Dataset Information

Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions.

ABSTRACT: Research on the dynamics of single-membrane proteins remains underdeveloped due to the lack of proper approaches that can probe in real time the protein's insertion depth in lipid bilayers. Here we report a single-molecule visualization method to track both vertical insertion and lateral diffusion of membrane proteins in supported lipid bilayers by exploiting the surface-induced fluorescence attenuation (SIFA) of fluorophores. The attenuation follows a d(-4) dependency, where d is the fluorophore-to-surface distance. The method is validated by observing the antimicrobial peptide LL-37 to transfer among five transmembrane positions: the surface, the upper leaflet, the centre, the lower leaflet and the bottom of the lipid bilayer. These results demonstrate the power of SIFA to study protein-membrane interactions and provide unprecedented in-depth understanding of molecular mechanisms of the insertion and translocation of membrane proteins.

SUBMITTER: Li Y

PROVIDER: S-EPMC5056435 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions.

Li Ying Y Qian Zhenyu Z Ma Li L Hu Shuxin S Nong Daguan D Xu Chunhua C Ye Fangfu F Lu Ying Y Wei Guanghong G Li Ming M

Nature communications 20160930

Research on the dynamics of single-membrane proteins remains underdeveloped due to the lack of proper approaches that can probe in real time the protein's insertion depth in lipid bilayers. Here we report a single-molecule visualization method to track both vertical insertion and lateral diffusion of membrane proteins in supported lipid bilayers by exploiting the surface-induced fluorescence attenuation (SIFA) of fluorophores. The attenuation follows a d<sup>-4</sup> dependency, where d is the f ...[more]

PMID: 27686409

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Project description:While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate for the altered interfacial interactions. Recently, we have developed a novel transmembrane peptide, termed GW(5,19)ALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide), which proves to be a well-behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW(7,17)ALP23) or outward (GW(3,21)ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane α-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state (2)H nuclear magnetic resonance experiments in varying lipid bilayer membrane environments. All of the GW(x,y)ALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC, and DLPC environments, even when the Trp residues are quite closely spaced, in GW(7,17)ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium-labeled tryptophan indole rings in the GW(x,y)ALP23 peptides provide additional insights into the behavior of the Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side chain torsion angles, permit the GW(x,y)ALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers with differing hydrophobic thicknesses.

Dataset Information

Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions.

Publications

Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions.

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