Polar and Hydrophobic Pores and Channels in Peptide Assemblies

I.L. Karle
Laboratory for Structure of Matter

Introduction: The formation of pores, channels, and nanotubes by the assembly of a number of individual like-molecules into larger entities results in properties that the individual molecules do not possess. These assemblies enable, for example, charged ions to pass through hydrophobic membranes, or permit the insertion of hydrophobic molecules into nonpolar pores to render them soluble in media in which they would be otherwise insoluble. The discussion and examples shown in this paper are limited to one class of natural peptides and to hybrid peptides in which macrocycles are formed from peptide segments that are interspersed with one or more organic scaffolds. Voltage-gated ion transport peptides, often functioning as antibiotics, have been found to occur in nature in the lower forms of life, such as fungi. They form ion channels in the shape of funnels or hourglasses by the assembly of three or more bent amphiphilic helices.¹ These channels have a polar interior wall containing carbonyl moieties and a hydrophobic exterior surface composed of methyl groups. An entirely different motif exists for forming channels or tubes from peptides or peptide hybrids in which the backbone is in the form of a macrocycle with 12 or more atoms in the ring. In this motif, the macrocycles stack over each other with their C=O and NH moieties in sufficient register to permit C=O---HN hydrogen bonds to be formed. Infinite, open-ended tubules are formed in the direction of the stacking in which the walls of the tubes are the hydrogen bonds.

Methods: The geometric parameters describing the structures of the molecules, as well as the folding, twisting, packing, and assembly of molecules are established by single crystal X-ray diffraction procedures.

Results and Discussion: Ion channel formers that occur in nature-Zervamicin and the related antiamoebin, 16-residue peptides found in spores in the soil,¹ transport K⁺ ions through lipophilic membranes by channel formation. Each molecule folds into a severely bent amphiphilic helix, Fig. 1(a). The hourglass-shaped channels are filled with water molecules that form hydrogen bonds among themselves and with the polar side chains or carbonyls that extend into the channel, such as the carbonyls of Hyp¹⁰ and Hyp¹³. ^* The channel formed by three helical molecules, Fig. 1(a), is closed at the waist-line by a trans-channel hydrogen bond between the terminal Ne atom of Gln^{11 **} in one helix and the O of the OH group of Hyp¹⁰ in another helix. This hydrogen bond occurs sequentially in a "closed" and "open" conformation for the passage of each K⁺ ion, Fig. 1(b). Remarkably, both positions have been found in the same crystal of zervamicin in an 80:20 ratio. Figure 1(b) is a schematic diagram of a possible path for a K⁺ ion that involves a double-gating mechanism. The chosen path has a continuous cross-section of at least 5.6 Å, a value that allows pairs of K⁺-O ligands at 2.8 Å to form fleetingly during the passage.

Construction of nonpolar tubules with model peptides - The construction of hollow tubules (such as indicated in the Introduction) has not been successful with cyclic peptides containing only α-amino acid residues and having the same chirality (all L- or all D-handed).^*** Tubules consisting only of cyclic peptides have been constructed successfully, and their crystal structures have been determined with the following motifs in the cyclic peptides: alternating aand β-amino acids, all β-amino acids and alternating D- and L- amino acids.

In an attempt to construct tubules with adjustable diameters, macrocycles were synthesized in which peptide segments were alternated with organic scaffolds. Successful tubule formers were the polymethylene- bridged cystine-based cyclo bisureas (Fig. 2(a)) and cyclo bisamides (Fig. 3). Molecules in these families formed relatively planar rings with C=O and NH moieties approximately perpendicular to the plane of the rings. In the crystals in Fig. 2, the molecules stack over each other and are held together by urea-type hydrogen bonding on either side of the ring. In the bisamides in Fig. 3, the hydrogen bonding is a linear repetition along a strand. Similar tubules are formed for members of each family. The tubules are characterized by three rigid walls consisting of opposing walls containing the hydrogen bonds and one connecting wall containing the disulfide moiety. The fourth wall consists of the polymethylene linker with a variable number of CH2 groups. It is the fourth wall that determines the size of the cavity in the tubule, depending on the length of the polymethylene chain that had been selected, from n = 4 to ~20 (Fig. 3). It should be noted that the polymethylene chain is quite flexible and usually has some disorder. The cavity does not collapse with longer polymethylene chains, but rather maintains a shape with empty space. This is an unusual occurrence considering that moderate and large macrocycles formed by cyclization of peptides have rarely been observed to form large open pores. More frequently, the interior space collapses to some minimum space, accompanied by folding of the backbone and formation of intracyclic NH-OC hydrogen bonds.

FIGURE 1
(a) Hourglass shaped channel, with a polar interior, of antibiotic molecules (only two are shown) that transports potassium ions through cell membranes. (b) Upon application of a small potential, the gate at residue 11 is opened by the rotation of the side chain at 11 to another position that allows partial passage of the K+ ion. The gate must close again before the K+ ion can continue its passage.

^*Hyp - hydroxyproline
^**Gln - glutamine
^***D - dextro (right-handed); L levo (left-handed)

FIGURE 2
Macrocycles that stack and self-assemble into infinite tubules by urea-type hydrogen bonding (crystal structure analysis). The upper wall of the molecule, composed of flexible methylene chains, may vary in length from 4 to ~20 CH2 groups.

Fig3 Image

FIGURE 3
Family of macrocycles that stack and self-assemble into infinite tubules by amide-type hydrogen bonding. The tubules differ from the ureatype shown in Fig. 2 by the removal of an NH group from each vertical side of cyclic backbones. Cavity in the tubules enlarges with the lengthening of the methylene chain.

No electron density is found inside the tubules, which attests to their empty state. Furthermore, unlike the channels in the peptide ionophores (above) these oval cavities have a hydrophobic lining. The interior behaves like an apolar organic solvent and can enhance the solubility in water of highly lipophilic substances by selective guest-host interactions. For example, the tubule with n = 20 can solubilize pyrene, perylene, and the dye Nile Red, whereas tubules of the same family, but with shorter polymethylene linkers and therefore smaller cavities cannot.²

Conclusions: This article describes synthetic hybrid peptide macrocycles with empty interior space and the propensity to stack, resulting in the formation of endless open tubules with NH-OC hydrogen bonds as important support struts in the tubule walls. The cystine residue appears to provide a sufficiently rigid scaffold for the remainder of the macrocycle to prevent the collapse of the empty space within the macrocycle. Flexibility in the long polymethylene chains in compounds forming the successful tubules, manifested by disorder in the crystal structures, may facilitate the entry process of hydrophobic guest molecules into the interior of the tubules.

Acknowledgements: I wish to acknowledge the collaboration of Dr. Darshan Ranganathan of Discovery Laboratory, Indian Institute of Chemical Technology, Hyderabad, India, for design and synthesis of the hybrid peptides, and Prof. P. Balaram of the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, for the isolation and crystallization of the antibiotic peptides.

[Sponsored by ONR and NIH]

References
¹I.L. Karle, M.A. Perozzo, V.K. Mishra, and P. Balaram, "Crystal Structure of the Channel-Forming Polypeptide Antiamoebin in a Membrane-Mimetic Environment," Proc. Natl. Acad. Sci. USA 95, 5501-5504 (1998).
² D. Ranganathan, V. Haridas, C.S. Sundari, D. Balasubramanian, K.P. Madhusudanan, R. Roy, and I.L. Karle, "Design, Synthesis, Crystal Structure, and Host-Guest Properties of Polymethylene- Bridged Cystine-Based Cyclobisamides: A Facile Entry into Hydrogen-Bonded Peptide Nanotubes," J. Org. Chem. 64, 9230-9240 (1999).