The structure of the peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been determined in crystals obtained from a dimethylsulfoxide-isopropanol mixture. Crystal parameters are as follows: C38H69N7O10.H2O.2C3H7OH, space group P2(1), a = 10.350 (2) A b = 26.084 (4) A, c = 10.395 (2) A, beta = 96.87 (12), Z = 2, R = 8.7% for 2686 reflections observed > 3.0 sigma (F). A single 5-->1 hydrogen bond is observed at the N-terminus, while two 4-->1 hydrogen bonds characteristic of a 3(10)-helix are seen in the central segment. The C-terminus residues, Ala(6) and Leu(7) are extended, while Val(5) is considerably distorted from a helical conformation. Two isopropanol molecules make hydrogen bonds to the C-terminal segment, while a water molecule interacts with the N-terminus. The structure is in contrast to that obtained for the same peptide in crystals from methanol-water [I. L. Karle, J. L. Flippen-Anderson, K. Uma, and P. Balaram (1990) Proteins: Structure, Function and Genetics, Vol. 7, pp. 62-73] in which two independent molecules reveal an almost perfect alpha-helix and a helix penetrated by a water molecule. A comparison of the three structures provides a snapshot of the progressive effects of solvation leading to helix unwinding. The fragility of the heptapeptide helix in solution is demonstrated by nmr studies in CDCl3 and (CD3)2SO. A helical conformation is supported in the apolar solvent CDCl3, whereas almost complete unfolding is observed in the strongly solvating medium (CD3)2SO.

The crystal structures of two helical peptides Boc-Val-Ala-Leu-Aib-Val-ala-Leu-OMe (VALU-7) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (VALU-8) have been determined to a resolution of 1.0 and 0.9 A, respectively. Both the seven and eight residue peptides crystallize with two conformers per asymmetric unit. The VALU-8 conformers are completely helical and differ only at the C-terminus by a sign reversal of the phi, psi angles of the last residue. One of the VALU-7 conformers occurs as a normal alpha-helix, whereas in the other, the N(7)--O(3) alpha-type hydrogen bond is ruptured by the entry of a water molecule (W) into the helix, which in turn makes hydrogen bonds N(7)...W = 2.97 A and W...O(3) = 2.77 A. The other side of the water molecule is surrounded by a hydrophobic pocket. These two conformers give a static representation of a step in a possible helix unwinding or folding process. In the VALU-8 crystal the helices aggregate in a parallel mode, whereas the aggregation is anti-parallel in the VALU-7 crystal. The crystal parameters are VALU-7, P2(1), a = 10.203 (3) A, b = 19.744 (6) A, c = 22.561 (6) A, beta = 96.76 degrees, Z = 4, C38H69N7O10.0.5H2O, R = 6.65% for 3674 reflections observed greater than 3 sigma (F); and VALU-8, P2(1), a = 10.593 (4) A, b = 27.57 (6) A, c = 17.745 (5) A, beta = 95.76 (3) degrees, Z = 4, C42H76N8O11.0.25 CH3OH, R = 6.63% for 4701 reflections observed greater than 3 sigma (F).

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ABSTRACT.Although the peptide Boc-Aibl-Ala2-Leu3-Aib4-Alas-Leu'-Aib7-Ala8-Leu9-Aib'0-OMe [with a t-butoxycarbonyl (Boc) blocking group at the amino terminus, a methyl ester (OMe) at the carboxyl terminus, and four a-aminoisobutyric (Aib) residues] has a 3-fold repeat of residues, the helix formed by the peptide backbone is irregular. The carboxyl-terminal half assumes an at-helical form with torsion angles ) and r of approximately -60°and -45°, respectively, whereas the amino-terminal half is distorted by an insertion of a water molecule between the amide nitrogen of Ala5 [N(5)] and the carbonyl oxygen of Ala2 [0(2)]. The water molecule W(1) acts as a bridge by forming hydrogen bonds N(5).W(1) (2.93 A) and W(1)---0(2) (2.86 A). The distortion of the helix exposes the carbonyl oxygens of Aib' and Aib4 to the outside environment, with the consequence that the helix assumes an amphiphilic character despite having all apolar residues. Neighboring helices in the crystal run in antiparallel directions. On one side of a helix there are only hydrophobic contacts with efficient interdigitation of leucine side chains with those from the neighboring helix. On the other side of the helix there are hydrogen bonds between protruding carbonyl oxygens and four water molecules that separate two neighboring helices. Along the helix axis the helices bind head-to-tail with a direct hydrogen bond N(2)-0(9) (3.00 A). Crystals grown from methanol/water solution are in space group P2, with a = 15.778 ± 0.004 A, b = 11.228 ± 0.002 A, c = 18.415 ± 0.003 A, = 102.10 ± 0.02ur and two formula units per cell for C49HON1003 2H2OCH3OH. The overall agreement factor R is 7.5% for 3394 reflections observed with intensities >3a(F), and the resolution is 0.90 A.Transmembrane channels formed by alamethicin and related fungal peptides are likely to arise by close association of peptide helices in bilayer membranes (1, 2). The role of specific side chains in promoting helix association is being studied by means of synthetic analogues., It has been observed that a-aminoisobutyric (Aib) residues, common components of membrane-active peptides, promote the formation of 310-helices in short peptides (3,4), and in longer peptides (>7 residues) if they comprise one-half or more of the peptide (ref. 5 and references therein). An a-helix has been favored in longer peptides when the Aib residues constitute one-third of the total. In the present study, the Aib content is 40%. The initial intent was to determine the type ofhelix formed and the mode of aggregation of these helices in the crystal. The serendipitous entry of a water molecule into the helix formed by the backbone atoms has produced unanticipated insights into the hydration and aggregation behavior of helices in peptides.EXPERIMENTAL PROCEDURES Boc-(Aib-Ala-Leu)3-Aib-OMe (where Boc is t-butoxycarbonyl) was synthesized by conventional solution-phase procedures and crystals were grown from CH3OH/H20 solution in the form of thin hexagonal plates. A crystal of size 0.20 X 0.35 x 0.08 mm was sealed in a t...