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

Fangfu Ye,Ying Li,Daguan Nong,Shuxin Hu,Ming Li,Chunhua Xu,Li Ma,Zhenyu Qian,Ying Lu,Guanghong Wei

doi:10.1038/ncomms12906

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.

Highlights

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
The technique is analogous to the fluorescence resonance energy transfer (FRET) exept that in surface-induced fluorescence attenuation (SIFA), energy is transferred from a point to a plane while in FRET, energy is transferred from a point to another point
We demonstrated that SIFA is a powerful tool to track the insertion depth of single-membrane proteins/peptides and to distinguish which part of a protein/peptide inserts how deeply into the supported lipid bilayer

Summary

Introduction

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. 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 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. We report a single-molecule imaging method to visualize threedimensional (3D) motions of membrane proteins in supported lipid bilayers, taking advantage of the surface-induced fluorescence attenuation (SIFA) of single emitting fluorophores[3,4,5,6]. SIFA enabled us to measure the transmembrane positions of the dye-labelled LL-37 with nanometre precision, revealing the existence of five transmembrane states of the peptide

Methods

Results

Conclusion