DOI: 10.5061/dryad.f1vhhmh7d
title: Data from: Bioinspired design rules for flipping across the lipid bilayer from systematic simulations of membrane protein segments

description: The orientation of integral membrane proteins (IMPs) with respect to the
 membrane is established during protein synthesis and insertion into the
 membrane. After synthesis, IMP orientation is thought to be fixed due to
 the thermodynamic barrier for “flipping” protein loops or helices across
 the hydrophobic core of the membrane in a process analogous to lipid
 flip-flop. A notable exception is EmrE, a homodimeric IMP with an
 N-terminal transmembrane helix that can flip across the membrane until
 flipping is arrested upon dimerization. Understanding the features of the
 EmrE sequence that permit this unusual flipping behavior would be valuable
 for guiding the design of synthetic materials capable of translocating or
 flipping charged groups across lipid membranes. To elucidate the molecular
 mechanisms underlying flipping in EmrE and derive bioinspired design
 rules, we employ atomistic molecular dynamics simulations and enhanced
 sampling techniques to systematically investigate the flipping of
 truncated segments of EmrE. Our results demonstrate that a
 membrane-exposed charged glutamate residue at the center of the N-terminal
 helix lowers the energetic barrier for flipping (from ~12.1 kcal mol-1 to
 ~5.4 kcal mol-1) by stabilizing water defects and minimizing membrane
 perturbation. Comparative analysis reveals that the marginal
 hydrophobicity of this helix, rather than the marginal hydrophilicity of
 its loop, is the key determinant of flipping propensity. Our results
 further indicate that interhelical hydrogen bonding upon dimerization
 inhibits flipping. These findings establish several bioinspired design
 principles to govern flipping in related materials: (1) marginally
 hydrophobic helices with membrane-exposed charged groups promote flipping,
 (2) modulating protonation states of membrane-exposed groups tunes
 flipping efficiency, and (3) interhelical hydrogen bonding can be
 leveraged to arrest flipping. These insights provide a foundation for
 engineering synthetic peptides, engineered proteins, and biomimetic
 nanomaterials with controlled flipping or translocation behavior for
 applications in intracellular drug delivery and membrane protein design.
description: # Data from: Bioinspired design rules for flipping across the lipid
 bilayer from systematic simulations of membrane protein segments Author:
 ByungUk Park Date: 7/22/2025 The following tarball file data.tar.gz
 contains all the data relevant to the simulations of truncated peptide
 systems introduced in this work
 ([https://doi.org/10.1039/D5ME00032G](https://doi.org/10.1039/D5ME00032G))
 and scripts for the analysis of the data. First six folders
 ('1.Nloop', '2.Npep_E14charge', '3.Cpep',
 '4.Npep_E14neutral', '5.monomer_E14charge',
 '6.Hbond_analysis') contain input and output files for the
 simulations of different peptide systems (e.g., 'index.ndx',
 '.mdp', '.top', '.tpr', '.xtc',
 etc). Each of these folders follows same organization (detailed
 explanation in '1.Nloop' section). The remaining two folders
 ('misc', 'scripts') contain scripts used to analyze
 the simulation outputs. All simulations were run with Gromacs 2021.5
 patched with PLUMED 2.8. * Simulation-related files * Some of them
 ('.gro', '.ndx', '.top', '.itp',
 '.mdp' and '.xvg') are text files that can be opened
 with typical text readers, while others ('.tpr',
 '.xtc') are binary files that require Gromacs software to read
 or run the files. * '.tpr': Simulation input (coordinates,
 parameters, etc.) * '.ndx': List of indices of atom/residue
 groups * '.top': System topology and parameters *
 '.itp': Modular components of a full topology (parameters, atom
 types, bonds, angles, dihedrals, etc.) * '.mdp': Simulation
 control parameters (simulation time, temperature, pressure, etc.) *
 '.gro': System structure (coordinates, atom type, box size) *
 '.xtc': Compressed trajectory * '.xvg': Plot/graph
 data generated by Gromacs built-in functions * Software & package
 prerequisites (tested version in parenthesis): Gromacs (2021.5), PLUMED
 (2.8), VMD (1.9), Python (3.8), mdtraj (1.9.7), numpy (1.23.5), pandas
 (2.0.3) 1.Nloop * This directory contains all simulation input and output
 files for the N-loop system, including '.gro', '.itp',
 '.mdp', and '.xtc' files. The folder is organized as
 follows: * 1.sys_prep: contains initial structure files generated by
 CHARMM-GUI * 2.smd: contains input and output files from steered molecular
 dynamics (SMD) in both (1) forwared (Flip) and (2) backward (Flop) pulling
 directions * 3.reus: contains output files from replica exchange umbrella
 samplings (REUS) simulations * 1.equilibration: output files for the
 equilibration step of REUS windows * 2.production: output files for
 production step of REUS windows * 4.us: present only in the
 '1.Nloop' directory, contains simple umbrella sampling (US)
 outputs generated from SMD pulling trajectories (both Flip, Flop) *
 1.equilibration: output files for equilibration step of US windows *
 2.production: output files for production step of US windows * mdp:
 parameter files ('.mdp') used to generate '.tpr' files
 for simulations * 'index.ndx', 'topol.top': index and
 topology files used to set up and run US simulations * output_files:
 contains post-simulation analyses * reus: weighted histogram analysis
 method (WHAM) outputs ('.xvg') from REUS using multiple sets of
 equilibration and production times * solvent_density: '.csv'
 files reporting time-averaged number densities of water and phosphorus (P)
 atoms across windows. Columns correspond to harmonic restraint positions
 along the z-axis; rows report densities at specific z-distances from the
 membrane midplane averaged over time and lateral dimensions. *
 trajectory_last_confs_concat: contains a concatenated trajectory file
 ('trajectory.xtc') of the final frame from each REUS window. *
 us: (only in '1.Nloop' directory) WHAM results for US windows
 from both pulling directions: * 1.forward_pull(Flip): results from US
 initialized from forward pulling SMD * 2.backward_pull(Flop): results from
 US initialized from backward pulling SMD * toppar: forcefield and topology
 parameters used for the simulations 2.Npep_E14charge * This directory
 contains all input and output files associated with simulations of the
 Npep_E14charge system. * The folder structure and file types follow the
 same organizational scheme as described for the '1.Nloop'
 directory. Only subdirectories and files not previously described are
 detailed below: * output_files * helicity: contains input and output files
 for analyzing time-resolved helicity of the Npep_E14charge peptide across
 window trajectories * cv_dat: output files ('.dat') that report
 time-series helicity scores for each REUS window * plumed_dat: input files
 ('.dat') used to compute helicity scores via PLUMED *
 'mcfile': configuration file used to execute the PLUMED helicity
 analysis * 'reference.pdb': reference structure used for
 helicity score calculations 3.Cpep * This directory contains all input and
 output files associated with simulations of the Cpep system. * The folder
 structure and file types follow the same organizational scheme as
 described for the '1.Nloop' and '2.Npep_E14charge'
 directories. 4.Npep_E14neutral * This directory contains all input and
 output files associated with simulations of the Npep_E14neutral system. *
 The folder structure and file types follow the same organizational scheme
 as described for the '1.Nloop' and '2.Npep_E14charge'
 directories. 5.monomer_E14charge * This directory contains all input and
 output files for simulations of N-terminal and C-terminal flipping in the
 EmrE monomer system. * The folder structure and file types follow the same
 organizational scheme as described for the '1.Nloop' and
 '2.Npep_E14charge' directories. Only subdirectories and files
 not previously described are detailed below: * c-term_flip: contains all
 input and output files for simulations of C-terminal flipping in the EmrE
 monomer * output_files/h-bond: output files from interhelical hydrogen
 bond analysis across REUS windows. Subsequent analyses and visualizations
 are generated using the 'reus_hbond_anal.ipynb' notebook in the
 'monomer_E14charge' directory. * n-term_flip: contains all input
 and output files for simulations of N-terminal flipping in the EmrE
 monomer * 'reus_hbond_anal.ipynb': jupyter notebook used to
 generate plots of average interhelical hydrogen bond counts per window,
 based on REUS trajectory data (e.g.,
 '5.monomer_E14charge/n-term_flip/output_files/h-bond')
 6.Hbond_analysis * This directory contains results from hydrogen bond
 (H-bond) analyses between transmembrane helices (TMHs) in EmrE monomer and
 dimer systems * input_files: contains simulation trajectories
 ('.xtc') and structure ('.gro') files for all systems
 evaluated in this study. File naming reflects: * System type:
 'emre_dimer' or 'emre_monomer' * E14 protonation
 state: 'E14-charged' or 'E14-neutral' * Simulation
 type: 'npt_500ns' (unbiased MD),
 'smd_backward_pull_posres', or
 'smd_forward_pull_posres' (steered MD in respective directions)
 * output_files: contains results of interhelical H-bond analysis for each
 system * For 'emre_dimer', H-bonds are categorized based on the
 helices involved: * btwPROA-PROB: interhelical H-bonds between chain A and
 chain B * PROA_helix_only: interhelical H-bonds within chain A *
 PROB_helix_only: interhelical H-bonds within chain B *
 'hbond_anal.ipynb': jupyter notebook for analyzing the
 time-averaged number of H-bonds between all helix pairs across different
 systems. Descriptions of key analysis parameters are provided within the
 code cells. misc * This directory contains scripts used for supplementary
 analyses, primarily presented in the Supporting Information (SI) *
 1.emre_sequence-based_hydropathy * 'hydropathy_plot.ipynb':
 jupyter notebook that generates a hydropathy plot for the EmrE sequence
 using Kyte-Doolittle hydropathy scores * 2.system_lipid_number_check *
 Subdirectories in the format '###_DMPC': contain simulation
 files and analysis results for systems with varying numbers of DMPC lipids
 * 'lipid_per_area.py': python script that generates summary
 ('.dat') and plot ('.eps') files of lipid number per
 area. To execute, set the 'root_dir' variable in the script to
 the target system directory and run: `$ python lipid_per_area.py` *
 3.helicity_analysis * 'COLVAR_datfile_anal_helicity.ipynb':
 jupyter notebook that generates helicity profiles for systematically
 truncated peptide systems as a function of window index scripts * scripts
 for multiple analysis of each evaluated system * density heatmap: contains
 scripts that generate number density heatmap of each system (for water
 & P atoms) * Please check README in the directory for detailed
 guidelines * reus: contains scripts that analyze the REUS results * Please
 check README in the directory for detailed guidelines