NMRlipids IV: Current status and reorganization of the manuscript

First of all, I want to thank again all the contributors for delivering significant amount of useful data about PE, PG and PS headgroups for the NMRlipids IV project.

In addition to simulation results, also experimental signs of headgroup and glycerol backbone order parameters were contributed for the POPS lipid. However, experimental signs for PE and PG are not yet known, which makes the comparison between simulations and experiments more ambiguous. Therefore, I have divided the manuscript into two parts. The first one contains results only from systems with PS lipids and the other contains results from systems with PE and PG lipids. This should also ease the management and completion of the projects. The manuscript about PS lipid systems should be more straightforward to finish and I have started to compile it towards a submittable version. Some of the current results and the most important open tasks are listed below.

Headgroup & glycerol backbone structures of PS lipid bilayers

Since the order parameter signs are known for the PS lipid headgroup and glycerol backbone, we can perform similar comparison between simulations and experiments as was done in NMRlipids I for the PC lipids. This is shown Fig. 1; the subjective quality assessment is also available. The tested models perform generally less well than the PC lipid models discussed in the NMRlipids I publication. Many relevant contributions have already been made to give structural insight to the differences, however, I think this deserves more attention (see the ToDo list below).

Figure 1: Headgroup and glycerol backbone order parameters of the PS lipids from different simulation models and experiments. 

Headgroups in mixtures of PS and PC lipids

Lipid mixtures are biologically relevant, and in fact experimental data for ion binding to PS lipid bilayers seems to be available only for PS/PC mixtures. Therefore, we also address the mutual interaction between PS and PC lipid headgroups. Figure 2 shows how the addition of POPS changes the order parameters of POPC (left column), as well as the changes of POPS order parameters with increasing amount of POPC (right column). It seems that the tested simulation models do not reproduce the interactions between PC and PS headgroups very well. However, the inaccuracies in counterion binding may also disturb the lipid headgroup structures when the amount on PS (and counterions) is increasing. I think that we need data with a few more different models before drawing general conclusions, and possibly also some data with different counterion concentrations (see the ToDo list below).

Figure 2: Headgroup order parameters from PC:PS mixtures from different simulation models and experiments. Left panel shows the PC headgroup order parameters and right panel shows the PS headgroup order parameters.

Interactions between cations and lipid bilayers containing PS

As already discussed in the opening post of NMRlipids IV project, molecular electrometer experiments show that the presence of negatively charged lipids enhance cation binding in lipid bilayers. The available experimental data for the lipid headgroup order parameters of POPC:POPS (5:1) mixture as a function of CaCl₂ concentration are shown in Fig. 3 together with the simulations ran with the MacRog model. In line with the NMRlipids II publication, the PC headgroup order parameters' decrease with increasing CaCl₂ concentration is overestimated in the MacRog simulations, indicating overestimated binding of Ca²⁺ to the lipid bilayer. It should, however, be noted that the point with the lowest CaCl₂ concentration is in better agreement with the experiments. Potential explanation could be an overestimated screening effect by the overbound counterions, but this requires further analysis. Order parameters of the POPS headgroup rapidly increase or decrease even with low CaCl₂ concentration and reach almost a plateau value above 100 mM. Also the order parameter changes of the POPS headgroup are overestimated in the MacRog simulations and a qualitative agreement for the alpha carbon is unclear. Simulation data of PC:PS mixtures with different CaCl₂ concentrations is required also from other than MacRog model for more general conclusions (see the ToDo list below).

Figure 3: Headgroup order parameters from PC:PS (5:1) mixtures with different CaCl₂ concentrations from the MacRog simulation model and experiments. Left column shows the PC headgroup order parameters and right column the POPS headgroup order parameters.

ToDo list

1. Structural relevance of the observed order parameter differences between different simulation models and experiments are now analyzed using dihedral distributions (see Fig. 13 in the manuscript) and we have pictures of the sampled conformations of glycerol backbone and phosphate. I think that we should apply the tools contributed by Pavel Buslaev to calculate dihedral distributions and to visualize the structural sampling also for other contributed models. Among these data we should then select the parts giving the best representation for structural differences related to order parameters. To do this in practice, we need more simulation trajectories available in Zenodo.
2. We need data for PC:PS mixtures without additional ions from few more simulation models. Some data from Berger model has already been delivered by Lukasz Cwiklik (see also data with calcium), but we still need simulations with counterions only. Simulations of PC:PS mixtures with the Slipids and CKP models may also be useful.
3. Simulations of POPC:POPS mixtures with different CaCl₂ concentrations are needed also from other force fields than MacRog. The above mentioned dataset with Berger force field complemented with simulations containing only counterions would be highly useful. I think that simulations with CHARMM36 is a must. Also simulations with the Slipids and CKP force fields may be worth of doing. Based on NMRlipids II, it is expected that cations will overbind to lipid bilayers in these simulations. However, the behavior of PS headgroup as a function of salt concentration in different force fields with respect to experiments is not yet known.