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Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

Krauson AJ, Hall OM, Fuselier T, Starr CG, Kauffman WB, Wimley WC - J. Am. Chem. Soc. (2015)

Bottom Line: Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state.While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes.The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine , New Orleans, Louisiana 70112, United States.

ABSTRACT
To better understand the sequence-structure-function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the "negative" peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

No MeSH data available.


Related in: MedlinePlus

Vesicle leakage.(A) Leakage of ANTS/DPX from 0.5 mM POPC vesiclesby peptide serially diluted from 50 to 0.024 μM. Each pointis the average of at least three independent measurements. Error barsare SEM. The gain- and loss-of-function derivatives of melittin (red)are indicated. The colors and symbols in panel A are used throughoutthis work. (B) Leakage of ANTS/DPX from 0.5 mM POPG vesicles by seriallydiluted peptide. Each point is the average of at least three independentmeasurements. Error bars are SEM. (C) Summary of leakage experiments.The midpoint of each leakage curve, expressed as 1/LIC50, is plotted against POPG content. The gain- and loss-of-functionderivatives of melittin (red) are indicated as in panel A.
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fig3: Vesicle leakage.(A) Leakage of ANTS/DPX from 0.5 mM POPC vesiclesby peptide serially diluted from 50 to 0.024 μM. Each pointis the average of at least three independent measurements. Error barsare SEM. The gain- and loss-of-function derivatives of melittin (red)are indicated. The colors and symbols in panel A are used throughoutthis work. (B) Leakage of ANTS/DPX from 0.5 mM POPG vesicles by seriallydiluted peptide. Each point is the average of at least three independentmeasurements. Error bars are SEM. (C) Summary of leakage experiments.The midpoint of each leakage curve, expressed as 1/LIC50, is plotted against POPG content. The gain- and loss-of-functionderivatives of melittin (red) are indicated as in panel A.

Mentions: In all of the experimentsthat follow, we study four closely related 26-residue, melittin-derivedpeptides in parallel: the parent sequence, melittin;the gain-of-function peptide, MelP5; the observed loss-of-functionsequence, MelN2; and the engineered loss-of-functionsequence, Mel L16G. All sequences are shown in Figure 2. The observed gain-of-functionand loss-of-function peptides, MelP5 and MelN2, both have chargesof +3 and differ by only five residues: an 81% identity. Melittinand Mel L16G have charges of +6 and differ by one residue. They are96% identical. With these four peptides, which are all highly solublein aqueous buffers, we measured peptide-induced leakage of the dyeANTS and its quencher DPX from zwitterionic POPC vesicles as a functionof peptide concentration. Results are shown in Figure 3. Melittin and MelP5 behave as reported elsewhere.30 We define the potency using the 50% leakage-inducingconcentration, or LIC50, the peptide-to-lipid ratio atwhich 50% leakage occurs. Melittin permeabilizes POPC vesicles, withLIC50 of 1:200, while MelP5, which is more potent, hasLIC50 of 1:1000. The behavior of the two loss-of-functionsequences are extraordinary; they have almost no effect on bilayerpermeability except at extremely high concentrations. Their LIC50 values are ≫1:10 (Figure 3A,C). Remarkably, even the single-residuechange, L16G, completely recapitulates the loss of function of MelN2,observed in the screen. We conclude that the L16G variation is likelyresponsible for the surprising abundance of loss-of-function variantsobserved in the screen, because 50% of library members have this variation(Figure 1).


Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

Krauson AJ, Hall OM, Fuselier T, Starr CG, Kauffman WB, Wimley WC - J. Am. Chem. Soc. (2015)

Vesicle leakage.(A) Leakage of ANTS/DPX from 0.5 mM POPC vesiclesby peptide serially diluted from 50 to 0.024 μM. Each pointis the average of at least three independent measurements. Error barsare SEM. The gain- and loss-of-function derivatives of melittin (red)are indicated. The colors and symbols in panel A are used throughoutthis work. (B) Leakage of ANTS/DPX from 0.5 mM POPG vesicles by seriallydiluted peptide. Each point is the average of at least three independentmeasurements. Error bars are SEM. (C) Summary of leakage experiments.The midpoint of each leakage curve, expressed as 1/LIC50, is plotted against POPG content. The gain- and loss-of-functionderivatives of melittin (red) are indicated as in panel A.
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fig3: Vesicle leakage.(A) Leakage of ANTS/DPX from 0.5 mM POPC vesiclesby peptide serially diluted from 50 to 0.024 μM. Each pointis the average of at least three independent measurements. Error barsare SEM. The gain- and loss-of-function derivatives of melittin (red)are indicated. The colors and symbols in panel A are used throughoutthis work. (B) Leakage of ANTS/DPX from 0.5 mM POPG vesicles by seriallydiluted peptide. Each point is the average of at least three independentmeasurements. Error bars are SEM. (C) Summary of leakage experiments.The midpoint of each leakage curve, expressed as 1/LIC50, is plotted against POPG content. The gain- and loss-of-functionderivatives of melittin (red) are indicated as in panel A.
Mentions: In all of the experimentsthat follow, we study four closely related 26-residue, melittin-derivedpeptides in parallel: the parent sequence, melittin;the gain-of-function peptide, MelP5; the observed loss-of-functionsequence, MelN2; and the engineered loss-of-functionsequence, Mel L16G. All sequences are shown in Figure 2. The observed gain-of-functionand loss-of-function peptides, MelP5 and MelN2, both have chargesof +3 and differ by only five residues: an 81% identity. Melittinand Mel L16G have charges of +6 and differ by one residue. They are96% identical. With these four peptides, which are all highly solublein aqueous buffers, we measured peptide-induced leakage of the dyeANTS and its quencher DPX from zwitterionic POPC vesicles as a functionof peptide concentration. Results are shown in Figure 3. Melittin and MelP5 behave as reported elsewhere.30 We define the potency using the 50% leakage-inducingconcentration, or LIC50, the peptide-to-lipid ratio atwhich 50% leakage occurs. Melittin permeabilizes POPC vesicles, withLIC50 of 1:200, while MelP5, which is more potent, hasLIC50 of 1:1000. The behavior of the two loss-of-functionsequences are extraordinary; they have almost no effect on bilayerpermeability except at extremely high concentrations. Their LIC50 values are ≫1:10 (Figure 3A,C). Remarkably, even the single-residuechange, L16G, completely recapitulates the loss of function of MelN2,observed in the screen. We conclude that the L16G variation is likelyresponsible for the surprising abundance of loss-of-function variantsobserved in the screen, because 50% of library members have this variation(Figure 1).

Bottom Line: Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state.While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes.The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine , New Orleans, Louisiana 70112, United States.

ABSTRACT
To better understand the sequence-structure-function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the "negative" peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

No MeSH data available.


Related in: MedlinePlus