Jakob P. Ulmschneider

United Atom Lipid Parameters for Combination with the Optimized Potentials for Liquid Simulations All-Atom Force Field.

We have developed a new united-atom set of lipid force field parameters for dipalmitoylphosphatidylcholine (DPPC) lipid bilayers that can be combined with the all-atom optimized potentials for liquid simulations (OPLS-AA) protein force field. For this, all torsions have been refitted for a nonbonded 1-4 scale factor of 0.5, which is the standard in OPLS-AA. Improved van der Waals parameters have been obtained for the acyl lipid tails by matching simulation results of bulk pentadecane against recently improved experimental measurements. The charge set has been adjusted from previous lipid force fields to allow for an identical treatment of the alkoxy ester groups. This reduces the amount of parameters required for the model. Simulation of DPPC bilayers in the tension-free NPT ensemble at 50 °C gives the correct area per lipid of 62.9 ± 0.1 Å(2), which compares well with the recently refined experimental value of 63.0 Å(2). Electron density profiles and deuterium order parameters are similarly well reproduced. The new parameters will allow for improved simulation results in microsecond scale peptide partitioning simulations, which have proved problematic with prior parametrizations.

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel’s full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.

Monte Carlo backbone sampling for polypeptides with variable bond angles and dihedral angles using concerted rotations and a Gaussian bias

An efficient concerted rotation algorithm for use in Monte Carlo statistical mechanics simulations of polypeptides is reported that includes flexible bond and dihedral angles. A Gaussian bias is applied with driver bond and dihedral angles to optimize the sampling efficiency. Jacobian weighting is required in the Metropolis test to correct for imbalances in resultant transition probabilities. Testing of the methodology includes Monte Carlo simulations for polyalanines with 8–14 residues and a 36-residue protein as well as a search to find the lowest-energy conformer of the pentapeptide Met-enkephalin. The results demonstrate the formal correctness and efficiency of the method.

In Silico Partitioning and Transmembrane Insertion of Hydrophobic Peptides under Equilibrium Conditions

Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning.

Mechanism and Kinetics of Peptide Partitioning into Membranes from All-Atom Simulations of Thermostable Peptides

Partitioning properties of transmembrane (TM) polypeptide segments directly determine membrane protein folding, stability, and function, and their understanding is vital for rational design of membrane active peptides. However, direct determination of water-to-bilayer transfer of TM peptides has proved difficult. Experimentally, sufficiently hydrophobic peptides tend to aggregate, while atomistic computer simulations at physiological temperatures cannot yet reach the long time scales required to capture partitioning. Elevating temperatures to accelerate the dynamics has been avoided, as this was thought to lead to rapid denaturing. However, we show here that model TM peptides (WALP) are exceptionally thermostable. Circular dichroism experiments reveal that the peptides remain inserted into the lipid bilayer and are fully helical, even at 90 degrees C. At these temperatures, sampling is approximately 50-500 times faster, sufficient to directly simulate spontaneous partitioning at atomic resolution. A folded insertion pathway is observed, consistent with three-stage partitioning theory. Elevated temperature simulation ensembles further allow the direct calculation of the insertion kinetics, which is found to be first-order for all systems. Insertion barriers are DeltaH(in)(double dagger) = 15 kcal/mol for a general hydrophobic peptide and approximately 23 kcal/mol for the tryptophan-flanked WALP peptides. The corresponding insertion times at room temperature range from 8.5 mus to 163 ms. High-temperature simulations of experimentally validated thermostable systems suggest a new avenue for systematic exploration of peptide partitioning properties.

Spontaneous transmembrane helix insertion thermodynamically mimics translocon-guided insertion

The favorable transfer free energy for a transmembrane (TM) α-helix between the aqueous phase and lipid bilayer underlies the stability of membrane proteins. However, the connection between the energetics and process of membrane protein assembly by the Sec61/SecY translocon complex in vivo is not clear. Here, we directly determine the partitioning free energies of a family of designed peptides using three independent approaches: an experimental microsomal Sec61 translocon assay, a biophysical (spectroscopic) characterization of peptide insertion into hydrated planar lipid bilayer arrays, and an unbiased atomic-detail equilibrium folding-partitioning molecular dynamics simulation. Remarkably, the measured free energies of insertion are quantitatively similar for all three approaches. The molecular dynamics simulations show that TM helix insertion involves equilibrium with the membrane interface, suggesting that the interface may play a role in translocon-guided insertion.

How reliable are molecular dynamics simulations of membrane active antimicrobial peptides

Membrane-active antimicrobial peptides (AMPs) are challenging to study experimentally, but relatively easy to investigate using molecular dynamics (MD) computer simulations. For this reason, a large number of MD studies of AMPs have been reported over recent years. Yet relatively little effort has focused on the validity of such simulations. Are these results reliable, and do they agree with what is known experimentally? And how much meaningful information can be obtained? To answer these questions, we demonstrate here some of the requirements and limitations of running MD simulations for several common AMPs: PGLa, melittin, maculatin and BP100. The two most important findings are: (a) simulation results depend strongly on force field parameters, making experimental verification of the simulations obligatory, and (b) slow orientational and conformational fluctuations mean that much longer sampling timescales (multi-μs) are needed if quantitative agreement between simulation averages and experimental data is to be achieved. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Reorientation and Dimerization of the Membrane-Bound Antimicrobial Peptide PGLa from Microsecond All-Atom MD Simulations

The membrane-active antimicrobial peptide PGLa from Xenopus laevis is known from solid-state (2)H-, (15)N-, and (19)F-NMR spectroscopy to occupy two distinct α-helical surface adsorbed states in membranes: a surface-bound S-state with a tilt angle of ~95° at low peptide/lipid molar ratio (P/L = 1:200), and an obliquely tilted T-state with a tilt angle of 127° at higher peptide concentration (P/L = 1:50). Using a rapid molecular-dynamics insertion protocol in combination with microsecond-scale simulation, we have characterized the structure of both states in detail. As expected, the amphiphilic peptide resides horizontally on the membrane surface in a monomeric form at a low P/L, whereas the T-state is seen in the simulations to be a symmetric antiparallel dimer, with close contacts between small glycine and alanine residues at the interface. The computed tilt angles and azimuthal rotations, as well as the quadrupolar splittings predicted from the simulations agree with the experimental NMR data. The simulations reveal many structural details previously inaccessible, such as the immersion depth of the peptide in the membrane and the packing of the dimerization interface. The study highlights the ability and limitations of current state-of-the-art multimicrosecond all-atom simulations of membrane-active peptides to complement experimental data from solid-state NMR.

Conformational States of Melittin at a Bilayer Interface

The distribution of peptide conformations in the membrane interface is central to partitioning energetics. Molecular-dynamics simulations enable characterization of in-membrane structural dynamics. Here, we describe melittin partitioning into dioleoylphosphatidylcholine lipids using CHARMM and OPLS force fields. Although the OPLS simulation failed to reproduce experimental results, the CHARMM simulation reported was consistent with experiments. The CHARMM simulation showed melittin to be represented by a narrow distribution of folding states in the membrane interface.

Folding Simulations of the Transmembrane Helix of Virus Protein U in an Implicit Membrane Model.

Vpu is an 81-amino-acid auxiliary membrane protein encoded by human immunodeficiency virus type 1 (HIV-1). One of its roles is to amplify viral release by self-assembling in homo-oligomers to form functional water-filled pores enabling the flux of ions across the membrane. Various NMR and CD studies have shown that the transmembrane domain of Vpu has a helical conformation. With a recently developed implicit membrane model and an efficient Monte Carlo (MC) algorithm using concerted backbone rotations, we simulate the folding of the transmembrane domain of Vpu at atomic resolution. The implicit membrane environment is based on the generalized Born theory and enables very long time scale events, such as folding to be observed using detailed all-atom representation of the protein. Such studies are currently computationally unfeasible with fully explicit lipid bilayer molecular dynamics simulations. The correct helical transmembrane structure of Vpu is predicted from extended conformations and remains stably inserted. Tilt and kink angles agree well with experimental estimates from NMR measurements. The experimentally observed change in tilt angle in membranes of varying hydrophobic width is accurately reproduced. The extensive simulation of a pentamer of the Vpu transmembrane domain in the implicit membrane gives results similar to the ones reported previously for fully explicit bilayer simulations.