Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

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 19F-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 19F-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.

Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions

“…Third, a deletion of a residue in the amphipathic segment has an effect of changing the amino acid register on the helical wheel, interspersing the hydrophobic and hydrophilic side chains. Several studies have shown that such mutations can alter the structure and/or topology of the peptides [58]. Based on these considerations, we anticipate that the naturally occurring PLN mutations at Arg9 to a hydrophobic residue may have a similar effect, with the regulatory cytoplasmic domain of the R9X mutants exhibiting altered dynamics relative to the wild type.…”

UV-CD12: synchrotron radiation circular dichroism beamline at ANKA

“…Like all the other 9 existing SRCD beamlines worldwide, UV-CD12 has a standard experimental setup for static liquidstate experiments on proteins/peptides/nucleic acids, which is the basic equipment for all structural biology related work. In addition, based on the specialized expertise at IBG-2 and long-term solid-state NMR experience in the structural characterization of membrane-active peptides and transmembrane proteins in macroscopically oriented lipid samples (Fanghä nel et al, 2014;Strandberg et al, 2013;Walther et al, 2010), a new in-house-built SR-OCD experimental station has been integrated. It is the only SR-OCD based setup worldwide for solid samples with automated rotational averaging and data acquisition, and it provides improved spectral data quality especially for unsaturated and long-chain lipid environments at wavelengths < 200 nm.…”

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

“…This can involve the formation of lipid domains, an increase of bilayer thickness and curvature, and even tubulation of lipid vesicles at high P:L ratios [57]. Similarly, circular dichroism spectra and simulations showed that transportan 10 is highly helical when bound to vesicles, and inserts its hydrophobic face into the bilayer[43,59,62–64]. After insertion into synthetic lipid vesicles, transportan interacts with lipid phosphates causing lipid rearrangement and changes in local curvature in the bilayer[25,47].…”