2]. Finally, the temperature dependence of the chemical shifts of aggregated melittin (Fig. 5) and of melittin b o u n d to dodecylphosphocholine miceUes [3] is very similar. The outstanding difference between the 1H-NMR spectra of aggregated and micelle-bound melittin is that the large high-field shifts observed for the Ile-2 methyl resonances in aggregated melittin (Table I) were not observed for micelle-bound melittin [2,3]. These high-field shifts are due to intra- or intermolecular proximity of Ile-2 and Trp-19 in aggregated melittin and the observed chemical shifts are therefore expected to be highly sensitive to local conformational features. This expectation is confirmed by the observation that in aggregated melittin the high-field shifts of the Ile-2 methyl resonances are greatly reduced at higher temperatures (Fig. 5). Overall, considering that the resonances in Table II arise from amino acid side-chain protons and hence may be particularly sensitive to the differing intermolecular environments present in aggregated melittin (peptide-peptide contacts) and in micelle-bound melittin (peptide-detergent contacts), the parallels between the spectral features observed for aggregated and micelle-bound melittin are striking and suggestive of similar conformations for melittin in these two states. A highly consistent picture would emerge with the hypothesis that the chemical shift variations between monomeric and aggregated or micelle-bound melittin arise primarily from the secondary and tertiary structure of the m o n o m e r units, with the exception of the high-field shifts of Ile-2 in tetrameric melittin, which would be caused by the packing interactions in the aggregate. We have previously suggested that the interaction of melittin with detergent or phospholipid micelles might be described as the formation of mixed micelles by two different types of amphiphilic molecules, each of which can selfassociate to form miceUes at sufficiently high concentrations [2]. The present results appear to support this hypothesis since each monomeric unit of tetrameric melittin appears to assume an amphiphilic three-dimensional conformation and since the melittin monomers appear to assume very similar conformations in the tetrameric and micelle-bound forms. Furthermore, the observation that self-association of melittin involves formation of a hydrophobic core and a charged, hydrophilic exterior is reminiscent of the organization of detergent micelles. However, melittin appears to differ from detergent or phospholipid molecules in that a suitable amphiphilic character is achieved only b y folding of extensive parts of the polypeptide chain into a defined spatial structure. This is presumably the reason w h y aggregates of melittin, in contrast to detergent or phospholipid micelles, involve only a small number of melittin molecules. Work on these questions is being further pursued. Acknowledgement Financial support by the Swiss National Science Foundation (project No. 3.004-0.76) is gratefully acknowledged.
244
References 1 Lauterwein, J., Brown, L.R. and W/ithrich, K. (1980) Biochim. Biophys. Acta 622, 219--230 2 Lauterwein, J., B~sch, Ch., Brown, L.R. and Wfithrich, K. (1979) Biochim. Biophys. Acta 556, 244-264 3 Brown, L.R. (1979) Biochim. Biophys. Acta 557, 135--148 4 Habermann, E. (1972) Science 177, 314--322 5 Gauldre, J., Hanson, J.M., Rumjanek, F.D., Shipolini, R. and Vernon, C.A. (1976) Eur. J. Bioehem. 61,369--376 6 Faucon, J.F., Dufourcq, J. and Lussan, C. (1979) FEBS Lett. 102, 187--190 7 Chu, B. (1974) Laser Light Scattering, Academic Press, New York 8 De Marco, A. (1977). J. Magn. Resonance 26, 527--528 9 De Marco, A. and Wiithrich, K. (1976) J. Magn. Resonance 24, 201--204 10 Wagner, G., Wfithrich, K. and Tschesche, H. (1978) Eur. J. Biochem. 86, 67--76 11 De Marco, A., Tschesche, H., Wagner, G. and Wilthrich, K. (1977) Biophys. Struct. Mech. 3, 303--315 12 Wagner, G. and Wfithrich, K. (1979) J. Magn. Resonance 33, 675--680 13 Talbot, J.C., Dufourcq, J. De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191--193 14 Bundi, A. and Wiithrich, K. (1979) Biopolymers 18, 285--297 15 Wiithrich, K. (1976) NMR in Biological Research: Peptides and Proteins, North Holland, A ms t e rda m 16 Dawson, C.R., Drake, A.F., HeUiweU, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75--86
Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 2 3 1 - - 2 4 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38405
H I G H - R E S O L U T I O N IH-NMR STUDIES OF S E L F - A G G R E G A T I O N OF MELITTIN IN A Q U E O U S SOLUTION
L A R R Y R. B R O W N , J U R G E N L A U T E R W E I N * a n d K U R T W O T H R I C H
Institut fiir Molekularbiologie und Biophysik, EidgeniSssische Technische Hochschule, 8093 Ziirich-H6nggerberg (Switzerland) ( R e c e i v e d A u g u s t 31st, 1 9 7 9 )
Key words: Melittin; Polypeptide conformation; Aggregation; 1H-NMR; (.4. mellifera)
Summary 4 mM melittin solution in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C was shown by ultracentrifugation to contain tetrameric melittin. Using the spectra of this species and the previously characterized monomeric melittin as references, high-resolution ~H-NMR at 360 MHz was used to investigate selfaggregation of melittin at variable temperatures, pH and ionic strength. The NMR parameters show that the spatial structure of aggregated melittin is different from monomeric melittin in aqueous solution but resembles closely the conformation adopted by melittin bound to detergent micelles. Comparison of melittin b o u n d to different detergent miceUes and self-aggregated melittin in different aqueous media indicates that the melittin monomers adopt similar conformations in all these systems. The present data suggest that melittin assumes an amphiphilic spatial structure which is stabilized both b y the formation of mixed micelles with detergents or b y self-aggregation.
Introduction
We have used high-resolution 'H-NMR to study conformational features of melittin b o u n d to a variety of detergent and phospholipid micelles [2,3]. To provide reference data for interpreting the influence of peptide-lipid interactions on conformational features of micelle-bound melittin, the accompanying paper [ 1] presents a ~H-NMR study of monomeric melittin in aqueous solution. With the present paper we attempt to further clarify the relationships between the conformations of monomeric, self-aggregated and micelle-bound melittin * Present address: Institut de Chimie Organique, Universitd de Lausanne, 2, Rue de la Barze, Lausanne, Switzerland.
232
[ 1--6]. In addition, the influence of a variety of solution conditions on aggregation of melittin and on conformational features of fully aggregated melittin have been studied in order to compare the nature of the peptide-peptide interactions in tetrameric melittin with the nature of the peptide-lipid interactions in mixed detergent-melittin micelles. Materials and Methods The preparation of melittin and the techniques used to record 360 MHz 1H-NMR spectra have been described in the accompanying paper [1] and elsewhere [8--12]. Ultracentrifugation experiments were performed on a Beckman LB-70 ultracentrifuge using Schlieren optics. Quasi-elastic light-scattering measurements utilized an argon laser (Spectra Physics) at 514.5 nm and a 96 channel correlator (Malvon). The autocorrelation function was fit with a single exponential function [ 7 ]. Results
Solution conditions which lead to self-association o f melittin Equilibrium ultracentrifugation experiments with 3 mM melittin * in 0.05 M sodium phosphate buffer at pH 7.0 and 20°C yielded an apparent molecular weight of 11 800 +- 600. Comparison with the molecular weight of 2840 calculated from the amino acid sequence [4] indicates that a tetrameric aggregate is most consistent with the observed molecular weight. This agrees with previous ultracentrifugation [ 5 ] and gel filtration [ 4,6 ] experiments indicating that at neutral or basic pH in the presence of various buffer salts, melittin probably forms tetrameric aggregates although pentamers could not be rigorously excluded [4--6]. In the accompanying paper we have shown that at acidic pH, low ionic strengths and melittiu concentrations up to 4 mM, melittin exists as a m o n o m e r [1]. Fig. l a shows a 360 MHz 1H-NMR spectrum of 4 mM melittin in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C. Under these conditions, the melittin IH-NMR spectrum was identical for melittin concentrations between 0.8 mM and 4.0 mM, indicating that melittin forms a tetrameric aggregate over this concentration range. In the accompanying paper [1], we have characterized the 1H-NMR spectrum of monomeric mehttin in considerable detail and have shown that monomeric melittin has a predominantly flexible, extended conformation. The 'H-NMR spectrum of tetrameric melittin (Fig. 1) is very different from the spectrum observed for monomeric melittin [1], which indicates that major conformational changes accompany self-association of mehttin. In the following, the differences between the 1H-NMR spectra of monomeric and tetrameric mehttin are used to investigate the influence of pH, ionic strength, melittin concentration and temperature upon the self-association of melittin and upon conformational features of aggregated melittin. As
* I n all t h e m e l i t t i n s a m p l e s u s e d in t h i s p a p e r t h e m i n o r f o r m o f m e l i t t i n a m i n o t e r m i n u s f o r m y l a t e d , h a d b e e n r e m o v e d (see Ref. 1).
found in bee venom,
with the
233
II
a
_ . . _
I 9
I 8
_
I 7
_
.
_
t ,5
I 4
I 3
I 2
I 1
I 0
PPM Fig. 1. 3 6 0 M H z 1 H - N M R s p e c t r a o f s e l f - a g g r e g a t e d m e l i t t i n . (a) T e t r a m e r i c m e l i t t i n in 4 m M m e l i t t i n s o l u t i o n in 2 H 2 0 c o n t a i n i n g 5 0 m M s o d i u m p h o s p h a t e b u f f e r a t p 2 H 7 . 0 a n d 3 0 ° C . All e x c h a n g e a b l e p r o t o n s w e r e p r e - e x c h a n g e d w i t h s o l v e n t d e u t e r o n s . (b) S p e c t r a l r e g i o n f r o m 6 t o 9 p p m o f a g g r e g a t e d m e l i t t i n in 2 H 2 0 r e c o r d e d in a 4 m M m e l i t t i n s o l u t i o n c o n t a i n i n g 1 . 5 M N a C l a t p 2 H 3 . 8 a n d 3 0 ° C 3 0 r a i n a f t e r d i s s o l v i n g t h e p e p t i d e in 2 H 2 0 . In a d d i t i o n t o t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f T r p - 1 9 several s l o w l y e x c h a n g i n g a m i d e p r o t o n s a r e o b s e r v e d . (c) S p e c t r a l r e g i o n f~om 6 t o 9 p p m o f a g g r e g a t e d m e l i t t i n in H 2 0 r e c o r d e d w i t h 6 m M m e l i t t i n in 9 0 % H 2 0 / 1 0 % 2 H 2 0 c o n t a i n i n g 1 . 5 M N a C l a t p H 3 . 8 a n d 3 0 ° C . T h e s p e c t r u m s h o w s t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f "£rp-19 a n d t h e labile p r o t o n s . T h e c o n d i t i o n s f o r e x p e r i m e n t s (b) a n d (c), i.e. 1 . 5 M NaC1, p H 3 . 8 , a n d 3 0 ° C , w e r e c h o s e n i n o r d e r t o o b t a i n slow exchange of amide NH protons [14]. The number of melittin molecules per aggregate has not been d e t e r m i n e d u n d e r t h e s e c o n d i t i o n s , b u t it a p p e a r s t h a t t h e p r e v a i l i n g s p e c i e s is t e t r a m e r i c m e l i t t i n (see text).
will be described in more detail below, the ~H-NMR spectra indicated that melittin is fully aggregated for the following conditions: (i) 4 mM melittin in 0.05 M sodium phosphate buffer at p2H 7.0 and 30°C; (ii) 4 mM melittin in 1.5 M NaC1 solution at p2H 3.0 and 30°C; (iii) 6 mM melittin in salt-free solution at p2H 9.0 and 30°C; (iv) 40 mM melittin in salt-free solution at p2H 3.4 and 30°C, and (v) 20 mM melittin in 0.15 M sodium phosphate buffer at p2H 7.0 between 30 and 90°C. Resonance assignments in the ~H-NMR spectrum o f tetrameric melittin Chemical shifts and spin-spin coupling constants for tetrameric melittin under the solution conditions of Fig. l a are shown in Table I. In the following the experiments used for the assignment of the resonances listed in Table I are described. These assignments were considerably simplified by the observation that each m o n o m e r contained in the tetrameric species appeared to give the same 1H-NMR spectrum. In Fig. l a this spectroscopic equivalence is most clearly seen for the aromatic region at 7--8 ppm, which contains a single set of resonances for the indole ring o f Trp-19. For melittin concentrations b e t w e e n 0.4 and 4.0 mM, the 1H-NMR spectrum of melittin in 50 mM sodium phosphate buffer at p2H 7.0 and at 60 or 80°C indicated that melittin dissociated at higher temperatures. This allowed correlations between monomeric and tetrameric melittin to be obtained by the following procedure. Resonances from tetrameric melittin in 50 mM sodium
234 TABLE I RESONANCE ASSIGNMENTS AND 1H-NMR PARAMETERS
FOR TETRAMERIC
MELITTIN
M e a s u r e d w i t h 4 m M m e l i t t i n in 5 0 m M s o d i u m p h o s p h a t e b u f f e r in 2 H 2 0 a t p 2 H 7 . 0 a n d 3 0 ° C . C h e m ical shifts (5) are relative t o s o d i u m 3 - t r i m e t h y l s i l y l - [ 2 , 2 , 3 , 3 , - 2 H ] p r o p i o n a t e a t p 2 H 7.0 [ 8 ] . V a l u e s in parenthesis correspond to chemical shifts where irradiation caused decoupling of resolved multiplets. A m i n o acid residue
Spin-spin coupling constants (+0.3 Hz)
6 (ppm) aCH
flCH
A l a I (4 o r 1 5 ) A l a II (4 o r 1 5 ) V a l I (5 o r 8)
4.146 4,306 3.636
1.487 1.576 (2.19)
V a l II (5 o r 8)
3.813
(2.25)
3.515 3.505
(2.02) (1.90) 4.261 4.351 3.450 3.679
ne-2 Ile I (17 Ile II ( 1 7 Thr I (10 T h r II ( 1 0 Trp-19
or or or or
20) 20) 11) 11)
L y s I ( 7 , 21 o r 2 3 ) L y s II (7, 21 o r 2 3 ) L y s III ( 7 , 21 o r 2 3 ) ArgI (22 or 24) Arg n (22 or 24) Gln-25 and -26
Others
~'CH 3
1.020 0.875 ~/CH 3 1 . 1 3 7 0.971 7CH 3 0.54 5CH 3 0.30 7CH 3 0.950 ~CH 3 0.827 7CH 3 1.298 7CH 3 1.316 C2H 7.422 C4H 7.541 C5H 7.046 C6H 7.000 C7H 7.455 NH 10.318 eCH2 a 2.624 2.37 b eCH 2 2.894 eCH 2 2.957 5CH 3 3.197 6CH 2 2.94 6CH2 2.43 b
3 Jc~fl
Others
7.3 7.3 9.3
3jfl,y 6 . 4
8.8
3J~37 6 . 4
3J87r 6.1 3Jf17 6.1
a For Lys I the two e-methylene protons were not equivalent. b At 30°C the resonance of one eCH 2 proteon of Lys I and the 6CH 2 resonances of the two glutamines give rise t o a g r o u p o f o v e r l a p p e d lines (Fig. 1). T h e a p p r o x i m a t e c h e m i c a l s h i f t s given w e r e o b t a i n e d f r o m Fig. 1 a n d f r o m o b s e r v a t i o n s a t h i g h e r t e m p e r a t u r e .
phosphate buffer at p2H 7.0 and 30°C were followed first at variable temperatures up to 85°C and then at variable phosphate concentration between 50 and 0 mM at 85°C and p2H 7.0 (Fig. 2). At p2H 7.0 and 85°C in the absence of phosphate, 4 mM melittin was monomeric [1]. By using these conditions of p2H, temperature and phosphate concentration, conditions were obtained where exchange between monomeric and aggregated melittin was rapid on the NMR time scale over the entire range of melittin association. Fig. 2 shows plots of the chemical shift versus solution condition obtained for the alanine, threonine, valine and isoleucine residues. To obtain more reliable correlations for a larger number of lines, the complete spin systems for the individual residues were identified b y spin-decoupling difference spectroscopy [11] at the different solution conditions. While assignments of the spin systems to particular positions in the amino acid sequence were obtained for only two residues (Table I), correspondences between spin systems in monomeric and tetrameric
235
[P04]" m M
5o2s6
PPM
'
a
i
'
i
b
4.5f-
4.0
S
I
3.5
I I
50 25 0
50 25 0
50 25 0
e
C
,
I
'
•
i
,
.
i
d
i I
I J I
2.0 I I I i
1.5
i
I
I
I
1.O
i , hi , e l 25
55
85
I I
I
i
i
I I
0.5
I r
I , ii
Iill,ll
25
55
85
25
TEMPERATURE
55
r I,
i i I
25
85
,I,
55
,I
85
°C
Fig. 2. C o r r e l a t i o n o f c o r r e s p o n d i n g r e s o n a n c e s in m o n o m e r i c a n d t e t r a m e r i c m e l i t t i n . T h e c h e m i c a l shifts o f s e l e c t e d r e s o n a n c e s o f 4 raM r a e l i t t i n in 50 m M s o d i u m p h o s p h a t e b u f f e r at p 2 H 7.0 a n d 3 0 ° C ( t e t r a m e r i c m e l i t t i n ) w e r e f o l l o w e d as a f u n c t i o n o f t e m p e r a t u r e u p t o 8 5 ° C ( l o w e r scale) a n d t h e n as a f u n c t i o n o f p h o s p h a t e c o n c e n t r a t i o n at 85 ° C ( u p p e r scale). A t 85 ° C a n d p 2 H 7 . 0 in t h e a b s e n c e o f p h o s p h a t e , 4 raM r a e l i t t i n w a s fully m o n o r a e r i c [ 1 ] . T h e a s ~ g n r a e n t s o f t h e r e s o n a n c e s are: (a) 0, [] a n d m c~CH,~3CH a n d "),CH3 of Val I (5 or 8); A ~ a n d v , ~ C H , ~ 3 C H a n d ~ , C H 3 o f V a l I I ( 5 o r 8 ) . ( b ) s, [ ] a n d 0, ~CH, ~CH a n d 7 C H 3 o f U e - 2 ; A Lx a n d v ~ C H , ~3CH a n d 7 C H 3 o f I l e I ( 1 7 o r 2 0 ) ; o o a n d o a C H , J3CH a n d 9"CH3 o f Ile II (17 o r 20). (c) A a n d V ~xCH,~CH 3 of A l a I (4 o r 1 5 ) ; m O, 0~CH.~CH 3 o f A l a I I (4 o r 15). (d) m 0, ~3CH, 7 C H 3 o f T h r I ( 1 0 o r 11); A v ~3CH, 7 C H 3 o f T h r II ( 1 0 o r 11). T h e riCH r e s o n a n c e s o f t h e v a l i n e a n d i s o l e u c i n e r e s i d u e s w e r e o b s e r v e d i n d i r e c t l y as t h e f r e q u e n c y at w h i c h t h e ~ a n d T-protons were decoupled.
melittin could thus be established for all the resonances in Table I. Truncated~Iriven nuclear Overhauser enhancement difference spectra [12] obtained with preirradiation at 0.3 ppm indicated that the two methyl groups observed at 0.30 and 0.54 ppm in tetrameric melittin were located near each other and near the indole ring of Trp-19. Since proximity to Trp-19 may, depending on the location in the amino acid sequence of the residue in question, impose rather stringent constraints on possible spatial structures of the melttin tetramer, individual assignments for the two high-field methyl lines were of particular interest. At 65°C, where these lines were at 0.58 and 0.71 ppm, the multiplet structures resolved after resolution enhancement were a triplet and a doublet. At 65°C the doublet was decoupled by irradiation at 1.70 ppm, and at 85°C and low phosphate concentration it was found to be a component of a spin system identical to that assigned to Ile-2 in monomeric melittin [1]. Because of the above-mentioned evidence for close proximity of the two m e t h y l groups observed at high field and since the two high-field lines showed nearly identical temperature dependences, the triplet resonance was tentatively assigned to 5CH3 of Ile-2. The assignment of the spin system of Ile-2
236
was further supported by experiments with 6 mM melittin in 9 mM sodium phosphate buffer at p:H 7.0 and 15°C. Fig. 3a shows that under these conditions the exchange between monomeric and aggregated melittin is slow and the NMR spectrum is a superposition of the spectra of monomeric and tetrameric melittin, with approx. 30% of the observed intensity corresponding to tetrameric melittin. Saturation transfer experiments established correlations between the line at 0.58 ppm and a doublet at 0.96 ppm, and between the line at 0.30 ppm and a triplet at 0.89 ppm (Fig. 3b and c). In corresponding experiments with fully tetrameric melittin the doublet resonance at 0.96 ppm and the triplet resonance at 0.89 ppm were not observed, which confirms that these
i=
I
I
b
t cs
d PPM
2
1
0
Fig. 3. 3 6 0 M H z 1 H - N M R spectra showing double-resonance experiments used to correlate the resonances o f the m e t h y l groups o f isoteucine 2 in m o n o m e r i e and tetrameric m e l i t t i n . The arrows denote the frequencies at w h i c h double-resonance i r r a d i a t i o n was applied. S, i r r a d i a t i o n applied p r i o r to data acquisition in o r d e r to s a t u r a t e t h e i r r a d i a t e d r e s o n a n c e . D, d e c o u p l i n g i r r a d i a t i o n a p p l i e d d u r i n g a c q u i s i t i o n . (a) H i g h - f i e l d r e g i o n o f t h e 1 H . N M R s p e c t r u m o f 6 m M m e l i t t i n in 9 m M s o d i u m p h o s p h a t e b u f f e r at p 2 H 7.0 a n d 1 5 ° C . T h e o b s e r v e d s p e c t r u m is a s u p e r p o s i t i o n o f t h e s p e c t r a o f m o n o m e r i c a n d t e t r a m e r i c melittin, with approx. 30% of the total intensity c o r r e s p o n d i n g to tetrameric melittin. The inset shows t h e 0 - - 0 . 7 p p m r e g i o n w i t h a 5-fold i n c r e a s e d v e r t i c a l scale. ( b ) D i f f e r e n c e b e t w e e n s p e c t r a t a k e n w i t h a n d w i t h o u t p r e s a t u r a t i o n o f t h e b r o a d m e t h y l r e s o n a n c e o f t e t r a m e r i c m e l i t t i n at 0 . 3 0 p p m . (c) D i f f e r ence b e t w e e n spectra t a k e n with and w i t h o u t presaturation of the broad m e t h y l resonance from tetram e r i c m e l t t i n at 0 . 5 8 p p m . ( d ) D i f f e r e n c e b e t w e e n s p e c t r a t a k e n w i t h a n d w i t h o u t p r e s a t u r a t i o n o f t h e m e t h y l r e s o n a n c e f r o m t e t r a r n e r i c m e l i t t i n at 0 . 5 8 p p m , w i t h s p i n d e c o u p l i n g at 1 . 9 2 p p m .
237 resonances correspond to monomeric melittin. The doublet at 0.96 p p m coincides with the previously identified doublet of Ile-2 in monomeric melittin [1] and was decoupled b y irradiation at the chemical shift previously determined for riCH o f Ile-2 (Fig. 3d). The experiments of Fig. 4 further resulted in the tentative jassignment of the triplet resonance at 0.89 p p m to ~CH3 of Ile-2 in monomeric melittin.
Effects o f p H and salt concentration on self-association o f melittin Table II lists 1H-NMR chemical shifts for numerous amino acid residues of melittin in aggregates formed in different aqueous solvents. That similar line widths for the NMR resonances were observed under all conditions indicates that aggregates containing a small number of melittin molecules prevail in all the preparations in Table II. This was independently supported by gel filtration
2
c
2
65
I 8
I
I 7
PPM
Fig. 4. 3 6 0 M H z 1 H . N M R s p e c t r a s h o w i n g t h e r e s o n a n c e s o f t h e i n d o l e r i n g o f T r p - 1 9 f o r 4 m M m e l i t t i n a t p 2 H 2 . 8 a n d 3 0 ° C as a f u n c t i o n o f NaC1 c o n c e n t r a t i o n . (a) s a l t free. (b) 0 . 0 8 M NaCI. (c) 0 . 2 M NaC1. (d) 1 . 2 M NaC1. T h e n u m b e r s i d e n t i f y i n d i v i d u a l i n d o l e r i n g p r o t o n lines. M e l i t t i n is m o n o m e r i c in (a) a n d f u l l y a g g r e g a t e d in (d)0 I n (b) a n d (c) m e l i t t i n is p a r t i a l l y a g g r e g a t e d a n d t h e o b s e r v e d s p e c t r a are s u p e r positions of resonances from monomeric and aggregated melittin.
C5H C6H C2H C7H C4H
eCH2 d
eCH 2 eCH 2 5CH 2 5CH 2 7CH~
T r p - 1 9 ring
L y s I (7, 21 or 23)
L y s II (7, 21 or 23) L y s I I I (7, 21 o r 23) Arg I (22 or 24) A r g II ( 2 2 or 24) G i n - 2 5 a n d -26
(2.62) (2.37) (2.89) (2.96) 3.197 (2.94) (2.43)
7.045 6.981 7.421 7.454 7.540
1.488 1.583 1.020 1.137 1.298 1.316 0.297
4 mM melittin, a 50 m M p h o s p h a t e p 2 H 7.0, 3 0 ° C
(2.43)
(2.90) 2.979 3.223
(2.62)
(7.02) (7.02) 7.438 7.458 7.533
1.507 1.572 1.017 1.128 1.307 1.307 0.334
4 mM melittin, b 1.5 M NaC1, p 2 H 3.0, 3 0 ° C
Self-aggregated melittin
6
(2.75) (2.64) (2.94) 2.999 3.200 (3.15) (2.45)
7.046 7.152 7.357 7.507 7.608
1.507 1.527 1.031 1.147 1.324 1.337 (0.40)
6 mM melittin, b n o salt, p 2 H 9.0, 3 0 ° C
(2.75) (2.60) (2.95) 3.005 3.216 3.070 (2.44)
7.048 7.086 7.420 7.491 7.585
1.522 1.588 1.040 1.162 1.334 1.334 0.421
40 mM melittin, b n o salt, p 2 H 3.4, 30OC
2.978 2.998 3.135 3.196 2.396
2.962
7.158 7.264 7.270 7.514 7.619
1.379 1.389 0.958 0.958 1.223 1.238 0.904
M o n o m e r i c melittin [1] 3 raM m e l i t t i n , n o salt, p 2 H 3.3, 3 0 ° C
0.13 0.27 0.06 0.05 0.03 0.21 0.02
0.03 0.17 0.08 0.05 0.08
0.03 0.06 0.02 0.03 0.04 0.03 0.12
ASA
--0.28 --0.43 --0.06 ---0.01 0.07 ---0.14 0.04
---0.12 --0.20 0.14 ---0.04 --0.05
0.13 0.18 0.07 0.19 0.09 0.09 --0.46
h A - - ~M
--0.13 --0.23 --0.04 --0.02 0.06 --0.04 0.02
--0.24 --O.18 0.09 --0.02 --0.03
0.18 0.18 0.18 0.23 0.12 0.12
~Mic
a F o r these c o n d i t i o n s m e ] i t t i n w a s s h o w n t o b e t e t r a m e r i c (see t e x t ) . b w h i l e c o r r e s p o n d e n c e s b e t w e e n i n d i v i d u a l spin s y s t e m s in m o n o m e r i c m e l i t t i n a n d in t h e species a w e r e e s t a b l i s h e d (see t e x t ) , o n l y c o r r e s p o n d e n c e s b e t w e e n t y p e s o f a m i n o acid residues w e r e d e t e r m i n e d f o r t h e o t h e r t h r e e s o l u t i o n c o n d i t i o n s . W h e n t h e r e w a s m o r e t h a n o n e a m i n o acid of a g i v e n t y p e t h e i n d i v i d u a l spin s y s t e m s w e r e c o r r e l a t e d so as to give g r e a t e s t s i m i l a r i t y in t h e s p e c t r a l p r o p e r t i e s for d i f f e r e n t s o l v e n t c o n d i t i o n s . c Only o n e 7 C H 3 of e a c h valine was r e s o l v e d in t h e s p e c t r a o f a g g r e g a t e d m e l i t t i n . d "rwn n n e - n r o t o n r e s o n a n c e s w e r e o b s e r v e d f o r t h e L y s I e C H 2 g r o u p .
~CH 3 ~CH 3 7CH3 c ~/CH3 c ~CH 3 "yCH3 5CH 3
Ala I (4 o r 15) Ala I I (4 or 15) Val I (5 o r 8) Val I I (5 or 8) T h r I ( 1 0 or 11) T h r II ( 1 0 or 11) ne-2
Resonance assignment
5 are t h e c h e m i c a l shifts relative to t h e r e f e r e n c e c o m p o u n d s o d i u m 3 - t r i m e t h y l s i l y l - [ 2 , 2 , 3 , 3 - 2 H ] p r o p i o n a t e at p 2 H 7.0 ( T o a c c o u n t f o r t h e t i t r a t i o n shift o f this r e f e r e n c e b e t w e e n p 2 H 3.0 a n d p 2 H 7.0 [ 8 ] , 0 . 0 1 9 p p m has b e e n a d d e d to t h e e x p e r i m e n t a l l y o b s e r v e d c h e m i c a l shifts f o r 4 m M m e l i t t i n in 1.5 M NaC1 a t p 2 H 3 . 0 a n d f o r 3 r a m m e l i t t i n at p 2 H 3.3 in t h e a b s e n c e of salt. C h e m i c a l shifts g i v e n in p a r e n t h e s i s are less a c c u r a t e , since t h e c o r r e s p o n d i n g r e s o n a n c e s w e r e b r o a d . ) A~ A is the m a x i m u m d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a of a g g r e g a t e d m e l i t t i n u n d e r t h e f o u r d i f f e r e n t s o l u t i o n c o n d i t i o n s c o n t a i n e d in this t a b l e . 6 A - 5 M is t h e a v e r a g e d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a o f a g g r e g a t e d m e l i t t i n u n d e r t h e f o u r d i f f e r e n t s o l u t i o n c o n d i t i o n s a n d m o n o m e r i c m e l i t t i n . 5M ic - - 5M is t h e a v e r a g e d i f f e r e n c e in c h e m i c a l shift b e t w e e n t h e s p e c t r a o f m e l i t t i n b o u n d t o five d i f f e r e n t t y p e s o f d e t e r g e n t m i c e n e s [ 2 ] a n d m o n o m e r i c melittin.
I N F L U E N C E OF S O L U T I O N C O N D I T I O N S ON T H E 1H-NMR C H E M I C A L SHIFTS OF A G G R E G A T E D M E L I T T I N
T A B L E II b0 f~ 00
239 experiments for high NaC1 concentrations [13] and by light-scattering studies of salt-free solutions. Table III summarizes the melittin and/or salt concentrations which were necessary to induce aggregation of melittin. Several interesting facets of melittin aggregation were revealed by these experiments. Firstly, for salt-free solution conditions at both p2H 3.4 and p2H 9.0, greater than 90% aggregation was achieved for about a 5-fold change in melittin concentration. This is not consistent with an equilibrium between m o n o m e r and aggregates containing a small number of molecules, such as a tetramer, unless a high degree of cooperativity is involved in melittin association. Since a small number of melittin molecules are involved in the aggregates discussed in Table II, the concentration dependence gives clear indication that self-association of melittin in salt-free solution involves cooperativity between individual melittin molecules. Table III also shows that under salt-free conditions, much higher melittin concentrations were necessary to achieve aggregation at p~H 3.4 as compared to p~H 9.0. p2H titration of 6 mM melittin in salt-free solution at 30°C showed that in aggregated melittin, as previously shown for monomeric melittin [1], the a-amino group of Gly-1 was the only ionizable group which titrated between p2H 3.0 and p2H 9.0. The present results on aggregation of melittin at p2H 3.4 and at p2H 9.0 (Table III) thus show that removal of the positive charge on the a-amino group of Gly-1 by deprotonation greatly enhances the tendency to aggregate. This is consistent with our previous observation that at acidic pH the minar c o m p o n e n t of melittin, where the positive charge on the a-amino group of Gly-1 is replaced by an uncharged formyl group, shows a more pronounced tendency to aggregate than the major c o m p o n e n t of melittin
[11. When monomeric mehttin was titrated with either NaCI or sodium phosphate to induce aggregation, slow exchange on the NMR time scale between
TABLE
nI
INFLUENCE
OF SOLUTION
CONDITIONS
ON AGGREGATION
OF MELITTIN
D e p e n d i n g o n w h e t h e r fast o r s l o w e x c h a n g e o n t h e N M R t i m e s c a l e p r e v a i l e d , t h e d e g r e e o f a g g r e g a t i o n was determined either from the observed chemical shifts or from the relative intensities of the monomer and aggregate resonances, respectively [15]. Solution
conditions
p2H 9.0, 30°C, no salt p2H 3.4, 30°C, no salt
Melittin conSalt conceneentration (raM) tration (M) 0.7 2 6 3 10 35
Degree of aggregation
Exchange rate on the N M R t i m e scale
----
monomer ca. 50% aggregated ~90% aggregated
fast
----
monomer ca. 50% aggregated ~'90% aggregated
fast
p2H 2.8, 30°C NaCI
4
-0.06 ~1
monomer ca. 5 0 % a g g r e g a t e d fully aggregated
slow
p2H 7.0, 30°C, NaH2PO 4/Na2HPO4
4
-0.01 0.05
monomer ca. 50% aggregated fully aggregated
slow
240
monomeric and aggregated melittin was observed (Table III). Since a superposition of monomeric melittin resonances and aggregated melittin resonances could be observed for salt concentrations where melittin was partially aggregated (Fig. 4), these experiments led to the observation that the chemical shifts observed for monomeric melittin were independent of salt concentration whereas the chemical shifts observed for aggregated melittin varied at intermediate salt concentrations. This behaviour suggests that the conformation of monomeric melittin is essentially independent of salt concentration whereas for aggregated melittin either the aggregate conformation or the number of molecules per aggregate changes as a function of salt concentration.
Temperature dependence of the 1H-NMR parameters of aggregated melittin The temperature dependence of the ~H-NMR chemical shifts of a 20 mM melittin solution in 150 mM sodium phosphate buffer at p2H 7.0 was measured. Evidence that under these conditions no appreciable change in the degree of melittin aggregation occurred between 20 and 90°C was obtained from the observation that 20 mM and 28 mM melittin showed identical 1H-NMR spectra over this temperature range. Below approx. 40°C the spectrum was virtually identical to the spectrum of 4 mM melittin in 50 mM sodium phosphate buffer at p2H 7.0, i.e. the reference tetramer state, suggesting that melittin is probably also tetrameric at the higher concentrations of melittin and buffer. Above approx. 40°C large changes in chemical shift were observed (Fig. 5) for the resonances of one lysine, one arginine, Ile-2 and the protons of the indole ring of Trp-19. In addition, the two protons of the 7CH2 group of each of the two glutamines in melittin, which were inequivalent below approx.
PPM 74 7.o
3.3 29
C
~
=
-"
=
_
"-
~
°
D
~
E
* ~
F
G
25 17 1.3 0.9
L M
0.5 ]
20
i
i
~0
i
i
t
I
60
TEMPERATURE
=
80
°C
Fig. 5. P l o t s o f t h e c h e m i c a l s h i f t s o b s e r v e d f o r s e l e c t e d r e s o n a n c e s o f 2 0 m M p h o s p h a t e b u f f e r a t p 2 H 7.0 as a f u n c t i o n o f t e m p e r a t u r e . T h e a s s i g n m e n t s T r p - 1 9 i n d o l e ring; F, A r g - 2 2 a n d -24 6 C H 2 ; G, Lys-7, -21 a n d -23 e C H 2 ; Ala-4 a n d -15 ~ C H 3 ; J , T h r - 1 0 a n d -11 7 C H 3 ; K, Val-5 a n d -8 7 C H 3 ( o n l y e a c h r e s i d u e w a s i d e n t i f i e d ) ; L a n d M, I l e - 2 7 C H 3 a n d 6 C H 3.
melittin in 150 mm sodium o f t h e r e s o n a n c e s are: A - - E ,
H, G l n - 2 5 a n d -26 ~/CH2; I, one methyl
resonance
from
241 40°C, indicating slow rotation about the C~--C~ bond, became equivalent above 40°C. At 90°C the NMR parameters of the lysine, arginine and glutamine residues were very similar to the parameters observed for monomeric melittin, whereas the NMR parameters of the alanine, threonine and valine residues were similar to the parameters observed for tetrameric melittin at 30°C. Thus qualitatively different behaviour is observed for residues located in the hydrophobic C-terminal or the hydrophilic N-terminal region of the melittin sequence. The chemical shifts of the protons of the indole ring of Trp-19 showed a different behaviour yet, i.e. at 90°C t h e y did not coincide with shifts of either the m o n o m e r or tetramer. Discussion The 1H-NMR spectrum of aggregated melittin in H20 solution (Fig. 1) revealed that a number of the labile protons have chemical shifts which differ appreciably from the chemical shifts of the labile protons in both monomeric melittin [1] and the model peptides H-Gly-Gly-X-Ala-OH [14]. Furthermore, in aggregated melittin approx. 12 labile protons per m o n o m e r were found to be stabilized against exchange with solvent deuterons (Fig. 1). Under the conditions of Fig. l b and c, half-times with respect to exchange of several hours were measured for these protons implying that, in contrast to monomeric melittin [1], aggregated melittin assumes a globular conformation where a sizeable portion of the backbone amide protons are shielded from solvent contact [15]. Since, in addition, a variety of 1H-NMR resonances from amino acid residues distributed t h r o u g h o u t the amino acid sequence were found to have the same chemical shifts for all four polypeptide chains in tetrameric melittin (Table I), it seems that the apparent s y m m e t r y of tetrameric melittin is based on the propensity of the melittin polypeptide chain to assume a particular nonrandom conformation. This is extremely interesting in view of our previous results indicating that melittin also assumes a well-defined, non-random conformation when bound to various types of detergent and phospholipid micelles [2]. In the following the ~H-NMR data on tetrameric and micelle-bound [2,3] melittin are used to compare conformational features of melittin in these two environments and to investigate the nature of melittin-melittin interactions in the aggregates and melittin
242 caused variations of the chemical shifts comparable to those observed upon aggregation (Table II). For the resonances of the indole ring of Trp-19, one of lysines, 7 21 or 23 and one of arginines 22 and 24, a third type of behaviour was observed. These resonances showed both appreciable changes in chemical shift to either high or low field upon aggregation of melittin and appreciable differences in the chemical shifts observed for aggregated melittin under various solution conditions (Table II). Trp-19, and possibly all three of these residues, are near the boundary between the hydrophobic and hydrophilic regions of the melittin amino acid sequence. It seems likely that the NMR parameters observed for these residues reflect changes in population of a limited number of conformation states in tetrameric melittin. The following qualitative picture of aggregated melittin seems to emerge from these observations. The hydrophobic N-terminal region of melittin assumes a definite conformation which includes intra- and/or interchain hydrogen bonding and which, by association, forms a hydrophobic core of the aggregate. It is presumably the spatial folding of this part of the polypeptide chain which gives rise to circular dichroism reminiscent of u-helical structure [2,16]. The hydrophilic C-terminal region of the melittin amino acid sequence lies at the surface of the aggregate and its spatial arrangement is highly sensitive to solvent interactions. These conclusions are supported by the temperature dependence studies of aggregated melittin. In the hydrophilic C-terminal region of melittin, changes in chemical shifts as a function of temperature were comparable to the chemical shift changes upon aggregation, whereas in the hydrophobic N-terminal region, the changes in chemical shift as a function of temperature were small compared to the chemical shift changes upon aggregation. Overall this picture suggests that the melittin monomer-aggregate equilibrium represents a balance between folding of the polypeptide chain into a spatial structure which is stabilized by self-aggregation, attractive interactions between the hydrophobic regions of this structure and electrostatic repulsion between the highly positively charged hydrophilic regions. This is supported by the observations that either reducing the charge on melittin by deprotonation of Gly-1 or shielding of the charges by addition of salt favours aggregation. Even though ultracentrifugation and quasi-elastic light-scattering experiments indicated that melittin binds as a m o n o m e r to dodecylphosphocholine miceUes and NMR experiments suggested that melittin is also monomeric when bound to a variety of other types of detergent and phospholipid micelles [2], the circular dichroism spectra of tetrameric and miceUe-bound melittin were very similar and would be compatible with an a-helical conformation [2]. On the other hand, the IH-NMR spectra of tetrameric and micelle-bound melittin showed appreciable differences [2]. Despite the differences in the 1H-NMR spectra, detailed comparison of the present NMR results on aggregated melittin under variety of solution conditions with our previous NMR results on melittin bound to a variety of different types of micelles [2] suggests a considerable degree of conformational similarity. Thus, for all of the resonances in Table II except Ile-2, which was not assigned in micelle-bound melittin, the average change in chemical shift when monomeric melittin aggregates under a variety of solution conditions (6A - - ~ M in Table II) are in the same direction as the aver-
243 age changes in chemical shift when monomeric melittin binds to a variety of different micelles (~Mic- ~M in Table II). Furthermore, while the chemical shifts observed for micelle-bound melittin show some variation with the type of micelle, the pattern of the variations for different amino acid residues was very similar to the pattern noted above for the chemical shift variations observed for aggregated melittin under various solution conditions [2]. Finally, the temperature dependence of the chemical shifts of aggregated melittin (Fig. 5) and of melittin b o u n d to dodecylphosphocholine miceUes [3] is very similar. The outstanding difference between the 1H-NMR spectra of aggregated and micelle-bound melittin is that the large high-field shifts observed for the Ile-2 methyl resonances in aggregated melittin (Table I) were not observed for micelle-bound melittin [2,3]. These high-field shifts are due to intra- or intermolecular proximity of Ile-2 and Trp-19 in aggregated melittin and the observed chemical shifts are therefore expected to be highly sensitive to local conformational features. This expectation is confirmed by the observation that in aggregated melittin the high-field shifts of the Ile-2 methyl resonances are greatly reduced at higher temperatures (Fig. 5). Overall, considering that the resonances in Table II arise from amino acid side-chain protons and hence may be particularly sensitive to the differing intermolecular environments present in aggregated melittin (peptide-peptide contacts) and in micelle-bound melittin (peptide-detergent contacts), the parallels between the spectral features observed for aggregated and micelle-bound melittin are striking and suggestive of similar conformations for melittin in these two states. A highly consistent picture would emerge with the hypothesis that the chemical shift variations between monomeric and aggregated or micelle-bound melittin arise primarily from the secondary and tertiary structure of the m o n o m e r units, with the exception of the high-field shifts of Ile-2 in tetrameric melittin, which would be caused by the packing interactions in the aggregate. We have previously suggested that the interaction of melittin with detergent or phospholipid micelles might be described as the formation of mixed micelles by two different types of amphiphilic molecules, each of which can selfassociate to form miceUes at sufficiently high concentrations [2]. The present results appear to support this hypothesis since each monomeric unit of tetrameric melittin appears to assume an amphiphilic three-dimensional conformation and since the melittin monomers appear to assume very similar conformations in the tetrameric and micelle-bound forms. Furthermore, the observation that self-association of melittin involves formation of a hydrophobic core and a charged, hydrophilic exterior is reminiscent of the organization of detergent micelles. However, melittin appears to differ from detergent or phospholipid molecules in that a suitable amphiphilic character is achieved only b y folding of extensive parts of the polypeptide chain into a defined spatial structure. This is presumably the reason w h y aggregates of melittin, in contrast to detergent or phospholipid micelles, involve only a small number of melittin molecules. Work on these questions is being further pursued. Acknowledgement Financial support by the Swiss National Science Foundation (project No. 3.004-0.76) is gratefully acknowledged.
244
References 1 Lauterwein, J., Brown, L.R. and W/ithrich, K. (1980) Biochim. Biophys. Acta 622, 219--230 2 Lauterwein, J., B~sch, Ch., Brown, L.R. and Wfithrich, K. (1979) Biochim. Biophys. Acta 556, 244-264 3 Brown, L.R. (1979) Biochim. Biophys. Acta 557, 135--148 4 Habermann, E. (1972) Science 177, 314--322 5 Gauldre, J., Hanson, J.M., Rumjanek, F.D., Shipolini, R. and Vernon, C.A. (1976) Eur. J. Bioehem. 61,369--376 6 Faucon, J.F., Dufourcq, J. and Lussan, C. (1979) FEBS Lett. 102, 187--190 7 Chu, B. (1974) Laser Light Scattering, Academic Press, New York 8 De Marco, A. (1977). J. Magn. Resonance 26, 527--528 9 De Marco, A. and Wiithrich, K. (1976) J. Magn. Resonance 24, 201--204 10 Wagner, G., Wfithrich, K. and Tschesche, H. (1978) Eur. J. Biochem. 86, 67--76 11 De Marco, A., Tschesche, H., Wagner, G. and Wilthrich, K. (1977) Biophys. Struct. Mech. 3, 303--315 12 Wagner, G. and Wfithrich, K. (1979) J. Magn. Resonance 33, 675--680 13 Talbot, J.C., Dufourcq, J. De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191--193 14 Bundi, A. and Wiithrich, K. (1979) Biopolymers 18, 285--297 15 Wiithrich, K. (1976) NMR in Biological Research: Peptides and Proteins, North Holland, A ms t e rda m 16 Dawson, C.R., Drake, A.F., HeUiweU, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75--86