Limits...
Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state.

Fanghänel S, Wadhwani P, Strandberg E, Verdurmen WP, Bürck J, Ehni S, Mykhailiuk PK, Afonin S, Gerthsen D, Komarov IV, Brock R, Ulrich AS - PLoS ONE (2014)

Bottom Line: Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide.On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration.The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry and DFG-Center for Functional Nanostructures (CFN), Karlsruhe, Germany.

ABSTRACT
Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

Show MeSH

Related in: MedlinePlus

Structure and aggregating behavior of TP10.(A) Monomeric structure of TP10 as determined from 19F-NMR in the membrane-bound state. The N-terminal galanin-derived region (green) is intrinsically unstructured, while the mastoparan-derived C-terminus (red) is folded as an α-helix. (B) When D-CF3-Bpg (black) is introduced into the flexible region, there is no conformational change. (C) However, when this stiff D-amino acid is placed into the helical C-terminal region, it leads to unfolding. At low concentration the monomers remain unstructured, but at high concentration surface-induced aggregation leads to the assembly of β-pleated amyloid-like fibrils. These cross-β-sheet assemblies must be distorted in the immediate vicinity of the D-amino acid. There is no indication if the β-sheets are parallel or antiparallel. As the β-sheets have no preferential orientation in the lipid bilayer (and they may be twisted), they give powder-type NMR spectra.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061077&req=5

pone-0099653-g009: Structure and aggregating behavior of TP10.(A) Monomeric structure of TP10 as determined from 19F-NMR in the membrane-bound state. The N-terminal galanin-derived region (green) is intrinsically unstructured, while the mastoparan-derived C-terminus (red) is folded as an α-helix. (B) When D-CF3-Bpg (black) is introduced into the flexible region, there is no conformational change. (C) However, when this stiff D-amino acid is placed into the helical C-terminal region, it leads to unfolding. At low concentration the monomers remain unstructured, but at high concentration surface-induced aggregation leads to the assembly of β-pleated amyloid-like fibrils. These cross-β-sheet assemblies must be distorted in the immediate vicinity of the D-amino acid. There is no indication if the β-sheets are parallel or antiparallel. As the β-sheets have no preferential orientation in the lipid bilayer (and they may be twisted), they give powder-type NMR spectra.

Mentions: We have shown that TP10 can assume three distinctly different conformations in the membrane-bound state (Figure 9). The monomeric structure has a well-defined α-helix in the C-terminal region, and a flexible N-terminus that may be regarded as intrinsically unstructured in the plane of the membrane (Figure 9A). This picture is consistent with an earlier NMR analysis in detergent micelles [47], and with a more recent MD study of TP10 in a POPC bilayer [93]. If the helix is perturbed, the peptide unfolds completely and starts to aggregate as amyloid-like fibrils in a concentration and time dependent manner. While the incorporation of L-CF3-Bpg as a 19F-NMR label does not significantly perturb the peptide, the sterically obstructive D-epimer can be used to dramatically shift the transition from the partially α-helical monomer ↔ unfolded monomer ↔ β-pleated aggregate. Remarkably, the tendency to aggregate depends distinctly on the position of the substitution. When D-CF3-Bpg is incorporated into the intrinsically flexible N-terminal region of TP10, the peptide maintains its usual partially α-helical structure (Figure 9B). A substitution in the C-terminal region, on the other hand, leads to unfolding and subsequent aggregation as β-sheets at high concentration (Figure 9C).


Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state.

Fanghänel S, Wadhwani P, Strandberg E, Verdurmen WP, Bürck J, Ehni S, Mykhailiuk PK, Afonin S, Gerthsen D, Komarov IV, Brock R, Ulrich AS - PLoS ONE (2014)

Structure and aggregating behavior of TP10.(A) Monomeric structure of TP10 as determined from 19F-NMR in the membrane-bound state. The N-terminal galanin-derived region (green) is intrinsically unstructured, while the mastoparan-derived C-terminus (red) is folded as an α-helix. (B) When D-CF3-Bpg (black) is introduced into the flexible region, there is no conformational change. (C) However, when this stiff D-amino acid is placed into the helical C-terminal region, it leads to unfolding. At low concentration the monomers remain unstructured, but at high concentration surface-induced aggregation leads to the assembly of β-pleated amyloid-like fibrils. These cross-β-sheet assemblies must be distorted in the immediate vicinity of the D-amino acid. There is no indication if the β-sheets are parallel or antiparallel. As the β-sheets have no preferential orientation in the lipid bilayer (and they may be twisted), they give powder-type NMR spectra.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061077&req=5

pone-0099653-g009: Structure and aggregating behavior of TP10.(A) Monomeric structure of TP10 as determined from 19F-NMR in the membrane-bound state. The N-terminal galanin-derived region (green) is intrinsically unstructured, while the mastoparan-derived C-terminus (red) is folded as an α-helix. (B) When D-CF3-Bpg (black) is introduced into the flexible region, there is no conformational change. (C) However, when this stiff D-amino acid is placed into the helical C-terminal region, it leads to unfolding. At low concentration the monomers remain unstructured, but at high concentration surface-induced aggregation leads to the assembly of β-pleated amyloid-like fibrils. These cross-β-sheet assemblies must be distorted in the immediate vicinity of the D-amino acid. There is no indication if the β-sheets are parallel or antiparallel. As the β-sheets have no preferential orientation in the lipid bilayer (and they may be twisted), they give powder-type NMR spectra.
Mentions: We have shown that TP10 can assume three distinctly different conformations in the membrane-bound state (Figure 9). The monomeric structure has a well-defined α-helix in the C-terminal region, and a flexible N-terminus that may be regarded as intrinsically unstructured in the plane of the membrane (Figure 9A). This picture is consistent with an earlier NMR analysis in detergent micelles [47], and with a more recent MD study of TP10 in a POPC bilayer [93]. If the helix is perturbed, the peptide unfolds completely and starts to aggregate as amyloid-like fibrils in a concentration and time dependent manner. While the incorporation of L-CF3-Bpg as a 19F-NMR label does not significantly perturb the peptide, the sterically obstructive D-epimer can be used to dramatically shift the transition from the partially α-helical monomer ↔ unfolded monomer ↔ β-pleated aggregate. Remarkably, the tendency to aggregate depends distinctly on the position of the substitution. When D-CF3-Bpg is incorporated into the intrinsically flexible N-terminal region of TP10, the peptide maintains its usual partially α-helical structure (Figure 9B). A substitution in the C-terminal region, on the other hand, leads to unfolding and subsequent aggregation as β-sheets at high concentration (Figure 9C).

Bottom Line: Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide.On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration.The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry and DFG-Center for Functional Nanostructures (CFN), Karlsruhe, Germany.

ABSTRACT
Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

Show MeSH
Related in: MedlinePlus