Limits...
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

Sequences of peptidesidentified in the screen: top line, melittin,from which the library was designed; second line, residue variationsin the combinatorial peptide library; and third line, MelP5, the bestgain-of-function sequence identified by us in another screen.25 The loss-of-function sequences were determinedby Edman degradation using 12 randomly selected negative library members.Blue columns are varied residues. Red amino acid codes represent changesin residues that were conserved in gain-of-function sequences. Redand green rows highlight two peptides tested for activity. In termsof the changes to glycine at sequence positions Val 8 and Leu 16,MelN1 is atypical, while MelN2 is typical. The bottom two rows showthe % conservation of native residue in the loss-of-function screen(this work) and the gain-of-function screen.25
© Copyright Policy - editor-choice
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697923&req=5

fig2: Sequences of peptidesidentified in the screen: top line, melittin,from which the library was designed; second line, residue variationsin the combinatorial peptide library; and third line, MelP5, the bestgain-of-function sequence identified by us in another screen.25 The loss-of-function sequences were determinedby Edman degradation using 12 randomly selected negative library members.Blue columns are varied residues. Red amino acid codes represent changesin residues that were conserved in gain-of-function sequences. Redand green rows highlight two peptides tested for activity. In termsof the changes to glycine at sequence positions Val 8 and Leu 16,MelN1 is atypical, while MelN2 is typical. The bottom two rows showthe % conservation of native residue in the loss-of-function screen(this work) and the gain-of-function screen.25

Mentions: We randomly selected 12 loss-of-functionlibrary members for sequencingby Edman degradation. We note that up to 1 or 2% of library beadsdo not release sufficient peptide for detection of activity (unpublishedobservation). Because the amount of peptide release is not known individuallyfor the 12 negative sequences in Figure 2, it is possible that some are false negatives,due to poor release. Compared to the gain-of-function sequences, thereis more overall variability in the loss-of-function sequences. Inthe identified negatives, the four cationic residues of the C-terminus,overall, were neither conserved nor changed to uncharged residuesmore often than expected by chance (p < 0.05).Similarly, other varied residues did not show statistically significantpreferences in the negatives, presumably because the sample size issmall. However, two residues, Val 8 and Leu 16, are simultaneously(i) mostly conserved in the gain-of-function sequences, and (ii) mostlychanged to glycine in the loss-of-function sequences (Figure 2). Because Val 8-to-Glywas also found in some validated gain-of-function sequences,25 we expect that its contribution to activityis complex. Here we focus on Leu 16, which was almost completelyconserved in the gain of function variants, and was almost completelychanged to glycine the loss-of-function variants.


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)

Sequences of peptidesidentified in the screen: top line, melittin,from which the library was designed; second line, residue variationsin the combinatorial peptide library; and third line, MelP5, the bestgain-of-function sequence identified by us in another screen.25 The loss-of-function sequences were determinedby Edman degradation using 12 randomly selected negative library members.Blue columns are varied residues. Red amino acid codes represent changesin residues that were conserved in gain-of-function sequences. Redand green rows highlight two peptides tested for activity. In termsof the changes to glycine at sequence positions Val 8 and Leu 16,MelN1 is atypical, while MelN2 is typical. The bottom two rows showthe % conservation of native residue in the loss-of-function screen(this work) and the gain-of-function screen.25
© Copyright Policy - editor-choice
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697923&req=5

fig2: Sequences of peptidesidentified in the screen: top line, melittin,from which the library was designed; second line, residue variationsin the combinatorial peptide library; and third line, MelP5, the bestgain-of-function sequence identified by us in another screen.25 The loss-of-function sequences were determinedby Edman degradation using 12 randomly selected negative library members.Blue columns are varied residues. Red amino acid codes represent changesin residues that were conserved in gain-of-function sequences. Redand green rows highlight two peptides tested for activity. In termsof the changes to glycine at sequence positions Val 8 and Leu 16,MelN1 is atypical, while MelN2 is typical. The bottom two rows showthe % conservation of native residue in the loss-of-function screen(this work) and the gain-of-function screen.25
Mentions: We randomly selected 12 loss-of-functionlibrary members for sequencingby Edman degradation. We note that up to 1 or 2% of library beadsdo not release sufficient peptide for detection of activity (unpublishedobservation). Because the amount of peptide release is not known individuallyfor the 12 negative sequences in Figure 2, it is possible that some are false negatives,due to poor release. Compared to the gain-of-function sequences, thereis more overall variability in the loss-of-function sequences. Inthe identified negatives, the four cationic residues of the C-terminus,overall, were neither conserved nor changed to uncharged residuesmore often than expected by chance (p < 0.05).Similarly, other varied residues did not show statistically significantpreferences in the negatives, presumably because the sample size issmall. However, two residues, Val 8 and Leu 16, are simultaneously(i) mostly conserved in the gain-of-function sequences, and (ii) mostlychanged to glycine in the loss-of-function sequences (Figure 2). Because Val 8-to-Glywas also found in some validated gain-of-function sequences,25 we expect that its contribution to activityis complex. Here we focus on Leu 16, which was almost completelyconserved in the gain of function variants, and was almost completelychanged to glycine the loss-of-function variants.

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