Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity.

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.

The solution structure of type II antifreeze protein reveals a new member of the lectin family.

A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system. The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein. The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H-¿15N¿ heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints. The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets. The structure confirms the proposed existence of five disulfide bonds. The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.

Lactam bridge stabilization of alpha-helices: the role of hydrophobicity in controlling dimeric versus monomeric alpha-helices.

A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.

Preferential heterodimeric parallel coiled-coil formation by synthetic Max and c-Myc leucine zippers: a description of putative electrostatic interactions responsible for the specificity of heterodimerization.

The oncoprotein c-Myc must heterodimerize with Max to bind DNA and perform its oncogenic activity. The c-Myc-Max heterodimer binds DNA through a basic helix-loop-helix leucine zipper (b-HLH-zip) motif and it is proposed that leucine zipper domains could, in concert with the HLH regions, provide the specificity and stability of the b-HLH-zip motif. In this context, we have synthesized the peptides corresponding to the leucine zipper domains of Max and c-Myc with a N-terminal Cys-Gly-Gly linker and studied their dimerization behavior using reversed-phase HPLC and CD spectroscopy. The preferential formation of a fully helical parallel c-Myc-Max heterodimeric coiled-coil was observed under air-oxidation and redox conditions at neutral pH. We show that the stability and the helicity of the disulfide-linked c-Myc-Max heterostranded coiled-coil is modulated by pH, with a maximum around pH 4.5, supporting the existence of stabilizing and specific interhelical electrostatic interactions. We present a molecular model of the c-Myc-Max heterostranded coiled-coil describing potential electrostatic interactions responsible for the specificity of the interaction, the main feature being putative buried electrostatic interactions between a histidine side-chain (in the Max leucine zipper) and two glutamic acid side-chains (in the c-Myc leucine zipper) at the heterodimer interface. This model is supported by the fact that the apparent pKa (as determined by [1H]-NMR spectroscopy) of this histidine side-chain at 25 degrees C is 0.42 (+/- 0.05) pKa units higher in the folded form than in the unfolded form. This indicates that the charged histidine side-chain contributes approximately 0.57 (+/- 0.07) kcal/mol (2.38 (+/- 0.30) kJ/mol) of stabilization free energy to the c-Myc-Max heterostranded coiled-coil through favorable electrostatic interaction.