NMRlipids III: Preliminary version of the manuscript

 [updated 19.9.2018]

I have now updated the progress made after NMRlipids III: Preliminary observations post into the manuscript. 

After extensive discussion about correct parameters to run CHARMM36 simulations it was concluded that the results from Gromacs 5 are consistent with other simulation packages and literature. Thus, the results from CHARMM gui parameters simulated with Gromacs 5 are currently used in the manuscript. However, there might still be some issues with parameters given by CHARMM gui for systems with cholesterol.

Current comparison for acyl chain order parameters between different simulation models and experiments is shown in Fig. 1. The main conclusion is that cholesterol ordering effect is overestimated in Berger/Holtje and MacRog models, while slight overestimation is observed also in CHARMM36 and Slipid models. The significance of overestimation in CHARMM36 and Slipid is yet to be analyzed (see Things to do list below).

FIG. 1: Order parameters from simulations and experiments for acyl chains of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without cholesterol.

Final comparison between form factors from simulations and experiments is yet to be done, but area per PC headgroups and per total number of molecules from different simulations are shown in Fig. 2. The difference between models do not seem excessively large, but Berger/Holtje and MacRog models predict strongest effect due to cholesterol in line with order parameter results.

FIG. 2: Area per molecules calculated from different simulation models as a function of cholesterol concentration. Solid marks are area per total amount of molecules (chol+PC) and empty marks are area per PC headgroups. Top figure shows absolute values and bottom figure shows changes respect to pure lipid system.

Things to be done:

• We need to analyze if overestimation of order parameters with cholesterol in CHARMM36 and Slipids is significant. There are at least two issues to be clarified: 
  1) Is the quantitative accuracy of experimental order parameters same for acyl chains as for glycerol backbone and headgroup (0.02) discussed in NMRlipids I ?
  2) Can the overestimation of order parameters in simulations be explained by missing undulations, as sometimes suggested? [update 19.9.2018] As discussed in the GitHub issue linked here, the undulations does not seem to make significant difference.
  
• Lipid 14 results from model by Madej et al. should be somehow included.
• Comparison of form factors between simulations and experiments should be finished.
  
  
  
  

NMRlipids IV: Headgroup & glycerol backbone structures, and cation binding in bilayers with PE, PG and PS lipids

In NMRlipids I and II projects, the goal was to find a MD model that would correctly reproduce NMR data (for lipid headgroup & glycerol backbone structures, and for cation binding) in PC bilayers. In NMRlipids IV project, we set the same goal for PE, PG and PS lipids in bilayers (pure or mixed with PC). The standard NMRlipids workflow and rules will be applied. The current version of the manuscript is available in the GitHub repository.

Currently, the manuscript is mainly a collection of relevant experimental data. For example, Fig. 1 compares the experimental headgroup and glycerol backbone order parameters between PC, PE, PG and PS lipids.

Fig.1 Absolute values of order parameters for headgroup and glycerol
backbone with different headgroups from experiments. For references and other details see the manuscript.

The conclusion based on this, together with some additional data, has been that the headgroup structures are similar for PC, PE and PG lipids, while PS headgroup is more rigid [Wohlgemuth et al, Buldt et al.]. On the other hand, the glycerol backbone structure has been considered to be similar in model systems and cells for all these lipids [Gally et al.].

Some preliminary comparison between experiments and simulations with CHARMM GUI parameters are shown in Figs. 2 and 3, suggesting that the model has some difficulties to reproduce the experimental order parameters for PS and PG headgroups. More detailed conclusions are difficult to draw only from these data, because experimentally the signs of order parameters for PS and PG are not available (as far as I know). However, the results from other models might help to draw some connections between order parameters and structural details, as was done in NMRlipids I for PC lipids.

Fig 2.  Order parameters for POPS headgroup and glycerol backbone
from simulations and experiments. For references and details see the manuscript. Absolute
values are shown for experimental data, because signs are not
known. Simulations values are -S_CH

Fig 3. Order parameters for PG headgroup and glycerol backbone
from simulations and experiments. For references and details see the manuscript. Absolute values are shown, because signs are not known for experimental data.

Experimental data on cation binding in PC bilayers mixed with of negatively charged PG and PS lipids is shown in Fig. 6. As expected, adding CaCl₂ causes a stronger decrease in the PC headgroup order parameters when the amount of negatively charged lipids is increased. According to the NMR electrometer concept (see NMRlipids II for discussion), this means that the amount of bound Ca²⁺ increases when negatively charged lipids are present in bilayers.

Fig. 6 Changes in the PC headgroup order parameter as a function of CaCl₂ concentration in bilayers containing various amounts of negatively charged lipids. For references and details see the manuscript.

A more specific interpretation of this kind of data has been that [Seelig]:

"(i) Ca²⁺ binds to neutral lipids (phosphatidylcholine, phosphatidylethanolamine) and negatively charged lipids (phosphatidylglycerol) with approximately the same binding constant of K = 10-20 M^-1;

(ii) the free Ca²⁺ concentration at the membrane interface is distinctly enhanced if the membrane carries a negative surface charge, either due to protein or to lipid;

(iii) increased interfacial Ca²⁺ also means increased amounts of bound Ca²⁺ at neutral and charged lipids;

(iv) the actual binding step can be described by a Langmuir adsorption isotherm with a 1_lipid_:_1_Ca²⁺ stoichiometry, provided the interfacial concentration C_M is used to describe the chemical binding equilibrium."

I believe that an MD simulation model correctly reproducing the cation binding in negatively charged lipids could further sharpen this interpretation.
The goal of this project will be to test if currently available models can be used for an such interpretation. This should also help the model development (if needed), however the actual improvement of force fields is beyond the scope of NMRlipids IV.

As in all NMRlipids projects, all types of contributions (data, comments, criticism, etc.) are welcomed from everyone. The authorship of the publication will be offered to all contributors and the final acceptance is based on self-assessment according to NMRlipids rules. The following contributions would be especially relevant at this stage:

1. Results from different simulation models. Simulations of bilayers containing PE, PG or PS almost under any conditions would be currently useful to map the behavior of different models. Direct delivery of calculated order parameters through GitHub or blog comments, or by making the simulation trajectories accessible (for example, through Zenodo) would be ideal ways of contributing. 
2. Order parameter signs for PE, PG and PS. The order parameter signs are very important for the structural interpretation. However, I am not aware of the order parameter sign measurements for other than PC lipids. If such data would be somehow available, this would be highly useful contribution.