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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

Library and screen. (A)The design of the 7776-member library isbased on the sequence of melittin (top line). The boxed and numberedpositions were varied using the residues indicated, which also includingthe native residue. (B) Results of 8000 library members screened atP:L = 1:20 using the two-step screen (see text) against POPC vesicles.Each point represents the results of a single library bead. Pointcolor is determined by local point density in rainbow order, withred representing the highest density. (C) Histogram of leakage activityfrom the library screen. The % leakage on the X-axisis the same measurement that is plotted on the Y-axisof panel B.
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fig1: Library and screen. (A)The design of the 7776-member library isbased on the sequence of melittin (top line). The boxed and numberedpositions were varied using the residues indicated, which also includingthe native residue. (B) Results of 8000 library members screened atP:L = 1:20 using the two-step screen (see text) against POPC vesicles.Each point represents the results of a single library bead. Pointcolor is determined by local point density in rainbow order, withred representing the highest density. (C) Histogram of leakage activityfrom the library screen. The % leakage on the X-axisis the same measurement that is plotted on the Y-axisof panel B.

Mentions: Inorder to learn moreabout the sequence features that modulate the activity of pore-formingpeptides, we screened for loss-of-function sequencesusing the same melittin-based library and the same lipid vesicles,made from 90% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) and 10% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol(POPG), that were used in the gain-of-function screen. We screened∼8000 individual library members at P:L = 1:20, a peptide concentrationthat is at least 10 times higher than the concentration at which melittinefficiently permeabilizes the same vesicles, and is at least 100 timeshigher than the concentration at which the best gain-of-function peptide,MelP5, efficiently permeabilizes these vesicles. The library designis shown in Figure 1A. The rationale for the 10 varied residues includes (i) modulationof conformational flexibility, (ii) changes in the angle subtendedby the polar face, (iii) disruption of a leucine zipper motif, and(iv) polarity and charge of the C-terminal tail.25 The 7776 member library explores a narrow sequence spacearound the parent sequence of melittin.


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)

Library and screen. (A)The design of the 7776-member library isbased on the sequence of melittin (top line). The boxed and numberedpositions were varied using the residues indicated, which also includingthe native residue. (B) Results of 8000 library members screened atP:L = 1:20 using the two-step screen (see text) against POPC vesicles.Each point represents the results of a single library bead. Pointcolor is determined by local point density in rainbow order, withred representing the highest density. (C) Histogram of leakage activityfrom the library screen. The % leakage on the X-axisis the same measurement that is plotted on the Y-axisof panel B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697923&req=5

fig1: Library and screen. (A)The design of the 7776-member library isbased on the sequence of melittin (top line). The boxed and numberedpositions were varied using the residues indicated, which also includingthe native residue. (B) Results of 8000 library members screened atP:L = 1:20 using the two-step screen (see text) against POPC vesicles.Each point represents the results of a single library bead. Pointcolor is determined by local point density in rainbow order, withred representing the highest density. (C) Histogram of leakage activityfrom the library screen. The % leakage on the X-axisis the same measurement that is plotted on the Y-axisof panel B.
Mentions: Inorder to learn moreabout the sequence features that modulate the activity of pore-formingpeptides, we screened for loss-of-function sequencesusing the same melittin-based library and the same lipid vesicles,made from 90% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) and 10% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol(POPG), that were used in the gain-of-function screen. We screened∼8000 individual library members at P:L = 1:20, a peptide concentrationthat is at least 10 times higher than the concentration at which melittinefficiently permeabilizes the same vesicles, and is at least 100 timeshigher than the concentration at which the best gain-of-function peptide,MelP5, efficiently permeabilizes these vesicles. The library designis shown in Figure 1A. The rationale for the 10 varied residues includes (i) modulationof conformational flexibility, (ii) changes in the angle subtendedby the polar face, (iii) disruption of a leucine zipper motif, and(iv) polarity and charge of the C-terminal tail.25 The 7776 member library explores a narrow sequence spacearound the parent sequence of melittin.

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