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

Solid-state NMR spectra of TP10:(A) 19F-NMR spectra of TP10 labeled with L-CF3-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B0 (indicated by an arrow). (B) Solid-state 31P-NMR spectra of the same samples as in (A), recorded before and after the corresponding 19F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state 19F-NMR spectra of the nine L-CF3-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF3-groups were obtained for the structure calculation. All experiments were performed at 40°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099653-g003: Solid-state NMR spectra of TP10:(A) 19F-NMR spectra of TP10 labeled with L-CF3-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B0 (indicated by an arrow). (B) Solid-state 31P-NMR spectra of the same samples as in (A), recorded before and after the corresponding 19F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state 19F-NMR spectra of the nine L-CF3-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF3-groups were obtained for the structure calculation. All experiments were performed at 40°C.

Mentions: To determine the three-dimensional structure of membrane-bound TP10, we carried out solid-state 19F-NMR experiments in oriented DMPC/DMPG (3∶1) bilayers. The L-epimeric peptides were used for this analysis, as L-CF3-Bpg has the same configuration as natural amino acids. This side chain does not usually perturb the peptide conformation when substituted for a bulky hydrophobic residue, as in the case of the labeled positions Leu4, Leu5, Ile8, Leu10, Leu13, Leu16, Ile20, and Leu21, (plus another label at Gly2). The structural compatibility of L-CF3-Bpg has been demonstrated above by CD, and was previously shown also for other membrane-active peptides [40], [52]. For the NMR structure analysis we first had to determine and stay below the concentration threshold at which the L-epimeric TP10 analogs would start to aggregate. The unique sensitivity of 19F-NMR makes it possible to detect even very low peptide concentrations [49], [56], [80], allowing us to readily screen P/L ratios of 1∶50, 1∶200, and 1∶400 (Figure 3A). Only at 1∶400 did we obtain sharp 19F-NMR spectra with no signs of aggregation (as explained below) for all TP10 L-epimers (Figure 3C), even after prolonged sample storage. Under these conditions also the 31P-NMR spectra of the phospholipids confirmed a high quality in terms of the degree of lipid orientation in the samples before and after the 19F-NMR measurements, with around 85% of the lipids being well-oriented (Figure 3B).


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)

Solid-state NMR spectra of TP10:(A) 19F-NMR spectra of TP10 labeled with L-CF3-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B0 (indicated by an arrow). (B) Solid-state 31P-NMR spectra of the same samples as in (A), recorded before and after the corresponding 19F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state 19F-NMR spectra of the nine L-CF3-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF3-groups were obtained for the structure calculation. All experiments were performed at 40°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099653-g003: Solid-state NMR spectra of TP10:(A) 19F-NMR spectra of TP10 labeled with L-CF3-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B0 (indicated by an arrow). (B) Solid-state 31P-NMR spectra of the same samples as in (A), recorded before and after the corresponding 19F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state 19F-NMR spectra of the nine L-CF3-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF3-groups were obtained for the structure calculation. All experiments were performed at 40°C.
Mentions: To determine the three-dimensional structure of membrane-bound TP10, we carried out solid-state 19F-NMR experiments in oriented DMPC/DMPG (3∶1) bilayers. The L-epimeric peptides were used for this analysis, as L-CF3-Bpg has the same configuration as natural amino acids. This side chain does not usually perturb the peptide conformation when substituted for a bulky hydrophobic residue, as in the case of the labeled positions Leu4, Leu5, Ile8, Leu10, Leu13, Leu16, Ile20, and Leu21, (plus another label at Gly2). The structural compatibility of L-CF3-Bpg has been demonstrated above by CD, and was previously shown also for other membrane-active peptides [40], [52]. For the NMR structure analysis we first had to determine and stay below the concentration threshold at which the L-epimeric TP10 analogs would start to aggregate. The unique sensitivity of 19F-NMR makes it possible to detect even very low peptide concentrations [49], [56], [80], allowing us to readily screen P/L ratios of 1∶50, 1∶200, and 1∶400 (Figure 3A). Only at 1∶400 did we obtain sharp 19F-NMR spectra with no signs of aggregation (as explained below) for all TP10 L-epimers (Figure 3C), even after prolonged sample storage. Under these conditions also the 31P-NMR spectra of the phospholipids confirmed a high quality in terms of the degree of lipid orientation in the samples before and after the 19F-NMR measurements, with around 85% of the lipids being well-oriented (Figure 3B).

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