Linear D,L-alternating peptides are a fascinating class of peptides with interesting and unique structures and properties. In contrast to conventional peptides, which consist of natural amino acids with L-configuration, the stereochemistry of the amino acids in the sequence of those peptides is strictly alternating. This difference in the primary structure enables D,L-alternating peptides to adopt unique secondary structures such as the E-helix (Figure 3). In this E-helix, the amino acid residues have conformations located in their respective E-regions and the hydrogen bonding is made as in E-structures (parallel or antiparallel, depending on the helix). One of the most popular D,L -alternating peptides is the naturally occurring antibiotic Gramicidin.
Figure 3: D-helical Structure of a L-amino acid sequence (left) in comparison to the E-helical structure of a D,L-alternating peptide. (Structures derived from protein data bank (PDB). PDB codes: 1ux8 (D-helix), 1mag (E-helix)).
2.2.1 Gramicidin
The antibiotic Gramicidin is produced by bacillus brevis as a mixture of Gramicidin A, B, C and S. Gramicidin S is a cyclic decapeptide. The Gramicidins A, B and C are D,L-alternating pentadecapeptides with hydrophobic sidechains, which were found to have antibiotic properties. They kill the bacterium by interrupting the synthesis of Adenosin-triphosphate (ATP) from ADP. They are doing so by simply drilling a hole into the cell membrane and allowing the exchange of charge carriers. In this case they are allowing monocations such as K+ or Na+ to pass through the membrane and thereby undoing the charge gradient, which is established in the processes of the respiratory chain and necessary for the formation of ATP. By this mechanism, they are toxic for procaryotic as well as for eucaryotic cells. This limits its therapeutic use to topological applications, since internal administration also leads to hemolysis.
The three Gramicidins differ in the amino acid in position 11 of the primary structure (Figure 4). Gramicidin A carries a tryptophane in this position, Gramicidin B a phenylalanine and Gramicidin C a tyrosine. Since Gramicidin A is the major product in this mixture, most of the investigations were focused on it.
First interests in the structure of Gramicidin can be traced back to 1941.[1]
Figure 4: Primary structure of Gramicidin.
In ion-conductance measurements through membranes, Hladky and Haydon experienced that the ion conductance through the membrane in the presence of Gramicidin was discrete, in accordance with the theory of a channel formation and not with the presence of a carrier molecule. [2] It was also postulated that the conducting species had to be a dimeric structure. The observed steps in the conduction, i.e. its commencement and termination, were attributed to the formation and dissociation of a dimer. The elucidation of the structure of this dimer was the main subject of an extensive research, which started in the 1970’s and still continues. The structure of Gramicidin was investigated in the solid state, in solution, and in membrane-resembling environments.
Gramicidin in the solid state is polymorph and its crystal structure strongly depends on the solvent, from which it is crystallized and on the presence of ions or lipids. Pioneering work in the determination of the Gramicidin crystal structures has been done by Wallace and Langs. [3-8] They found out that the two main species are a channel and a pore structure (Figure 5).
Figure 5: Schematic representation of the parallel and antiparallel double helical pore structures (left and middle) and the end-to-end dimerized channel structure (right) of Gramicidin (taken from [4])
In the crystal, the pore structure is a left handed antiparallel double helix with varying size and inner diameter, depending on the presence or absence of
incorporated ions. The channel structure is an end-to-end helical dimer, which is only obtained in a lipid complex of Gramicidin.[9] In both cases, the structures areE-helical and hollow and would meet the requirements for an ion-conducting molecule.
In solution, several interconverting structures are present, making the structure elucidation far more complex. Intensive work on this subject has been done by Urry and Veatch and Blout. Urry was the first one who suggested the existence of a new type of helix after CD- and NMR-experiments with Gramicidin A.[10,11]
The existence of the two double helical pore structures has been derived by Veatch and Blout by means of CD-, NMR- and IR-measurements.[12,13]
Antiparallel and parallel double-helices are interconverting via a monomeric helix.
In membrane-resembling environment, extensive work had been carried out by Urry. He observed ion-conducting activity after a covalent head-to-head dimerization of deformyl-Gramicidin with malonic acid and therefore could prove that the dimeric channel structure was an active form of Gramicidin A.[14]
Several other conformations depending on the experiment conditions were also observed.[15-17]
In summary, Gramicidin can adopt a variety of different structures, depending on its environment. In the solid state, the antiparallel double-helix occurs, in solution, antiparallel and parallel double-helices are interconverting, whereas in membrane-resembling environment, the head-to-head dimer of the single-stranded E-helix is one active structure.
2.2.2 Synthetic Gramicidin-mimicking Peptides
The fascinating structural variety of Gramicidin gave rise to the synthesis of model compounds for a deeper understanding of the structural behavior of D,L -alternating peptides. Extensive research on the synthesis and structures of hydrophobic homo-D,L-alternating oligopeptides has been carried out by Lorenzi and coworkers.[18-30] In some of their works, they describe the racemization-free synthesis and structure elucidation of oligo-D-(alt)-L-valines and oligo-D-(alt)-L -phenylalanines with variable lengths. The most detailled studied model peptide was the oligo-D-(alt)-L-valine-system. Di Blasio and Lorenzi were able to solve
the crystal structure of Boc-(L-Val-D-Val)4-OMe and provided detailed conformational parameters of an antiparallel double-stranded E-helix.[26] NMR- and CD studies in solution were carried out with members of the series Boc-(D -Val)m-(L-Val-D-Val)(n-m)/2-OMe, with m = 0 or 1 and n = 7, 8, 9, 12, 15, 16.[19] It could be demonstrated that in CHCl3 (in some cases, solvent mixtures with CH2Cl2 or cyclohexane were used), the oligovalines occured as E-helix. In dependence on the chain length and on the stereochemistry of the last residue in the chain, different types of E-helix could be observed. The four occurring helix-species were the right- and left-handed monomeric E4.4-helices (P)E4.4 and (M)E4.4 with 4.4 residues per turn and the left-handed antiparallel and parallel double helices E5.6 with 5.6 residues per turn. In the monomeric E4.4-helix, the number of possible hydrogen bonds is smaller than in the E5.6-helix (n-4 for (P)E4.4, n-3 for (M)E4.4 and n-1 for both E5.6), rendering the double helix generally more favorable. On the other hand, the steric repulsion of the residues in the tighter E4.4-helix is smaller than in the E5.6-helix. Since the steric conflicts among the side chains augment on increasing chain length, whereas the differences in the number of hydrogen bonds that can be established in both helices remain constant, the tendency to form E4.4-helices should increase with increasing chain length. So only with shorter oligovalines with even n the double helix conformation occured in an observable degree, whereas oligovalines with oddn and longer ones had the tendency to form exclusively the single stranded E4.4-helix (Figure 6).
Figure 6: Helix conformation of oligo-D-(alt)-L-valines in dependence on chain length. (x-axis: number of residues, y-axis: amount of double E-helix (E5.6) divided by the amount of monomeric left- and right-handed E-helix (E4.4)). (taken from [19])
The helix twist sense of the E4.4-helix was reported to be overwhelmingly left-handed for odd n and prevailingly right-left-handed when n was even, but this preference was leveling off with increasing chain length. For the left-handed double stranded E5.6-helix, the population of the antiparallel E5.6-helix was about three times higher than that of the parallel E5.6-helix.
Later 2D-NMR-studies treating the solution structure of a D,L-alternating oligonorleucines were carried out by Celda and Navarro.[31-33] In combination with molecular dynamics calculations, they identified the antiparallel double stranded E5.6-helix to be the major conformation of these peptides, which is in equilibrium with the single stranded E4.4-helix.
These unique structural properties of D,L-alternating oligopeptides make the D -(alt)-L-motif very attractive for polypeptides as well, opening the door to a new class of polymers with new properties and application fields.
2.2.3 D,L-Alternating Polypeptides
Inspired by the Gramicidin motif of a strictly D,L-alternating amino acid sequence, D-(alt)-L-polypeptides had been synthesized and investigated, expecting a new class of polymers with just as remarkable structures. The most famous representative of this polymer class is poly(J-benzyl-D-L-glutamate).
Most of the work has been done by Lotz, Heitz, and Spach.[34-37] They investigated poly(J-benzyl-D-L-glutamate) in the solid state by IR, X-ray, and electron diffraction and found a family of double-helices for poly(J-benzyl-D-L -glutamate), strongly depending on the conditions they applied to the polymer (Figure 7).
Figure 7: Helix transitions of poly(J-benzyl-D-L-glutamate) (taken from [34]).
A fresh sample of poly(J-benzyl-D-L-glutamate), dissolved and recast from chloroform at room temperature was in the D-helical conformation. When the same sample was heated to 130 °C, and cooled to room temperature, it was found to exist in a monomeric, single stranded E4.4-conformation. After further heating to 220 °C to 230 °C and cooling to room temperature again, the dimeric, double stranded E5.6-conformation was obtained, which was only stable in the absence of solvent. When the same sample was dissolved and recast from methylene chloride, the double stranded E7.2-conformation was obtained. When dissolved and recast from chloroform or dioxane, the double stranded E9.0 -conformation and when dissolved and recast from collidine, the double stranded E10.8-conformation was obtained. It appears that the size and shape of the E7.2-, the E9.0-, and the E10.8-helix was determined by the solvent, which was included in the hollow core of the helix.