| Our laboratory has investigated the relationship between amino acid sequence and stability requirements for peptide folding into two-stranded alpha-helical coiled-coil structures, a common structural motif in many naturally occurring proteins. In particular, we examined a 14-residue 'consensus trigger sequence' that was found in several different coiled-coil sequences, as it relates to folding and stability. We demonstrated that the presence of a 'consensus trigger sequence' does not guarantee folding of two-stranded coiled-coils, and is not essential for folding but rather, any sequence that stabilizes coiled-coil structure beyond a minimum threshold level will suffice. The high-resolution X-ray crystal structure of a de novo-designed coiled-coil that lacks the 'consensus trigger sequence' reveals numerous stabilizing factors including a complex interchain ion-pairing and hydrogen-bonding network across a destabilizing central asparagine residue located in the hydrophobic core of the structure, as well as stabilization through hydrophobic interactions of leucine residues located at the hydrophobic interface (positions e and g of the heptad repeat, [abcdefg]n).;We have also performed structure-activity studies on 14-residue cyclic cationic peptides to elucidate the key design features responsible for their selective toxicity towards microbial organisms vs. mammalian cells, i.e. a high therapeutic index. Based on the structural template of GS14, an amphipathic (nonpolar and polar residues localized to opposite faces of the molecule) beta-sheet cyclic cationic peptide, a diastereomeric analog with D-Lys at position 4 (peptide GS14K4) showed greatly improved microbial specificity with little toxicity to human red blood cells over GS14 (Kondejewski et al. 1999, J. Biol. Chem 274: 13181--92) and was selected to carry out further studies. The D-Lys was positioned on the nonpolar face (vs. L-Lys on the polar face in GS14), disrupting beta-sheet structure and decreasing peptide amphipathicity.;Using the GS 14K4 template, we investigated the effect of hydrophobicity on microbial specificity by altering the amino acid sequence composition of the residues comprising the nonpolar face. Analogs that were less hydrophobic than GS 14K4 had alanine residues substituted for the three valine and three leucine residues found in the native sequence, while more hydrophobic analogs had phenylalanine residues substituted for tyrosine and/or leucine residues substituted for valines. Increased hydrophobicity tended to increase both antimicrobial and hemolytic activity, while decreased hydrophobicity decreased both antimicrobial and hemolytic activity relative to GS14K4.;A novel method that we call "temperature profiling in reversed-phase chromatography" has been used to identify self-association of our 14-residue peptides in aqueous solution. The technique consists of analyzing peptides on a reversed-phase HPLC column at discrete temperature intervals over a range of 5 to 80°C. Loading onto the reversed-phase matrix concentrates the sample and promotes intermolecular interactions causing self-association. We hypothesize that self-association can be observed by an increase in HPLC retention time with increased temperature, and that larger increases in retention times indicate a greater extent of self-association. By studying a series of linear and cyclic peptide analogs, we show that high and low degrees of self-association, as monitored by this technique, are undesirable for optimal microbial specificity in these peptides. The most efficacious peptide, GS14K4, showed an intermediate degree of self-association as noted by the transition temperature, Tp, of 20°C; in comparison, the least efficacious peptide, GS14 had a transition temperature of 55°C. |
