Structural characterization of de novo designed L5K5W model peptide isomers with potent antimicrobial and varied hemolytic activities

Abstract

In an effort to develop short antimicrobial peptides with simple amino acid compositions, we generated a series of undecapeptide isomers having the L(5)K(5)W formula. Amino acid sequences were designed to be perfectly amphipathic when folded into a helical conformation by converging leucines onto one side and lysines onto the other side of the helical axis. The single tryptophans, whose positions were varied in the primary structures, were located commonly at the critical amphipathic interface in the helical wheel projection. Helical conformations and the tryptophanyl environments of the 11 L(5)K(5)W peptides were confirmed and characterized by circular dichroism, fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers exhibited a potent, broad-spectrum of antibacterial activity with just a slight variance in individual potency, whereas their hemolytic activities against human erythrocytes were significantly diversified. Interestingly, helical dispositions and fluorescence blue shifts of the peptides in aqueous trifluoroethanol solutions, rather than in detergent micelles, showed a marked linear correlation with their hemolytic potency. These results demonstrate that our de novo design strategy for amphipathic helical model peptides is effective for developing novel antimicrobial peptides and their hemolytic activities can be estimated in correlation with structural parameters.

Figure 1

Peptide design and conformational validation. Amino acid sequence of each L₅K₅Wⁿ model peptide (left panel) is labeled with the serial number on the left and the peptide name as a formula on the right. An example of the helical wheel diagram (middle panel) is depicted for the W7 peptide. Far-UV CD spectra (right panel) of the W7 peptide were measured in 10 mM phosphate buffer (pH 6.5) containing 50% TFE, 10 mM SDS micelles and 2 mM DPC micelles, respectively.

Figure 2

Hemolytic activities of the L₅K₅Wⁿ model peptides. Human red blood cells were treated with individual peptides at various concentrations and hemolysis extents (%) are plotted along the peptide concentrations. Amino acid sequences are represented by the corresponding model numbers (refer to Figure 1).

Figure 3

CD analysis of the L₅K₅Wⁿ model peptides. (A) Far-UV CD spectra of individual peptides in 10 mM phosphate buffer at pH 6.5 are superimposed; (B and C) CD spectra of the W10 peptide in phosphate buffer containing various concentrations of TFE and detergents (SDS and DPC) are represented, respectively; (D) Absolute values of mean residue molar ellipticity at 222 nm ([θ]₂₂₂; upper panel) and 208 nm ([θ]₂₀₈; lower panel) are plotted for individual peptides in three different solutions.

Figure 4

NMR analysis of the L₅K₅Wⁿ model peptides. (A) Selected regions from the 2D-COSY spectrum of the W8 peptide in 50% TFE solution are aligned to present the ¹H^α-¹H^β2/β3 connectivity of the tryptophan residue (dotted line); (B) Tryptophan ¹H^α chemical shifts of individual peptides in the 50% TFE solution are plotted.

Figure 5

Intrinsic tryptophan fluorescence of the L₅K₅Wⁿ model peptides. (A) Fluorescence emission spectra of NATA (thin lines) and the W2 peptide (thick lines), in 75% TFE (blue), 10 mM SDS (red) and 2 mM DPC (green) solutions are represented; (B) Emission λ_max in each solution is plotted for individual peptides and NATA (denoted by ‘c’, control).

Figure 6

Relationships between hemolytic activity and structural parameters of the L₅K₅Wⁿ model peptides. (A) Hemolysis extents (%) by individual peptides at a 128 μg/mL concentration were extracted from Figure 2; (B) Structural and spectroscopic parameters of individual peptides in 50% (for CD) or 75% (for fluorescence) TFE solutions. Predicted helical contents ([α]; upper panel) and the [θ]₂₂₂ × [θ]₂₀₈ values (middle panel) were derived from the CD data in Figure 3. Differences in λ_max (|Δλ_max|; bottom panel) between peptides and NATA were derived from fluorescence emission data in Figure 5. Light gray bars correspond to the light symbols in C; (C) Linear correlation between hemolytic activity and structural parameters in 50% (for CD) or 75% (for fluorescence) TFE solutions. The hemolysis % is plotted along the [θ]₂₂₂ × [θ]₂₀₈ (filled circles with bottom coordinate; left panel) and [α] (empty circles with upper coordinate; middle panel), respectively, with the corresponding regression lines determined by linear least square fitting. The coefficients of determination R² were 0.9022 (solid line) and 0.7321 (dotted line), respectively. The hemolysis % is also plotted along the |Δλ_max| (diamonds; right panel). The linear regression lines were derived using all 11 data points (dotted line; R² = 0.7776) and 9 selected data points (solid line with bold diamond symbols; R² = 0.9518), respectively.