Understanding the free-energy landscape of phase separation in lipid bilayers using molecular dynamics.

Liquid-liquid phase separation inside the cell often results in biological condensates that can critically affect cell homeostasis. Such phase separation events occur in multiple parts of cells, including the cell membranes, where the "lipid raft" hypothesis posits the formation of ordered domains floating in a sea of disordered lipids. The resulting lipid domains often have functional roles. However, the thermodynamics of lipid phase separation and their resulting mechanistic effects on cell function and dysfunction are poorly understood. Understanding such complex phenomena in cell membranes, with their diverse lipid compositions, is exceptionally difficult. For these reasons, simple model systems that can recapitulate similar behavior are widely used to study this phenomenon. Despite these simplifications, the timescale and length scales of domain formation pose a challenge for molecular dynamics (MD) simulations. Thus, most MD studies focus on spontaneous lipid phase separation-essentially measuring the sign (but not the amplitude) of the free-energy change upon separation-rather than directly interrogating the thermodynamics. Here, we propose a proof-of-concept pipeline that can directly measure this free energy by combining coarse-grained MD with enhanced sampling protocols using a novel collective variable. This approach will be a useful tool to help connect the thermodynamics of phase separation with the mechanistic insights already available from MD simulations.

Understanding the Free Energy Landscape of Phase Separation in Lipid Bilayers using Molecular Dynamics.

Liquid-liquid phase separation (LLPS) inside the cell often results in biological condensates that can critically impact cell homeostasis. Such phase separation events occur in multiple parts of cells, including the cell membranes, where the so-called "lipid raft" hypothesis posits the formation of ordered domains floating in a sea of disordered lipids. The resulting lipid domains often have functional roles. However, the thermodynamics of lipid phase separation and their resulting mechanistic effects on cell function and dysfunction are poorly understood. Understanding such complex phenomena in cell membranes, with their diverse lipid compositions, is exceptionally difficult. For this reasons, simple model systems that can recapitulate similar behavior are widely used to study this phenomenon. Despite these simplifications, the timescale and and length scales of domain formation pose a challenge for molecular dynamics (MD) simulations. Thus, most MD studies focus on spontaneous lipid phase separation - essentially measuring the sign (but not the amplitude) of the free energy change upon separation - rather than directly interrogating the thermodynamics. Here, we propose a proof-of-concept pipeline that can directly measure this free energy by combining coarse-grained MD with enhanced sampling protocols using a novel collective variable. This approach will be a useful tool to help connect the thermodynamics of phase separation with the mechanistic insights already available from molecular dynamics simulations. SIGNIFICANCE: Standard molecular dynamics simulations can determine the sign the free energy change upon phase separation, but not the amplitude. We present a new method to determine the phase separation free energy for lipid membranes, based on a enhanced sampling using the weighted ensemble method combined with a novel collective variable, validated using coarse-grained simulations applied to several simple systems. The new method will be valuable as a way to develop models that connect molecular-level structural features to the thermodynamics of phase separation.

Enthalpic and entropic contributions to interleaflet coupling drive domain registration and antiregistration in biological membrane.

Biological membrane is a complex self-assembly of lipids, sterols, and proteins organized as a fluid bilayer of two closely stacked lipid leaflets. Differential molecular interactions among its diverse constituents give rise to heterogeneities in the membrane lateral organization. Under certain conditions, heterogeneities in the two leaflets can be spatially synchronized and exist as registered domains across the bilayer. Several contrasting theories behind mechanisms that induce registration of nanoscale domains have been suggested. Following a recent study showing the effect of position of lipid tail unsaturation on domain registration behavior, we decided to develop an analytical theory to elucidate the driving forces that create and maintain domain registry across leaflets. Towards this, we formulated a Hamiltonian for a stacked lattice system where site variables capture the lipid molecular properties such as the position of unsaturation and various other interactions that could drive phase separation and interleaflet coupling. We solve the Hamiltonian using Monte Carlo simulations and create a complete phase diagram that reports the presence or absence of registered domains as a function of various Hamiltonian parameters. We find that the interleaflet coupling should be described as a competing enthalpic contribution due to interaction of lipid tail termini, primarily due to saturated-saturated interactions, and an interleaflet entropic contribution from overlap of unsaturated tail termini. A higher position of unsaturation is seen to provide weaker interleaflet coupling. Thermodynamically stable nanodomains could also be observed for certain points in the parameter space in our bilayer model, which were further verified by carrying out extended Monte Carlo simulations. These persistent noncoalescing registered nanodomains close to the lower end of the accepted nanodomain size range also point towards a possible "nanoscale" emulsion description of lateral heterogeneities in biological membrane leaflets.

Nanoscale structures and mechanics of peptide nucleic acids.

Peptide nucleic acids (PNAs) are charge-neutral polyamide oligomers having extremely favorable thermal stability and high affinity to cell membranes when coupled with cationic cell-penetrating peptides (CPPs), as well as the encouraging antisense and antigene activity in cell-free systems. The study of the mechanical properties of short PNA molecules is rare both in experiments and theoretical calculations. Here, we studied the microscopic structures and elastic properties; namely, persistence length, stretch modulus, twist-stretch coupling, and structural crookedness of double-stranded PNA (dsPNA) and their hybrid derivatives using all-atom MD simulation and compared them with those of double-stranded DNA (dsDNA) and double-stranded RNA (dsRNA). The stretch modulus of the dsPNA is found to be ∼160 pN, an order of magnitude lower than that of dsDNA and smaller than dsRNA, respectively. Similarly, the persistence length of dsPNA is found to be ∼35 nm, significantly smaller than those of dsDNA and dsRNA. The PNA-DNA and PNA-RNA hybrid duplexes have elastic properties lying between that of dsPNA and dsDNA/dsRNA. We argue that the neutral backbones of the PNA make it less stiff than dsDNA and dsRNA molecules. Measurement of structural crookedness and principal component analysis additionally support the bending flexibility of dsPNA. Detailed analysis of the helical-rise coupled to helical-twist indicates that the PNA-DNA hybrid over-winds like dsDNA, while PNA-PNA and PNA-RNA unwind like dsRNA upon stretching. Because of the highly flexible nature of PNA, it can bind other biomolecules by adopting a wide range of conformations and is believed to be crucial for future nanobiotechnology research studies.