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

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.


Details of the atomistic MD simulations for the LL-37 monomer system.
We All of the MD simulations were carried out in the isothermal-isobaric (NPT) ensemble with GROMACS 4.5.3 software package 8 . The LL-37 peptide was described using GROMOS87 force field 9 . The force-field parameters of DMPG were based on Berger et al. 10 , with those for the glycerol group taken from Elmore 11 , as done previously by us 2,3,12,13 and other groups [14][15][16] . To justify the use of the modified GROMOS87 force field here instead of more recent united-atom GROMOS96-based force fields, we performed four additional independent MD simulations using the GROMOS53A6/GROMOS-CKP force field 17 .
The integration time step for MD simulations is 2 fs. Peptide bonds were constrained by the LINCS algorithm 18 and water geometries were constrained by SETTLE method 19 . The pressure was maintained at 1 bar using a semi-isotropic scheme in which the lateral and perpendicular pressures were coupled separately with a coupling constant of 1.0 ps and a compressibility of 4.5 × 10 −5 bar −1 using isotropic Parrinello-Rahman's method 20,21 . The temperature was maintained at 310 K using Nose-Hoover's method 22,23 . Long-range electrostatic interaction was calculated using the Particle Mesh Ewald (PME) method 24 with a real space cutoff of 1.2 nm, as recommended for membrane simulations, especially for those involving charged lipids. The van der Waals interaction was calculated using a cutoff of 1.4 nm.

Details of the coarse-grained MD simulations.
We performed twenty 1-μs coarse-grained (CG) MD simulations for the 8-mer LL-37 toroidal pore and four 1-μs CG-MD simulations for the 10-mer pore in DMPG lipid bilayer. All MD simulations were carried out in the isothermal-isobaric (NPT) ensemble using GROMACS 4.5.3 software package 8 . We used the MARTINI coarse-grained model [25][26][27] to simulate the lipids, amino acids and water molecules, as done in previous studies on membrane proteins [28][29][30][31][32][33]  (type Na), and the saturated fatty acid by three apolar beads (type C1) each tail 25 . Thus a DMPG lipid has one negative net charge.
System setup for LL-37 toroidal pores for the coarse-grained MD simulations.
Neutron in-plane scattering experimental studies reported that the water channel radius of the transmembrane pore formed by LL-37 is ~3.3 nm at the peptide/lipid molar ratio of 1/50. 37 As previous studies suggest LL-37 forms a toroidal pore [37][38][39][40] , an 8-membered pore model (inner radius3.2 nm) is built with the hydrophilic face of LL-37 exposed to water solution, and placed into the DMPG bilayer by the program INFLATEGRO from Tieleman's group 41 with lipids inside the pore removed. Counterions (Na+) are added to neutralize the system. This system is energy minimized and solvated with water, followed by energy minimization and a protein-position-restrained simulation for 100 ns, to obtain a well-equilibrated toroidal pore. In this work, the peptide/lipid ratio is 8/458, above the experimentally-determined peptide/lipid molar ratio of 1/50 for transmembrane pore formation 37 . We also carry out MD simulations in other peptide/lipid ratios, and find that a stable toroidal structure cannot exist when the number of peptides is less than six.

System setup for LL-37 toroidal pores for the atomistic MD simulation.
We also performed one 350-ns atomistic MD simulation in order to probe the structural stability of a more realistic LL-37 pore in DMPG bilayer. This simulation started from an octamer in which each of the eight peptide chain is in an α-helical structure and perpendicular to the normal of the DMPG bilayer. The LL-37 peptide and the DMPG lipids are described respectively using the GROMOS87 force field 9 and the modified Berger parameters 10,11 . Similar to the CG-MD simulations, the peptide/lipid ratio is 8/458. There are 127,509 atoms in this atomistic LL-37 octamer-membrane system. It took about 2 months for a 350-ns MD simulation using 96-cores on a PC-cluster. It can be seen from Supplementary Figure 8 that the LL-37 pore remained stable during the full period MD simulation and it gradually changed into a toroidal shape. In addition, each LL-37 peptide kept α-helical conformation.