Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2602-2606, July 1977
Chemistry
Arrangement of water molecules in cavities and channels of the lattice of [Phe4Val6]antamanide dodecahydrate (bound water/solvent channels/hydrogen bonds/direct Roentgen-ray analysis/membrane models)
ISABELLA L. KARLE* AND EILEEN DUESLERt * Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375; and t Department of Chemistry, University of Illinois, Urbana, Illinois 61801
Communicated by Bernhard Witkop, April 19, 1977
ABSTRACT The synthetic, biologically active, [Phe4Val6J analog of natural antamanide has been crystallized from a solution containing calcium nitrate, acetone, and acetonitrile. The crystal does not contain any Ca2+ ions but does contain 12 water molecules per peptide molecule. The conformation of this dodecahydrate is identical to the trihydrate crystallized from nhexane/methyl acetate. The packing in both crystals is very similar, governed by parallel bands of phenyl and pyrrolidine ring stacking and by continuous channels for the solvent molecules, water in this case and n-hexane/methyl acetate in the previous study. The two structures are not ideally isomorhous, since the c cell parameter differs by more than 1.OAin the two cells. There are three functions for H20 molecules: the three intrinsic H20 molecules in the interior of the peptide ring, the H20 bound to the exposed C=.O groups, and the HI0 molecules that fill space in the solvent channels. There are no direct hydrogen bonds between neighboring peptide molecules and there are only two intramolecular NHmO=C bonds (of the 5 - 1 type).
category of gas hydrates where the hydrogen-bonded water molecules form host lattices with polyhedral voids that are occupied by the hydrocarbon molecules (11) or in the formation of hydrogen-bonded sheets of water molecules that separate layers of organic molecules (12). Of prime interest at present is the structure of water surrounding protein molecules, which is slowly being resolved in such crystals as insulin (13), rubredoxin (14), and myoglobin (15). An analysis of the water structure in a smaller peptide may give further insights into the relationship of water to peptides in macromolecules and membranes, particularly since the ratio of observed reflections to number of atoms is about 50:1 in this decapeptide as compared to about 6.5:1 in protein crystals, thus permitting a more definitive determination of atomic positions.
The conformation of peptide molecules, linear as well as cyclic, appears to be a function of a number of factors, including the peptide sequence, complexation with ions, polarity of the solvent, and crystal packing forces, such as hydrogen bonding and van der Waals' attraction. Natural antamanide and the biologically active synthetic analog [Phe4Val6]antamanide (ref. 1 and references therein) present an excellent opportunity for conformational studies by x-ray diffraction, since crystals can be grown from a. large variety of solvents. Furthermore, conformational models have been proposed on the basis of spectroscopic data and minimum energy calculations (refs. 2-5 and references therein) that can be evaluated by comparison with the results of the crystal structure analyses. Aside from Li+ antamanide-CH3CN (6, 7) and Na+[Phe4Val6]antamanideC2H5OH (6, 8), which are isostructural despite different alkali metal ions, different side chains, different solvents, and different crystal packing, the structure and conformation of alkali metal-free [Phe4Val6]antamanide-3H20, crystallized from a mixture of n-hexane and methyl acetate, has been reported recently (9, 10). Except for the two pairs of Pro-Pro linkages, the conformation of [Phe4Val6Jantamanide-3H20 is totally different from the alkali metal complexes of antamanide and
solution of [Phe4Val6]antamanide with the sequence
EXPERIMENTAL
Colorless prisms were obtained at room temperature from a 8
9
10
1
2
Pro-Phe-Phe-Val-Pro
I
Pro-Val-Phe-Phe-Pro 7
6
5
4
3
in a mixture of acetone and CH3CN containing calcium nitrate. A crystal of dimensions 0.70 X 0.75 X 0.98 mm was sealed in a thin-walled, boron glass capillary with a drop of mother liquor. The lattice collapses if the crystal is allowed to dry. The [110] axis was parallel to the walls of the capillary. Diffraction data were measured on an automatic four-circle diffractometer with CuKa radiation to a maximum scattering angle of 20 = 1260 for a total of 3340 independent reflections. The space group is P21212 with a = 15.084(4) A, b = 21.920(6) A, c11.015(3) A, V = 3642 A3, and calculated density of 1.27 g/cm3 based on a molecular weight of 1391.6 for C66H82N10Oo12H20 and two molecules in the unit cell. The cyclic peptide molecule must contain a 2-fold axis of rotation coincident with the c axis of the cell. A comparison of the cell parameters of the present compound with those of [Phe4Val6]antamanide-3H20, crystallized from n-hexane/methyl acetate, shows that the a and b cell parameters are very close, i.e., 15.08 compared to 14.85 A and 21.92 compared to 21.77 A, respectively, and that the hOl reflections are very similar in intensity for both compounds. However, there is a decrease of 1.05 A in the c parameter for the cell in the present investigation. Nevertheless, phases based on the coordinates of [Phe4Val6]antamanide-3H20 and refined by the tangent formula (16) led directly to the present structure. Difference maps revealed 16 sites for H20 molecules with
[Phe4Val6]antamanide.
This manuscript is concerned with [Phe4Val6]antamanide crystallized from a solution containing Ca(NO3)2, acetone, and acetonitrile. The initial intention was to crystallize a complex containing the Ca2+ ion. The structure analysis shows that the Ca2+ ions were not incorporated into the lattice; however, the equivalent of 12 water molecules are associated with each peptide molecule. Hence, the effect of the nature of the solvent, very polar in this case and nonpolar in the previous investigation (9, 10), can be assessed.
W(i)-W(vdii) at full occupancy and W(ix)-W(xvd) at ap-
proximately half occupancy, thus having an equivalent of 12
The manner in which water becomes involved in organic crystals is extremely versatile, for example, in the clathrate
H20 molecules associated with each peptide molecule. At 2602
Table 2. Bond lengths (A) and angles (deg.)
Table 1. Fractional coordinates and thermal parameters for [Phe4Val6lantamaniden12H20 Atom
Ni Cal C,1 01
CO' C'Yll C712 N2 C"2
C'2 02 Ct2 C72
C62 N3 Ca3 C'3 03
C#3 C73
C63 N4 Ca4 C'4 04
C#4 C74
C641 C642 C'41 C'42 Ct4 N5
C"5 C'5 05
C65 Ct5 C151
C652 C151 C052
Cr5 wi wii Wiv WV WVii Wixt Wxit Wxiit Wxiiit WxVt *
x
0.3601 0.2667 0.2697 0.3352 0.2257 0.2102 0.2816 0.1918 0.1875 0.2012 0.1843 0.0900 0.0513 0.1064 0.2315 0.2450 0.3429 0.3609 0.2245 0.2546 0.2328 0.3994 0.4949 0.5321 0.5997 0.5482 0.5198 0.5103 0.5014 0.4847 0.4758 0.4651 0.4923 0.5153 0.6160 0.6604 0.4988 0.3996 0.3667 0.3475 0.2758 0.2523 0.2203 0.5000 0.3981 0.5000 0.6507 0.8570 0.1778 0.0000 0.0000 0.0967 0.0642
y
z
-0.0514 0.0631 -0.0308 0.0841 0.0379 0.1083 0.0646 0.1451 -0.0652 0.1973 -0.1321 0.1627 -0.0595 0.3132 0.0671 0.0933 0.1325 0.1287 0.1391 0.2643 0.0967 0.3365 0.1502 0.0943 0.0983 0.0228 0.0417 0.0480 0.1923 0.3056 0.2484 0.2300 0.2547 0.1854 0.3006 0.1268 0.2994 0.3294 0.2761 0.4440 0.2043 0.4384 0.2105 0.2132 0.2207 0.1886 0.1813 0.0841 0.1991 0.0320 0.2106 0.3023 0.2566 0.4007 0.3181 0.3807 0.2322 0.5191 0.3584 0.4688 0.2734 0.6126 0.3360 0.5899 0.1280 0.0585 0.0925 -0.0489 0.0703 -0.0417 0.0703 -0.1354 0.1248 -0.1734 0.1416 -0.1847 0.1962 -0.1309 0.0974 -0.2430 0.2097 -0.1367 0.1139 -0.2479 0.1693 -0.1961 0.0000 0.2306 0.0987 0.3867 0.0000 0.4978 0.0476 0.6064
0.0743 0.0069 0.0000 0.0000 0.0030 0.0136
0.8179 0.5460 0.7132 0.3300 0.4379 0.7124
B22
B33
B12
B13
B23
3.61 3.27 4.80 4.02 3.78 3.90 5.80 3.61 3.70 4.32 4.11 4.61 5.83 4.37 3.99 3.57 3.74 4.89 4.28 8.69 5.89 3.75 4.64 3.13 3.89 4.21 5.56 4.70 6.81 6.56 7.71 8.29 3.70 3.82 3.30 6.86 7.60 6.26 5.60 10.17 9.08 10.55 9.93
4.17 5.73 4.19 5.43 7.15 10.58 6.89 6.34 5.47 5.78 4.82 8.83 20.05 9.76 4.93 5.87 4.45 6.42 9.75 5.84 4.52 4.03 4.16 3.79 5.36 3.85 3.99 4.92 4.94 5.93 4.70 5.49 3.82 4.44 4.00 5.09 4.30 4.02 6.61 10.79 10.77 12.13 11.74
0.61 0.60 0.41 -0.57 0.71 -0.52 1.17 0.08 -0.08 0.50 -0.14 1.62 0.75 0.28 0.02 0.12 -0.20 0.17 0.53 -1.25 0.44 0.18 -0.65 0.12 -0.34 0.33 -0.49 -0.49 -0.45 -1.08 -0.80 -1.77 0.33 0.84 0.01 1.39 1.26 0.87 0.83 -1.48 2.90 -0.68 1.54
0.56 0.43 0.62 -0.26 2.14 1.46 1.28 -0.54 -0.10 1.10 1.44 -1.46 -3.27 -1.62 0.50 0.52 -0.07 0.37 2.11 0.41 0.19 -0.40 0.69 -0.16 1.35 0.11
-0.75 -0.84 0.21 -0.02 -0.17 -0.29 1.05 -0.34 0.35 -1.21 1.36 -0.20 -4.50 -0.89 -0.49 -1.26 -1.37 1.99 -1.86 -2.27 -1.24 -0.25 -0.64 -0.01 -0.49 -0.20 0.13
-0.44 1.99 2.25 -5.22 2.48 5.82 -5.17 -0.30 0.22 3.99
-0.96
-3.02
0.92
8.96 5.90 -3.32 5.47 9.04 0.58 18.33 12.70 -3.66
0.87 0.27 5.72
0.95 1.17 -0.49
1.96 1.39
11.84 -1.23
6.35 2.20
Bil 3.28 3.33 3.03 2.76 4.40 5.66 6.21 2.52 3.47 3.10 5.75 4.00 5.02 2.82 2.85 2.89 2.86 3.51 4.99 5.07 4.74 2.52 2.81 3.39 3.26 3.51 3.17 4.70 5.29 5.14 6.51 5.63 2.63 2.72 2.81 4.27 3.36 4.69 5.21 7.17 5.99 7.75 7.37 9.29* 11.57 11.52* 19.99 8.92 11.79 11.61* 14.35* 7.62 16.39
9.35 10.78
2.09 26.55 15.45 15.93
-0.46 0.57 -0.20
0.38 -0.49 -0.52 0.00 -0.78 0.21 0.23 -0.25 -0.72
Isotropic value.
present,
Val 1
Pro 2
Pro 3
Phe
i
4
Phe 5
Avg.
Ni Cia CitaCi' Ci ' Oi Ci' Ni+l Cia Ciad
1.499 1.530 1.218 1.348 1.583
1.485 1.516 1.249 1.332 1.567
1.500 1.561 1.227 1.325 1.595
1.482 1.543 1.234 1.345 1.504
1.458 1.598 1.230 1.279 1.563
1.485 1.550 1.231 1.326 1.562
Cift-ci-Y
1.531 1.502 1.435 1.542 1.546 11.535 1.518 1.608 11.373 11.405 11.437 1.424 1.368 1.405 11.423 1.481 1.404 1.383 11.452 1.427 1.490 1.487
Bonds
Ci,
Ci6
Ci- Ni Angles
Cjj'NjCjc,
118.3 114.0 118.7 118.1 123.1 110.1 110.2
107.7 107.1 108.1 104.5 Ci~
1123.9 1116.7
Ni+,Ci'Oi Ci'Ci acio NiCiaCio
CjaCj'6Cj-Y
108| 1113.7
CjaCj'Nj+1 CiC'Ci Oi
Ci'YCibNj
120.6 110.8 116.7 119.7 123.5 109.9
109.9 109.4 120.3 114.5
103.2 102.4 114.2 113.5 128.1 119.7
Table 3. Conformational angles for
[Phe4Val6lantamanide-12H20
rameters
Peptide Molecule. The conformation of the peptide molecule in the crystal grown from the polar solvent is the same as that for the [Phe4Val6]antamanide.3H20 grown from anhydrous n-hexane/methyl acetate (9, 10). The stereodiagram in Fig. 1 shows the conformation of the molecule and the ellipsoids associated with the thermal motion of each atom. The thermal
121.9 110.4 114.3 118.4 127.2 109.4 115.6
ellipsoids are smaller in the present crystal, reflecting a much more rigid arrangement due to extensive hydrogen bonding to the water molecules in the cell. In the crystal derived from the n-hexane/methyl acetate solvent mixture, there were only weak van der Waals' contacts with a disordered solvent. In both crystals there are three H20 molecules, W(i)-W(Mi), intimately associated with the interior of the peptide ring that are not removed by extensive drying (17, 18). In the crystal from nhexane/methyl acetate, the sites for W(ii) and W(iM) are only partially occupied, thus allowing the phenyl rings on Phe4 and Phe9 to rotate toward the sites of W(fl) and W(iMi) when the H20 molecules are absent. In the present crystal, these sites are
the agreement factor is 9.0% for all reflections with
RESULTS
7
Ci6NiCia Ci6NjCj-1'
FoI > 5.0, a total of 2259 reflections. Anisotropic thermal pa-
for the C, N, and 0 atoms have been refined, but hydrogen atoms have not yet been introduced into the structure factor calculations. Fractional coordinates are listed in Table 1, bond lengths and angles are shown in Table 2, and the conformational angles are in Table 3.
117.6 110.2 117.8 121.9 120.3 110.0 102.5
125.1 112.0 118.1 116.5 125.3 109.8 99.6
120.1 107.2 114.8 -123.6 121.4 110.1 110.1
NiCiaC~i'
-0.95 0.33 -1.96 0.78 -1.95 0.42 -0.68 -0.47 0.05 1.44 0.82
t One-half occupancy.
I
2603
Proc. Nati. Acad. Sci. USA 74 (1977)
Chemistry: Karle and Duesler
Oj(N-Ca)
,i (Ca- C') Wi (C' Ni+ 1)
Gil
Oi2
Val
Pro
Pro
Phe
Phe
1,6
2,7
3,8
4,9
5,10
-113 161 173
-65 156 6
-96 3 -170
-110
63 42 -178
1
-10
33
-62
-59
-36
r-48 -48 1+131
(83 83 1-93
19
-26 171
The convention followed is that proposed by the IUPAC-IUB Commission on Biochemical Nomenclature (1970) Biochemistry 9, 3471. In the fully extended chain Xi = {i = wi = 1800.
2604
Chemistry: Karle and Duesler
Proc. Natl. Acad. Sci. USA 74 (1977)
FIG. 1. Stereodiagram of [Phe4Val6]antamanide.3H20 drawn from experimentally determined coordinates. The peptide backbone is outlined by darkened bonds; the Arabic numbers label the 10 Ca atoms. Water molecules are labeled with Roman numerals; thin lines represent hydrogen bonding. The ellipsoids represent the thermal motion at a 50% probability level.
fully occupied and, consequently, the positions of the phenyl rings in Phe4 and Phe9 are localized. Conformational angles shown in Table 3 are within 40 of those found for the crystal from n-hexane/methyl acetate, except for Phe4 and Phe9 where they differ by 6-140. The differences may be caused in part by the rotation of the phenyl groups in Phe4 and Phe9 in the crystal from the nonpolar solvent and in part by tighter packing in the crystal from the polar solvent. Crystal Packing. The arrangement of the peptide molecules is quite similar in both crystals. A schematic representation is shown in Fig. 2 and the comparable stereodiagram is shown in Fig. 3. Parallel to the a axis and at the 1/4 and 3/4 positions of the b axis, pairs of phenyl and pyrrolidine rings from one molecule interleave with those of neighboring molecules to create a continuous stacking pattern of lipophilic groups. On the other hand, parallel to the a axis and at the 0 and 1/2 positions of the b axis, each crystal has a continuous channel. In Table 4. Hydrogen bonding*
Type Intramolecular Intrinsic H20
Bound H20
Donor
Acceptor
Separation,A
W(ii) W(i) W(v)
0(1) W(i) W(ii) 0(1) W(ii) 0(5)
2.91 3.02 311 2.92 3.16 2.89
W(vii) W(vii)
0(5) 0(3)
3.01 2.81
N(5) N(1) N(4)
W(ix)t W(xii)t W(xiii)t W(ii) W(iv) W(vi) W(ix)t W(xi)t W(xiii)t W(xv)t W(xv)t W(xV)t
3.03 0(2) 3.49t 0(2) 2.69 0(2) Intrawater W(iv) 2.92 2.73 W(v) 2.92 W(ix)t 3.26 W(xi)t 2.94 W(vii) 2.92 W(xiv)t 2.54 W(viii) 3.07 W(xiii)t 2.51 W(ix)t * The following pairs of atoms are related by a 2-fold rotation axis (1 - x, 5y, z) or (x, 5y, z): W(ii) and W(iii); W(v) and W(vi); W(vii) and W(viii); W(ix) and W(x); W(xiii) and W(xiv); W(xv) and W(xvi). In the peptide, all atoms i are related to i + 5 by the 2-fold
rotation axis. t Occupancy in cell approximately one-half. W(xii) probably not on c axis but disordered between two positions relatively near the c axis.
the dodecahydrate, this channel is occupied by water molecules that are hydrogen-bonded to the peptide molecules and to each other to form an uninterrupted column of polar atoms. In the crystal from n-hexane/methyl acetate, the channel is larger by 1.0 A in the c direction and occupied by unordered and nonpolar n-hexane and/or methyl acetate molecules. Thus the packing of the crystal is governed by the van der Waals' attraction between phenyl and pyrrolidine rings, and the solvent molecules, whether H20 or n-hexane, perform a space-filling function. WateriStructure. There are three distinct roles for the water molecules in this structure: (a) Three water molecules, W(i) -W(i), are an integral part of the peptide molecule in that they reside in the interior of the molecule and by forming hydrogen-bonded bridges between N(1), N(6), N(4), and N(9), they stabilize the conformation of the 30-membered ring. These three H20 molecules are represented in both structures that have been obtained from the polar solvent as well as from nonpolar solvents. The hydrogen bond lengths are listed in Table 4. (b) Water molecules W(v)-W(x) and W(xii)-W(iv) are hydrogen-bonded to the exterior carbonyl oxygens 0(2), 0(7), 0(3), 0(8), 0(5), and 0(10). "Bound water" may be a misnomer, since the peptide molecule maintains its conformation in the absence of these water molecules in the crystal obtained from nonpolar solvents. (c) Water molecules W(iv), W(xi), W(xv), and W(xvi) are hydrogen bonded only to other 1/4
0
1/2
3/4
Pro
Pe Pro
IPh A
> b
FIG. 2. Schematic diagram of the arrangement of [Phe4Val6Jantamanide molecules in the crystal showing the stacking of phenyl and pyrrolidine rings and the location of the solvent channels. The c axis is directed up from the page. This diagram can be compared directly with Fig. 3.
Proc. Natl. Acad. Sci. USA 74 (1977)
Chemistry: Karle and Duesler
2605
FIG. 3. Stereodiagram of the crystal packing. The shaded atoms represent water molecules. The axes intersect in the center of each figure and are directed with a b, b A, and c up from the page.
FIG. 4. Two [Phe4Val6jantamanide molecules related by a translation along the c axis. The view is perpendicular to that in Figs. 1 and 3. The six water molecules (labeled with Roman numerals) are part of a continuous channel containing water molecules shown in Figs. 2 and 3.
water molecules and occupy the space near the central core of the water channel. Some of the details of the hydrogen-bonding scheme can be seen in Fig. 4. Here the peptide molecules are depicted in the direction of the 2-fold rotation axis. The two molecules are related by a translation of one cell length and the water channel is perpendicular to the view. The six water sites shown have full occupancy. W(v) and W(vi) make additional hydrogen bonds along the direction of the channel to W(ix) and W(x). The remainder of the water molecules have a more complicated hydrogen bonding scheme since water sites W(ix)-W(xvi) are occupied only about half the time. The distances between the water sites exclude the simultaneous occupation of W(ix) and W(xiii) or W(x) and W(xiv) or W(xi), W(xv), and W(xvi). A few of the numerous different possible hydrogen-bonding schemes are depicted in Fig. 5. Scheme (1) has the maximum
number of hydrogen bonds and is the scheme shown in Fig. 3.
The six NH groups are turned inward in the peptide ring, whereas eight of the 10 carbonyl oxygens are directed to the outer surface of the molecule. All are involved in hydrogen bonding to water molecules except for a pair of intratnolecular NH ..O=C bonds of the 5 - 1 type and atoms 0(4) and 0(9). These latter two oxygen atoms are involved only in van der Waals' contacts to carbon atoms of Pro and Phe side groups from neighboring molecules such as 0(4)...C(51) at 3.52 A and 0(4)... C:(2) at 3.59 A. DISCUSSION The significant result of this investigation is that the conformation of a cyclic decapeptide has remained constant, independent of crystallization from polar or nonpolar solvents,
(3) FIG. 5. Three of several possible hydrogen-bonding schemes in the water channels parallel to the a axis. Atoms W(ix)-W(xvi), representing the oxygen atoms of water molecules, are present in the cell only at about one-half occupancy; therefore, different water sites are occupied from cell to cell and the hydrogen-bonding scheme varies from cell to cell. Scheme (1) represents the hydrogen bonding shown in Fig. 3.
(1)
(2)
2606
Chemistry: Karle and Duesler
contrary to the indications from nuclear magnetic resonance experiments of several different conformations in solution (2-4). None of the conformations proposed from solution data is similar to those established in the crystalline state so far. Four other crystalline variations of antamanide and analogs are being analyzed (10). A further significant result is the absence of intramolecular hydrogen bonding of the familiar 4 - 1 type, or any transannular NH...O=C bonds, except for a pair of 5 - 1 hydrogen bonds that involve the Pro2(css)Pro3Phe4 and Pro7(cis)Pro8Phe9 sequences. The remaining four NH moieties, although directed toward the interior of the peptide ring, make hydrogen bonds with water molecules that bridge the ring. Ivanov (19) has reported 2:1 complexes of antamanide and Ca2+ in solution, based on circular dichroism and nuclear magnetic resonance studies. He proposed a sandwich complex using the conformation established for the Na+ complex of antamanide (6, 8). An alternate possibility for a sandwich complex in solution would be the configuration shown in Fig. 4 with W(iv) replaced by a Ca2+ ion. In that respect two antamanide molecules are suitably stacked so that the remaining five water molecules would provide an appropriate environment for a calcium complex. The ability of antamanide to protect hepatocytes from the toxic effects of phalloidin points to membrane affinity, association, or possibly membrane penetration. Gramicidin A, a linear pentadecapeptide with the highest "hydrophobic index," is a well-known "channel former" in membranes. Other "transmembrane proteins" possess hydrophobic peptide sequences that are correlated with association or penetration of membranes (20). The highly lipophilic antamanide with the novel organization of water of three different modes of association in its channels may well serve as a model for the unusual properties and arrangements of water molecules in proteins and membranes. Note Added in Proof. We understand that J. T. Edsall is completing a monograph on the subject of water and proteins. The New York Academy of Sciences sponsored a conference entitled "Electrical
Proc. Natl. Acad. Sci. USA 74 (1977) Properties of Biological Polymers, Water and Membranes", Chairman, Siro Takashima, the proceedings of which will be published in the fall of 1977. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Wieland, Th. (1972) in Chemistry and Biology ofPeptides, ed. Meienhofer, J. (Ann Arbor Science Publishers, Ann Arbor, MI), pp. 377-396. 2. Patel, D. J. (1973) Biochemistry 12, 667-676. 3. Tonelli, A. E. (1973) Biochemistry 12,689-692. 4. Ovchinnikov, Yu. A. & Ivanov, V. T. (1975) Tetrahedron 31, 2177-2209. 5. Gromov, E. P., Pletnov, V. A. & Popov, E. M. (1976) Bioorg. Khim. 2, 19-27 (in Russian). 6. Karle, I. L., Karle, J., Wieland, Th., Burgermeister, W., Faulstich, H. & Witkop, B. (1973) Proc. Natl. Acad. Sci. USA 70, 18361840. 7. Karle, I. L. (1974) J. Am. Chem. Soc. 96,4000-4006. 8. Karle, I. L. (1974) Biochemistry 13,2155-2162. 9. Karle, L. L., Karle, J., Wieland, Th., Burgermeister, W. & Witkop, B. (1976) Proc. Natl. Acad. Sci. USA 73, 1782-1785. 10. Karle, I. L. (1977) J. Am. Chem. Soc., in press. 11. Jeffrey, G. A. (1969) Acc. Chem. Res. 2,344-52. 12. Karle, I. L. & Karle, J. (1971) Acta Crystallogr. Sect. B, 27, 1891-1898. 13. Hodgkin, D. (1977) Am. Crystallogr. Assoc. Meeting, Asilomar, Abstr. Al, p. 11. 14. Watenpaugh, K. D., Sieker, L. C. & Jensen, L. H. (1977) Am. Crystallogr. Assoc. Meeting, Asilomar, Abstr. EA6, p. 17. 15. Takano, T. (1977) J. Mol. Biol. 110, 537-565. 16. Karle, J. & Hauptman, H. (1956) Acta Crystallogr. 9, 635657. 17. Wieland, T., Birr, C., Burgermeister, W., Trietsch, P. & Rohr, G. (1974) Justus Liebigs Ann. Chem. 1974, 24-26. 18. Wieland, T., Faesel, J. & Konz, W. (1969) Justus Liebigs Ann. Chem. 722, 197-209. 19. Ivanov, V. T. (1975) Ann. N.Y. Acad. Sci. 264,221-243. 20. Segrest, J. P. & Feldmann, R. J. (1974) J. Mol. Biol. 87, 853858.
Chemistry
Arrangement of water molecules in cavities and channels of the lattice of [Phe4Val6]antamanide dodecahydrate (bound water/solvent channels/hydrogen bonds/direct Roentgen-ray analysis/membrane models)
ISABELLA L. KARLE* AND EILEEN DUESLERt * Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375; and t Department of Chemistry, University of Illinois, Urbana, Illinois 61801
Communicated by Bernhard Witkop, April 19, 1977
ABSTRACT The synthetic, biologically active, [Phe4Val6J analog of natural antamanide has been crystallized from a solution containing calcium nitrate, acetone, and acetonitrile. The crystal does not contain any Ca2+ ions but does contain 12 water molecules per peptide molecule. The conformation of this dodecahydrate is identical to the trihydrate crystallized from nhexane/methyl acetate. The packing in both crystals is very similar, governed by parallel bands of phenyl and pyrrolidine ring stacking and by continuous channels for the solvent molecules, water in this case and n-hexane/methyl acetate in the previous study. The two structures are not ideally isomorhous, since the c cell parameter differs by more than 1.OAin the two cells. There are three functions for H20 molecules: the three intrinsic H20 molecules in the interior of the peptide ring, the H20 bound to the exposed C=.O groups, and the HI0 molecules that fill space in the solvent channels. There are no direct hydrogen bonds between neighboring peptide molecules and there are only two intramolecular NHmO=C bonds (of the 5 - 1 type).
category of gas hydrates where the hydrogen-bonded water molecules form host lattices with polyhedral voids that are occupied by the hydrocarbon molecules (11) or in the formation of hydrogen-bonded sheets of water molecules that separate layers of organic molecules (12). Of prime interest at present is the structure of water surrounding protein molecules, which is slowly being resolved in such crystals as insulin (13), rubredoxin (14), and myoglobin (15). An analysis of the water structure in a smaller peptide may give further insights into the relationship of water to peptides in macromolecules and membranes, particularly since the ratio of observed reflections to number of atoms is about 50:1 in this decapeptide as compared to about 6.5:1 in protein crystals, thus permitting a more definitive determination of atomic positions.
The conformation of peptide molecules, linear as well as cyclic, appears to be a function of a number of factors, including the peptide sequence, complexation with ions, polarity of the solvent, and crystal packing forces, such as hydrogen bonding and van der Waals' attraction. Natural antamanide and the biologically active synthetic analog [Phe4Val6]antamanide (ref. 1 and references therein) present an excellent opportunity for conformational studies by x-ray diffraction, since crystals can be grown from a. large variety of solvents. Furthermore, conformational models have been proposed on the basis of spectroscopic data and minimum energy calculations (refs. 2-5 and references therein) that can be evaluated by comparison with the results of the crystal structure analyses. Aside from Li+ antamanide-CH3CN (6, 7) and Na+[Phe4Val6]antamanideC2H5OH (6, 8), which are isostructural despite different alkali metal ions, different side chains, different solvents, and different crystal packing, the structure and conformation of alkali metal-free [Phe4Val6]antamanide-3H20, crystallized from a mixture of n-hexane and methyl acetate, has been reported recently (9, 10). Except for the two pairs of Pro-Pro linkages, the conformation of [Phe4Val6Jantamanide-3H20 is totally different from the alkali metal complexes of antamanide and
solution of [Phe4Val6]antamanide with the sequence
EXPERIMENTAL
Colorless prisms were obtained at room temperature from a 8
9
10
1
2
Pro-Phe-Phe-Val-Pro
I
Pro-Val-Phe-Phe-Pro 7
6
5
4
3
in a mixture of acetone and CH3CN containing calcium nitrate. A crystal of dimensions 0.70 X 0.75 X 0.98 mm was sealed in a thin-walled, boron glass capillary with a drop of mother liquor. The lattice collapses if the crystal is allowed to dry. The [110] axis was parallel to the walls of the capillary. Diffraction data were measured on an automatic four-circle diffractometer with CuKa radiation to a maximum scattering angle of 20 = 1260 for a total of 3340 independent reflections. The space group is P21212 with a = 15.084(4) A, b = 21.920(6) A, c11.015(3) A, V = 3642 A3, and calculated density of 1.27 g/cm3 based on a molecular weight of 1391.6 for C66H82N10Oo12H20 and two molecules in the unit cell. The cyclic peptide molecule must contain a 2-fold axis of rotation coincident with the c axis of the cell. A comparison of the cell parameters of the present compound with those of [Phe4Val6]antamanide-3H20, crystallized from n-hexane/methyl acetate, shows that the a and b cell parameters are very close, i.e., 15.08 compared to 14.85 A and 21.92 compared to 21.77 A, respectively, and that the hOl reflections are very similar in intensity for both compounds. However, there is a decrease of 1.05 A in the c parameter for the cell in the present investigation. Nevertheless, phases based on the coordinates of [Phe4Val6]antamanide-3H20 and refined by the tangent formula (16) led directly to the present structure. Difference maps revealed 16 sites for H20 molecules with
[Phe4Val6]antamanide.
This manuscript is concerned with [Phe4Val6]antamanide crystallized from a solution containing Ca(NO3)2, acetone, and acetonitrile. The initial intention was to crystallize a complex containing the Ca2+ ion. The structure analysis shows that the Ca2+ ions were not incorporated into the lattice; however, the equivalent of 12 water molecules are associated with each peptide molecule. Hence, the effect of the nature of the solvent, very polar in this case and nonpolar in the previous investigation (9, 10), can be assessed.
W(i)-W(vdii) at full occupancy and W(ix)-W(xvd) at ap-
proximately half occupancy, thus having an equivalent of 12
The manner in which water becomes involved in organic crystals is extremely versatile, for example, in the clathrate
H20 molecules associated with each peptide molecule. At 2602
Table 2. Bond lengths (A) and angles (deg.)
Table 1. Fractional coordinates and thermal parameters for [Phe4Val6lantamaniden12H20 Atom
Ni Cal C,1 01
CO' C'Yll C712 N2 C"2
C'2 02 Ct2 C72
C62 N3 Ca3 C'3 03
C#3 C73
C63 N4 Ca4 C'4 04
C#4 C74
C641 C642 C'41 C'42 Ct4 N5
C"5 C'5 05
C65 Ct5 C151
C652 C151 C052
Cr5 wi wii Wiv WV WVii Wixt Wxit Wxiit Wxiiit WxVt *
x
0.3601 0.2667 0.2697 0.3352 0.2257 0.2102 0.2816 0.1918 0.1875 0.2012 0.1843 0.0900 0.0513 0.1064 0.2315 0.2450 0.3429 0.3609 0.2245 0.2546 0.2328 0.3994 0.4949 0.5321 0.5997 0.5482 0.5198 0.5103 0.5014 0.4847 0.4758 0.4651 0.4923 0.5153 0.6160 0.6604 0.4988 0.3996 0.3667 0.3475 0.2758 0.2523 0.2203 0.5000 0.3981 0.5000 0.6507 0.8570 0.1778 0.0000 0.0000 0.0967 0.0642
y
z
-0.0514 0.0631 -0.0308 0.0841 0.0379 0.1083 0.0646 0.1451 -0.0652 0.1973 -0.1321 0.1627 -0.0595 0.3132 0.0671 0.0933 0.1325 0.1287 0.1391 0.2643 0.0967 0.3365 0.1502 0.0943 0.0983 0.0228 0.0417 0.0480 0.1923 0.3056 0.2484 0.2300 0.2547 0.1854 0.3006 0.1268 0.2994 0.3294 0.2761 0.4440 0.2043 0.4384 0.2105 0.2132 0.2207 0.1886 0.1813 0.0841 0.1991 0.0320 0.2106 0.3023 0.2566 0.4007 0.3181 0.3807 0.2322 0.5191 0.3584 0.4688 0.2734 0.6126 0.3360 0.5899 0.1280 0.0585 0.0925 -0.0489 0.0703 -0.0417 0.0703 -0.1354 0.1248 -0.1734 0.1416 -0.1847 0.1962 -0.1309 0.0974 -0.2430 0.2097 -0.1367 0.1139 -0.2479 0.1693 -0.1961 0.0000 0.2306 0.0987 0.3867 0.0000 0.4978 0.0476 0.6064
0.0743 0.0069 0.0000 0.0000 0.0030 0.0136
0.8179 0.5460 0.7132 0.3300 0.4379 0.7124
B22
B33
B12
B13
B23
3.61 3.27 4.80 4.02 3.78 3.90 5.80 3.61 3.70 4.32 4.11 4.61 5.83 4.37 3.99 3.57 3.74 4.89 4.28 8.69 5.89 3.75 4.64 3.13 3.89 4.21 5.56 4.70 6.81 6.56 7.71 8.29 3.70 3.82 3.30 6.86 7.60 6.26 5.60 10.17 9.08 10.55 9.93
4.17 5.73 4.19 5.43 7.15 10.58 6.89 6.34 5.47 5.78 4.82 8.83 20.05 9.76 4.93 5.87 4.45 6.42 9.75 5.84 4.52 4.03 4.16 3.79 5.36 3.85 3.99 4.92 4.94 5.93 4.70 5.49 3.82 4.44 4.00 5.09 4.30 4.02 6.61 10.79 10.77 12.13 11.74
0.61 0.60 0.41 -0.57 0.71 -0.52 1.17 0.08 -0.08 0.50 -0.14 1.62 0.75 0.28 0.02 0.12 -0.20 0.17 0.53 -1.25 0.44 0.18 -0.65 0.12 -0.34 0.33 -0.49 -0.49 -0.45 -1.08 -0.80 -1.77 0.33 0.84 0.01 1.39 1.26 0.87 0.83 -1.48 2.90 -0.68 1.54
0.56 0.43 0.62 -0.26 2.14 1.46 1.28 -0.54 -0.10 1.10 1.44 -1.46 -3.27 -1.62 0.50 0.52 -0.07 0.37 2.11 0.41 0.19 -0.40 0.69 -0.16 1.35 0.11
-0.75 -0.84 0.21 -0.02 -0.17 -0.29 1.05 -0.34 0.35 -1.21 1.36 -0.20 -4.50 -0.89 -0.49 -1.26 -1.37 1.99 -1.86 -2.27 -1.24 -0.25 -0.64 -0.01 -0.49 -0.20 0.13
-0.44 1.99 2.25 -5.22 2.48 5.82 -5.17 -0.30 0.22 3.99
-0.96
-3.02
0.92
8.96 5.90 -3.32 5.47 9.04 0.58 18.33 12.70 -3.66
0.87 0.27 5.72
0.95 1.17 -0.49
1.96 1.39
11.84 -1.23
6.35 2.20
Bil 3.28 3.33 3.03 2.76 4.40 5.66 6.21 2.52 3.47 3.10 5.75 4.00 5.02 2.82 2.85 2.89 2.86 3.51 4.99 5.07 4.74 2.52 2.81 3.39 3.26 3.51 3.17 4.70 5.29 5.14 6.51 5.63 2.63 2.72 2.81 4.27 3.36 4.69 5.21 7.17 5.99 7.75 7.37 9.29* 11.57 11.52* 19.99 8.92 11.79 11.61* 14.35* 7.62 16.39
9.35 10.78
2.09 26.55 15.45 15.93
-0.46 0.57 -0.20
0.38 -0.49 -0.52 0.00 -0.78 0.21 0.23 -0.25 -0.72
Isotropic value.
present,
Val 1
Pro 2
Pro 3
Phe
i
4
Phe 5
Avg.
Ni Cia CitaCi' Ci ' Oi Ci' Ni+l Cia Ciad
1.499 1.530 1.218 1.348 1.583
1.485 1.516 1.249 1.332 1.567
1.500 1.561 1.227 1.325 1.595
1.482 1.543 1.234 1.345 1.504
1.458 1.598 1.230 1.279 1.563
1.485 1.550 1.231 1.326 1.562
Cift-ci-Y
1.531 1.502 1.435 1.542 1.546 11.535 1.518 1.608 11.373 11.405 11.437 1.424 1.368 1.405 11.423 1.481 1.404 1.383 11.452 1.427 1.490 1.487
Bonds
Ci,
Ci6
Ci- Ni Angles
Cjj'NjCjc,
118.3 114.0 118.7 118.1 123.1 110.1 110.2
107.7 107.1 108.1 104.5 Ci~
1123.9 1116.7
Ni+,Ci'Oi Ci'Ci acio NiCiaCio
CjaCj'6Cj-Y
108| 1113.7
CjaCj'Nj+1 CiC'Ci Oi
Ci'YCibNj
120.6 110.8 116.7 119.7 123.5 109.9
109.9 109.4 120.3 114.5
103.2 102.4 114.2 113.5 128.1 119.7
Table 3. Conformational angles for
[Phe4Val6lantamanide-12H20
rameters
Peptide Molecule. The conformation of the peptide molecule in the crystal grown from the polar solvent is the same as that for the [Phe4Val6]antamanide.3H20 grown from anhydrous n-hexane/methyl acetate (9, 10). The stereodiagram in Fig. 1 shows the conformation of the molecule and the ellipsoids associated with the thermal motion of each atom. The thermal
121.9 110.4 114.3 118.4 127.2 109.4 115.6
ellipsoids are smaller in the present crystal, reflecting a much more rigid arrangement due to extensive hydrogen bonding to the water molecules in the cell. In the crystal derived from the n-hexane/methyl acetate solvent mixture, there were only weak van der Waals' contacts with a disordered solvent. In both crystals there are three H20 molecules, W(i)-W(Mi), intimately associated with the interior of the peptide ring that are not removed by extensive drying (17, 18). In the crystal from nhexane/methyl acetate, the sites for W(ii) and W(iM) are only partially occupied, thus allowing the phenyl rings on Phe4 and Phe9 to rotate toward the sites of W(fl) and W(iMi) when the H20 molecules are absent. In the present crystal, these sites are
the agreement factor is 9.0% for all reflections with
RESULTS
7
Ci6NiCia Ci6NjCj-1'
FoI > 5.0, a total of 2259 reflections. Anisotropic thermal pa-
for the C, N, and 0 atoms have been refined, but hydrogen atoms have not yet been introduced into the structure factor calculations. Fractional coordinates are listed in Table 1, bond lengths and angles are shown in Table 2, and the conformational angles are in Table 3.
117.6 110.2 117.8 121.9 120.3 110.0 102.5
125.1 112.0 118.1 116.5 125.3 109.8 99.6
120.1 107.2 114.8 -123.6 121.4 110.1 110.1
NiCiaC~i'
-0.95 0.33 -1.96 0.78 -1.95 0.42 -0.68 -0.47 0.05 1.44 0.82
t One-half occupancy.
I
2603
Proc. Nati. Acad. Sci. USA 74 (1977)
Chemistry: Karle and Duesler
Oj(N-Ca)
,i (Ca- C') Wi (C' Ni+ 1)
Gil
Oi2
Val
Pro
Pro
Phe
Phe
1,6
2,7
3,8
4,9
5,10
-113 161 173
-65 156 6
-96 3 -170
-110
63 42 -178
1
-10
33
-62
-59
-36
r-48 -48 1+131
(83 83 1-93
19
-26 171
The convention followed is that proposed by the IUPAC-IUB Commission on Biochemical Nomenclature (1970) Biochemistry 9, 3471. In the fully extended chain Xi = {i = wi = 1800.
2604
Chemistry: Karle and Duesler
Proc. Natl. Acad. Sci. USA 74 (1977)
FIG. 1. Stereodiagram of [Phe4Val6]antamanide.3H20 drawn from experimentally determined coordinates. The peptide backbone is outlined by darkened bonds; the Arabic numbers label the 10 Ca atoms. Water molecules are labeled with Roman numerals; thin lines represent hydrogen bonding. The ellipsoids represent the thermal motion at a 50% probability level.
fully occupied and, consequently, the positions of the phenyl rings in Phe4 and Phe9 are localized. Conformational angles shown in Table 3 are within 40 of those found for the crystal from n-hexane/methyl acetate, except for Phe4 and Phe9 where they differ by 6-140. The differences may be caused in part by the rotation of the phenyl groups in Phe4 and Phe9 in the crystal from the nonpolar solvent and in part by tighter packing in the crystal from the polar solvent. Crystal Packing. The arrangement of the peptide molecules is quite similar in both crystals. A schematic representation is shown in Fig. 2 and the comparable stereodiagram is shown in Fig. 3. Parallel to the a axis and at the 1/4 and 3/4 positions of the b axis, pairs of phenyl and pyrrolidine rings from one molecule interleave with those of neighboring molecules to create a continuous stacking pattern of lipophilic groups. On the other hand, parallel to the a axis and at the 0 and 1/2 positions of the b axis, each crystal has a continuous channel. In Table 4. Hydrogen bonding*
Type Intramolecular Intrinsic H20
Bound H20
Donor
Acceptor
Separation,A
W(ii) W(i) W(v)
0(1) W(i) W(ii) 0(1) W(ii) 0(5)
2.91 3.02 311 2.92 3.16 2.89
W(vii) W(vii)
0(5) 0(3)
3.01 2.81
N(5) N(1) N(4)
W(ix)t W(xii)t W(xiii)t W(ii) W(iv) W(vi) W(ix)t W(xi)t W(xiii)t W(xv)t W(xv)t W(xV)t
3.03 0(2) 3.49t 0(2) 2.69 0(2) Intrawater W(iv) 2.92 2.73 W(v) 2.92 W(ix)t 3.26 W(xi)t 2.94 W(vii) 2.92 W(xiv)t 2.54 W(viii) 3.07 W(xiii)t 2.51 W(ix)t * The following pairs of atoms are related by a 2-fold rotation axis (1 - x, 5y, z) or (x, 5y, z): W(ii) and W(iii); W(v) and W(vi); W(vii) and W(viii); W(ix) and W(x); W(xiii) and W(xiv); W(xv) and W(xvi). In the peptide, all atoms i are related to i + 5 by the 2-fold
rotation axis. t Occupancy in cell approximately one-half. W(xii) probably not on c axis but disordered between two positions relatively near the c axis.
the dodecahydrate, this channel is occupied by water molecules that are hydrogen-bonded to the peptide molecules and to each other to form an uninterrupted column of polar atoms. In the crystal from n-hexane/methyl acetate, the channel is larger by 1.0 A in the c direction and occupied by unordered and nonpolar n-hexane and/or methyl acetate molecules. Thus the packing of the crystal is governed by the van der Waals' attraction between phenyl and pyrrolidine rings, and the solvent molecules, whether H20 or n-hexane, perform a space-filling function. WateriStructure. There are three distinct roles for the water molecules in this structure: (a) Three water molecules, W(i) -W(i), are an integral part of the peptide molecule in that they reside in the interior of the molecule and by forming hydrogen-bonded bridges between N(1), N(6), N(4), and N(9), they stabilize the conformation of the 30-membered ring. These three H20 molecules are represented in both structures that have been obtained from the polar solvent as well as from nonpolar solvents. The hydrogen bond lengths are listed in Table 4. (b) Water molecules W(v)-W(x) and W(xii)-W(iv) are hydrogen-bonded to the exterior carbonyl oxygens 0(2), 0(7), 0(3), 0(8), 0(5), and 0(10). "Bound water" may be a misnomer, since the peptide molecule maintains its conformation in the absence of these water molecules in the crystal obtained from nonpolar solvents. (c) Water molecules W(iv), W(xi), W(xv), and W(xvi) are hydrogen bonded only to other 1/4
0
1/2
3/4
Pro
Pe Pro
IPh A
> b
FIG. 2. Schematic diagram of the arrangement of [Phe4Val6Jantamanide molecules in the crystal showing the stacking of phenyl and pyrrolidine rings and the location of the solvent channels. The c axis is directed up from the page. This diagram can be compared directly with Fig. 3.
Proc. Natl. Acad. Sci. USA 74 (1977)
Chemistry: Karle and Duesler
2605
FIG. 3. Stereodiagram of the crystal packing. The shaded atoms represent water molecules. The axes intersect in the center of each figure and are directed with a b, b A, and c up from the page.
FIG. 4. Two [Phe4Val6jantamanide molecules related by a translation along the c axis. The view is perpendicular to that in Figs. 1 and 3. The six water molecules (labeled with Roman numerals) are part of a continuous channel containing water molecules shown in Figs. 2 and 3.
water molecules and occupy the space near the central core of the water channel. Some of the details of the hydrogen-bonding scheme can be seen in Fig. 4. Here the peptide molecules are depicted in the direction of the 2-fold rotation axis. The two molecules are related by a translation of one cell length and the water channel is perpendicular to the view. The six water sites shown have full occupancy. W(v) and W(vi) make additional hydrogen bonds along the direction of the channel to W(ix) and W(x). The remainder of the water molecules have a more complicated hydrogen bonding scheme since water sites W(ix)-W(xvi) are occupied only about half the time. The distances between the water sites exclude the simultaneous occupation of W(ix) and W(xiii) or W(x) and W(xiv) or W(xi), W(xv), and W(xvi). A few of the numerous different possible hydrogen-bonding schemes are depicted in Fig. 5. Scheme (1) has the maximum
number of hydrogen bonds and is the scheme shown in Fig. 3.
The six NH groups are turned inward in the peptide ring, whereas eight of the 10 carbonyl oxygens are directed to the outer surface of the molecule. All are involved in hydrogen bonding to water molecules except for a pair of intratnolecular NH ..O=C bonds of the 5 - 1 type and atoms 0(4) and 0(9). These latter two oxygen atoms are involved only in van der Waals' contacts to carbon atoms of Pro and Phe side groups from neighboring molecules such as 0(4)...C(51) at 3.52 A and 0(4)... C:(2) at 3.59 A. DISCUSSION The significant result of this investigation is that the conformation of a cyclic decapeptide has remained constant, independent of crystallization from polar or nonpolar solvents,
(3) FIG. 5. Three of several possible hydrogen-bonding schemes in the water channels parallel to the a axis. Atoms W(ix)-W(xvi), representing the oxygen atoms of water molecules, are present in the cell only at about one-half occupancy; therefore, different water sites are occupied from cell to cell and the hydrogen-bonding scheme varies from cell to cell. Scheme (1) represents the hydrogen bonding shown in Fig. 3.
(1)
(2)
2606
Chemistry: Karle and Duesler
contrary to the indications from nuclear magnetic resonance experiments of several different conformations in solution (2-4). None of the conformations proposed from solution data is similar to those established in the crystalline state so far. Four other crystalline variations of antamanide and analogs are being analyzed (10). A further significant result is the absence of intramolecular hydrogen bonding of the familiar 4 - 1 type, or any transannular NH...O=C bonds, except for a pair of 5 - 1 hydrogen bonds that involve the Pro2(css)Pro3Phe4 and Pro7(cis)Pro8Phe9 sequences. The remaining four NH moieties, although directed toward the interior of the peptide ring, make hydrogen bonds with water molecules that bridge the ring. Ivanov (19) has reported 2:1 complexes of antamanide and Ca2+ in solution, based on circular dichroism and nuclear magnetic resonance studies. He proposed a sandwich complex using the conformation established for the Na+ complex of antamanide (6, 8). An alternate possibility for a sandwich complex in solution would be the configuration shown in Fig. 4 with W(iv) replaced by a Ca2+ ion. In that respect two antamanide molecules are suitably stacked so that the remaining five water molecules would provide an appropriate environment for a calcium complex. The ability of antamanide to protect hepatocytes from the toxic effects of phalloidin points to membrane affinity, association, or possibly membrane penetration. Gramicidin A, a linear pentadecapeptide with the highest "hydrophobic index," is a well-known "channel former" in membranes. Other "transmembrane proteins" possess hydrophobic peptide sequences that are correlated with association or penetration of membranes (20). The highly lipophilic antamanide with the novel organization of water of three different modes of association in its channels may well serve as a model for the unusual properties and arrangements of water molecules in proteins and membranes. Note Added in Proof. We understand that J. T. Edsall is completing a monograph on the subject of water and proteins. The New York Academy of Sciences sponsored a conference entitled "Electrical
Proc. Natl. Acad. Sci. USA 74 (1977) Properties of Biological Polymers, Water and Membranes", Chairman, Siro Takashima, the proceedings of which will be published in the fall of 1977. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Wieland, Th. (1972) in Chemistry and Biology ofPeptides, ed. Meienhofer, J. (Ann Arbor Science Publishers, Ann Arbor, MI), pp. 377-396. 2. Patel, D. J. (1973) Biochemistry 12, 667-676. 3. Tonelli, A. E. (1973) Biochemistry 12,689-692. 4. Ovchinnikov, Yu. A. & Ivanov, V. T. (1975) Tetrahedron 31, 2177-2209. 5. Gromov, E. P., Pletnov, V. A. & Popov, E. M. (1976) Bioorg. Khim. 2, 19-27 (in Russian). 6. Karle, I. L., Karle, J., Wieland, Th., Burgermeister, W., Faulstich, H. & Witkop, B. (1973) Proc. Natl. Acad. Sci. USA 70, 18361840. 7. Karle, I. L. (1974) J. Am. Chem. Soc. 96,4000-4006. 8. Karle, I. L. (1974) Biochemistry 13,2155-2162. 9. Karle, L. L., Karle, J., Wieland, Th., Burgermeister, W. & Witkop, B. (1976) Proc. Natl. Acad. Sci. USA 73, 1782-1785. 10. Karle, I. L. (1977) J. Am. Chem. Soc., in press. 11. Jeffrey, G. A. (1969) Acc. Chem. Res. 2,344-52. 12. Karle, I. L. & Karle, J. (1971) Acta Crystallogr. Sect. B, 27, 1891-1898. 13. Hodgkin, D. (1977) Am. Crystallogr. Assoc. Meeting, Asilomar, Abstr. Al, p. 11. 14. Watenpaugh, K. D., Sieker, L. C. & Jensen, L. H. (1977) Am. Crystallogr. Assoc. Meeting, Asilomar, Abstr. EA6, p. 17. 15. Takano, T. (1977) J. Mol. Biol. 110, 537-565. 16. Karle, J. & Hauptman, H. (1956) Acta Crystallogr. 9, 635657. 17. Wieland, T., Birr, C., Burgermeister, W., Trietsch, P. & Rohr, G. (1974) Justus Liebigs Ann. Chem. 1974, 24-26. 18. Wieland, T., Faesel, J. & Konz, W. (1969) Justus Liebigs Ann. Chem. 722, 197-209. 19. Ivanov, V. T. (1975) Ann. N.Y. Acad. Sci. 264,221-243. 20. Segrest, J. P. & Feldmann, R. J. (1974) J. Mol. Biol. 87, 853858.
