Inverse Conformational Selection in Lipid-Protein Binding.

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses.

On the Effect of the Various Assumptions and Approximations used in Molecular Simulations on the Properties of Bio-Molecular Systems: Overview and Perspective on Issues.

Computer simulations of molecular systems enable structure-energy-function relationships of molecular processes to be described at the sub-atomic, atomic, supra-atomic or supra-molecular level and plays an increasingly important role in chemistry, biology and physics. To interpret the results of such simulations appropriately, the degree of uncertainty and potential errors affecting the calculated properties must be considered. Uncertainty and errors arise from (1) assumptions underlying the molecular model, force field and simulation algorithms, (2) approximations implicit in the interatomic interaction function (force field), or when integrating the equations of motion, (3) the chosen values of the parameters that determine the accuracy of the approximations used, and (4) the nature of the system and the property of interest. In this overview, advantages and shortcomings of assumptions and approximations commonly used when simulating bio-molecular systems are considered. What the developers of bio-molecular force fields and simulation software can do to facilitate and broaden research involving bio-molecular simulations is also discussed.

Molecular Study of Ultrasound-Triggered Release of Fluorescein from Liposomes.

Several investigations have suggested that ultrasound triggers the release of drugs encapsulated into liposomes at acoustic pressures low enough to avoid cavitation or high hyperthermia. However, the mechanism leading to this triggered release as well as the adequate composition of the liposome membrane remains unknown. Here, we investigate the ultrasound-triggered release of fluorescein disodium salt encapsulated into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-distearoylphosphatidyl-ethanolamine (DSPC) lipids with various concentrations of cholesterol (from 0 to 44 mol %). The passive release of encapsulated fluorescein was first characterized. It was observed to be higher when the membrane is in a fluid phase and increased with temperature but decreased upon addition of cholesterol. Next, the release of fluorescein was measured at different acoustic frequencies (0.8, 1.1, and 3.3 MHz) and peak-to-peak pressures (0, 2, 2.5, 5, and 8 MPa). Measurements were performed at temperatures where DOPC and DSPC liposomes were, respectively, in the fluid or gel phase. We found that the release rate did not depend on the ultrasound frequency. For DOPC liposomes, the ultrasound-triggered release of fluorescein decreased with increasing concentration of cholesterol in liposomes, while the behavior was more complex for DSPC liposomes. Overall, the triggered release from DSPC liposomes was up to ten times less than DOPC liposomes. Molecular dynamics simulations performed on a pure DOPC membrane showed that a membrane experiences, under a directional pressure of ±2.4 MPa, various changes in properties such as the area per lipid (APL). An increase in the APL was notably observed when the simulation box was laterally stretched or perpendicularly compressed, which was accompanied by an increase in the number of water molecules crossing the membrane. This suggests that ultrasound most probably enhances the diffusion of encapsulated molecules at small acoustic pressures by increasing the distance between lipids.

Biophysical Insight on the Membrane Insertion of an Arginine-Rich Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this peptide toward zwitterionic lipids.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

Interdigitation between Triglycerides and Lipids Modulates Surface Properties of Lipid Droplets.

Intracellular lipid droplets (LDs) are the main cellular site of metabolic energy storage. Their structure is unique inside the cell, with a core of esterified fatty acids and sterols, mainly triglycerides and sterol esters, surrounded by a single monolayer of phospholipids. Numerous peripheral proteins, including several that were previously associated with intracellular compartments surrounded by a lipid bilayer, have been recently shown to target the surface of LDs, but how they are able to selectively target this organelle remains largely unknown. Here, we use atomistic and coarse-grained molecular dynamics simulations to investigate the molecular properties of the LD surface and to characterize how it differs from that of a lipid bilayer. Our data suggest that although several surface properties are remarkably similar between the two structures, key differences originate from the interdigitation between surface phospholipids and core neutral lipids that occurs in LDs. This property is extremely sensitive to membrane undulations, unlike in lipid bilayers, and it strongly affects both lipid-packing defects and the lateral pressure profile. We observed a marked change in overall surface properties for surface tensions >10 mN/m, indicative of a bimodal behavior. Our simulations provide a comprehensive molecular characterization of the unique surface properties of LDs and suggest how the molecular properties of the surface lipid monolayer can be modulated by the underlying neutral lipids.

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Simulating Bilayers of Nonionic Surfactants with the GROMOS-Compatible 2016H66 Force Field.

Polyoxyethylene glycol alkyl ether amphiphiles (CiEj) are important nonionic surfactants, often used for biophysical and membrane protein studies. In this work, we extensively test the GROMOS-compatible 2016H66 force field in molecular dynamics simulations involving the lamellar phase of a series of CiEj surfactants, namely C12E2, C12E3, C12E4, C12E5, and C14E4. The simulations reproduce qualitatively well the monitored structural properties and their experimental trends along the surfactant series, although some discrepancies remain, in particular in terms of the area per surfactant, the equilibrium phase of C12E5, and the order parameters of C12E3, C12E4, and C12E5. The polar head of the CiEj surfactants is highly hydrated, almost like a single polyethyleneoxide (PEO) molecule at full hydration, resulting in very compact conformations. Within the bilayer, all CiEj surfactants flip-flop spontaneously within tens of nanoseconds. Water-permeation is facilitated, and the bending rigidity is 4 to 5 times lower than that of typical phospholipid bilayers. In line with another recent theoretical study, the simulations show that the lamellar phase of CiEj contains large hydrophilic pores. These pores should be abundant in order to reproduce the comparatively low NMR order parameters. We show that their contour length is directly correlated to the order parameters, and we estimate that they should occupy approximately 7-10% of the total membrane area. Due to their highly dynamic nature (rapid flip-flops, high water permeability, observed pore formation), CiEj surfactant bilayers are found to represent surprisingly challenging systems in terms of modeling. Given this difficulty, the results presented here show that the 2016H66 parameters, optimized independently considering pure-liquid as well as polar and nonpolar solvation properties of small organic molecules, represent a good starting point for simulating these systems.

Overlay databank unlocks data-driven analyses of biomolecules for all.

Tools based on artificial intelligence (AI) are currently revolutionising many fields, yet their applications are often limited by the lack of suitable training data in programmatically accessible format. Here we propose an effective solution to make data scattered in various locations and formats accessible for data-driven and machine learning applications using the overlay databank format. To demonstrate the practical relevance of such approach, we present the NMRlipids Databank-a community-driven, open-for-all database featuring programmatic access to quality-evaluated atom-resolution molecular dynamics simulations of cellular membranes. Cellular membrane lipid composition is implicated in diseases and controls major biological functions, but membranes are difficult to study experimentally due to their intrinsic disorder and complex phase behaviour. While MD simulations have been useful in understanding membrane systems, they require significant computational resources and often suffer from inaccuracies in model parameters. Here, we demonstrate how programmable interface for flexible implementation of data-driven and machine learning applications, and rapid access to simulation data through a graphical user interface, unlock possibilities beyond current MD simulation and experimental studies to understand cellular membranes. The proposed overlay databank concept can be further applied to other biomolecules, as well as in other fields where similar barriers hinder the AI revolution.

Amphipathic lipid packing sensor motifs: probing bilayer defects with hydrophobic residues.

Sensing membrane curvature allows fine-tuning of complex reactions that occur at the surface of membrane-bound organelles. One of the most sensitive membrane curvature sensors, the Amphipathic Lipid Packing Sensor (ALPS) motif, does not seem to recognize the curved surface geometry of membranes per se; rather, it recognizes defects in lipid packing that arise from membrane bending. In a companion paper, we show that these defects can be mimicked by introducing conical lipids in a flat lipid bilayer, in agreement with experimental observations. Here, we use molecular-dynamics (MD) simulations to characterize ALPS binding to such lipid bilayers. The ALPS motif recognizes lipid-packing defects by a conserved mechanism: peptide partitioning is driven by the insertion of hydrophobic residues into large packing defects that are preformed in the bilayer. This insertion induces only minor modifications in the statistical distribution of the free packing defects. ALPS insertion is severely hampered when monounsaturated lipids are replaced by saturated lipids, leading to a decrease in packing defects. We propose that the hypersensitivity of ALPS motifs to lipid packing defects results from the repetitive use of hydrophobic insertions along the monotonous ALPS sequence.

Conical lipids in flat bilayers induce packing defects similar to that induced by positive curvature.

In biological membranes, changes in lipid composition or mechanical deformations produce defects in the geometrical arrangement of lipids, thus allowing the adsorption of certain peripheral proteins. Here, we perform molecular dynamics simulations on bilayers containing a cylindrical lipid (PC) and a conical lipid (DOG). Profiles of atomic density and lateral pressure across the bilayer show differences in the acyl chain region due to deeper partitioning of DOG compared to PC. However, such analyses are less informative for the interfacial region where peripheral proteins adsorb. To circumvent this limitation, we develop, to our knowledge, a new method of membrane surface analysis. This method allows the identification of chemical defects, where hydrocarbon chains are accessible to the solvent, and geometrical defects, i.e., voids deeper than the glycerol backbone. The size and number of both types of defects increase with the number of monounsaturated acyl chains in PC and with the introduction of DOG, although the defects do not colocalize with the conical lipid. Interestingly, the size and probability of the defects promoted by DOG resemble those induced by positive curvature, thus explaining why conical lipids and positive curvature can both drive the adsorption of peripheral proteins that use hydrophobic residues as membrane anchors.

Amphipathic-Lipid-Packing-Sensor interactions with lipids assessed by atomistic molecular dynamics.

The Amphipathic-Lipid-Packing-Sensor (ALPS) motif targets the protein ArfGAP1 to curved membranes during vesicle formation in the Golgi apparatus. ALPS specifically recognizes lipid packing defects due to the positive curvature of budding vesicles. In this work we assessed the microscopic interactions between ALPS and two phospholipid membranes at different degrees of lipid packing by explicit molecular dynamics (MD). Simulations were performed within loosely packed membranes composed of a mixture of dioleoylphosphatidylcholine (DOPC)/dioleoylglycerol (DOG) at a molar ratio 85:15. Some other simulations were performed in pure DOPC for which lipid packing is tighter. We show that the presence of DOG causes packing defects at the phosphate level and thereby modifies some properties of the bilayer. This leads to a higher hydration of the lipid headgroups. When embedded in a membrane with such defects, ALPS displays a higher degree of conformational flexibility than in a more packed membrane. We propose that lipid packing sensing by ALPS may have an entropic origin and that its flexibility is a key feature.

Anionic lipids induce a fold-unfold transition in the membrane-translocating Engrailed homeodomain.

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.

Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin.

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.

Cooperation of Conical and Polyunsaturated Lipids to Regulate Initiation and Processing of Membrane Fusion.

The shape of lipids has long been suspected to be a critical determinant for the control of membrane fusion. To experimentally test this assertion, we used conical and malleable lipids and measured their influence on the fusion kinetics. We found that, as previously suspected, both types of lipids accelerate fusion. However, the implicated molecular mechanisms are strikingly different. Malleable lipids, with their ability to change shape with low energy cost, favor fusion by decreasing the overall activation energy. On the other hand, conical lipids, with their small polar head relative to the area occupied by the hydrophobic chains, tend to make fusion less energetically advantageous because they tend to migrate towards the most favorable lipid leaflet, hindering fusion pore opening. They could however facilitate fusion by generating hydrophobic defects on the membranes; this is suggested by the similar trend observed between the experimental rate of fusion nucleation and the surface occupied by hydrophobic defects obtained by molecular simulations. The synergy of dual-process, activation energy and nucleation kinetics, could facilitate membrane fusion regulation in vivo.

Interpretation of 2H-NMR experiments on the orientation of the transmembrane helix WALP23 by computer simulations.

Orientation, dynamics, and packing of transmembrane helical peptides are important determinants of membrane protein structure, dynamics, and function. Because it is difficult to investigate these aspects by studying real membrane proteins, model transmembrane helical peptides are widely used. NMR experiments provide information on both orientation and dynamics of peptides, but they require that motional models be interpreted. Different motional models yield different interpretations of quadrupolar splittings (QS) in terms of helix orientation and dynamics. Here, we use coarse-grained (CG) molecular dynamics (MD) simulations to investigate the behavior of a well-known model transmembrane peptide, WALP23, under different hydrophobic matching/mismatching conditions. We compare experimental (2)H-NMR QS (directly measured in experiments), as well as helix tilt angle and azimuthal rotation (not directly measured), with CG MD simulation results. For QS, the agreement is significantly better than previously obtained with atomistic simulations, indicating that equilibrium sampling is more important than atomistic details for reproducing experimental QS. Calculations of helix orientation confirm that the interpretation of QS depends on the motional model used. Our simulations suggest that WALP23 can form dimers, which are more stable in an antiparallel arrangement. The origin of the preference for the antiparallel orientation lies not only in electrostatic interactions but also in better surface complementarity. In most cases, a mixture of monomers and antiparallel dimers provides better agreement with NMR data compared to the monomer and the parallel dimer. CG MD simulations allow predictions of helix orientation and dynamics and interpretation of QS data without requiring any assumption about the motional model.

Influence of the Treatment of Nonbonded Interactions on the Thermodynamic and Transport Properties of Pure Liquids Calculated Using the 2016H66 Force Field.

The effect of different treatments of the nonbonded interactions in simulations employing the recently introduced GROMOS-compatible 2016H66 force field is evaluated based on calculations carried out with the GROMACS software. This is done considering four thermodynamic and transport properties (pure liquid density, vaporization enthalpy, surface-tension coefficient, and self-diffusion constant) of 58 organic liquids representative of the chemical groups alcohol, ether, aldehyde, ketone, carboxylic acid, ester, amine, amide, thiol, sulfide, disulfide, and aromatic compounds, also including water (SPC model). A dipalmitoylphosphatidylcholine bilayer system is considered as well. The simulated properties are found to be very sensitive to the treatment of the long-range dispersion interactions, notably for the least polar systems. In general, the treatment of the long-range electrostatic or Lennard-Jones interactions using homogeneous correction terms or lattice-sum approaches yield similar results, with punctual discrepancies. The combination of a lattice-sum approach for the electrostatic interactions with a straight-cutoff truncation of the Lennard-Jones interactions at a distance of at least 1.2 nm is found to represent a good compromise setup within GROMACS for achieving compatibility with the reference results obtained using GROMOS as well as a comparable level of agreement with the experimental data. This study also reveals two potential issues with the GROMACS software, related to an incorrect calculation of the pressure when using LINCS in version 4.0.7 and an inadequate implementation of the twin-range scheme in version 5.1.2.

Effect of Dimethyl Sulfoxide on the Binding of 1-Adamantane Carboxylic Acid to ß- and γ-Cyclodextrins.

Most therapeutic targets are proteins whose binding sites are hydrophobic cavities. For this reason, the majority of drugs under development are hydrophobic molecules exhibiting low solubility in water. To tackle this issue, a few percent of cosolvent, such as dimethyl sulfoxide (DMSO), is usually employed to increase drug solubility during the drug screening process. However, the few published studies dealing with the effect of adding DMSO showed that the affinity of hydrophobic ligands is systematically underestimated. To better understand the effect of DMSO, there is a need of studying its effect on a large range of systems. In this work, we used ß- and γ-cyclodextrins (made of 6 and 7 α-d-glucopyranoside units, respectively) as models of hydrophobic cavities to investigate the effect of the addition 5% DMSO on the affinity of 1-adamantane carboxylic acid (ADA) to these cyclodextrins. The two systems differ by the size of the cyclodextrin cavity. The evaluation of binding constants was performed using ultrasound velocimetry, nuclear magnetic resonance spectroscopy, and molecular simulations. All techniques show that the presence of 5% DMSO does not significantly modify the affinity of ADA for γ-cyclodextrin, while the affinity is dramatically reduced for ß-cyclodextrin. The bias induced by the presence of DMSO is thus more important when the ligand volume better fits the cyclodextrin cavity. Our work also suggests that free energy calculations provide a sound alternative to experimental techniques when dealing with poorly water-soluble drugs.

On the orientation of a designed transmembrane peptide: toward the right tilt angle?

The orientation of the transmembrane peptide WALP23 under small hydrophobic mismatch has been assessed through long-time-scale molecular dynamics simulations of hundreds of nanoseconds. Each simulation gives systematically large tilt angles (>30 degrees). In addition, the peptide visits various azimuthal rotations that mostly depend on the initial conditions and converge very slowly. In contrast, small tilt angles as well as a well-defined azimuthal rotation were suggested by recent solid-state 2H NMR studies on the same system. To optimally compare our simulations with NMR data, we concatenated the different trajectories in order to increase the sampling. The agreement with 2H NMR quadrupolar splittings is spectacularly better when these latter are back-calculated from the concatenated trajectory than from any individual simulation. From these ensembled-average quadrupolar splittings, we then applied the GALA method as described by Strandberg et al. (Biophys J. 2004, 86, 3709-3721), which basically derives the peptide orientation (tilt and azimuth) from the splittings. We find small tilt angles (6.5 degrees), whereas the real observed tilt in the concatenated trajectory presents a higher value (33.5 degrees). We thus propose that the small tilt angles estimated by the GALA method are the result of averaging effects, provided that the peptide visits many states of different azimuthal rotations. We discuss how to improve the method and suggest some other experiments to confirm this hypothesis. This work also highlights the need to run several and rather long trajectories in order to predict the peptide orientation from computer simulations.

A GROMOS-Compatible Force Field for Small Organic Molecules in the Condensed Phase: The 2016H66 Parameter Set.

This article reports on the calibration and validation of a new GROMOS-compatible parameter set 2016H66 for small organic molecules in the condensed phase. The calibration is based on 62 organic molecules spanning the chemical functions alcohol, ether, aldehyde, ketone, carboxylic acid, ester, amine, amide, thiol, sulfide, and disulfide, as well as aromatic compounds and nucleic-acid bases. For 57 organic compounds, the calibration targets are the experimental pure-liquid density ρliq and the vaporization enthalpy ΔHvap, as well as the hydration free energy ΔGwat and the solvation free energy ΔGche in cyclohexane, at atmospheric pressure and at (or close to) room temperature. The final root-mean-square deviations (RMSD) for these four quantities over the set of compounds are 32.4 kg m(-3), 3.5 kJ mol(-1), 4.1 kJ mol(-1), and 2.1 kJ mol(-1), respectively, and the corresponding average deviations (AVED) are 1.0 kg m(-3), 0.2 kJ mol(-1), 2.6 kJ mol(-1), and 1.0 kJ mol(-1), respectively. For the five nucleic-acid bases, the parametrization is performed by transferring the final 2016H66 parameters from analogous organic compounds followed by a slight readjustment of the charges to reproduce the experimental water-to-chloroform transfer free energies ΔGtrn. The final RMSD for this quantity over the five bases is 1.7 kJ mol(-1), and the corresponding AVED is 0.8 kJ mol(-1). As an initial validation of the 2016H66 set, seven additional thermodynamic, transport, and dielectric properties are calculated for the 57 organic compounds in the liquid phase. The agreement with experiment in terms of these additional properties is found to be reasonable, with significant deviations typically affecting either a specific chemical function or a specific molecule. This suggests that in most cases, a classical force-field description along with a careful parametrization against ρliq, ΔHvap, ΔGwat, and ΔGche results in a model that appropriately describes the liquid in terms of a wide spectrum of its physical properties.