Heberle Frederick A, Doktorova Milka
Department of Chemistry, University of Tennessee Knoxville, Knoxville, TN 37916, USA.
Department of Biochemistry and Biophysics, Stockholm University, Science for Life Laboratory, Solna 171 65, Sweden.
Faraday Discuss. 2025 May 8. doi: 10.1039/d4fd00200h.
Biological membranes have two leaflets that can differ in both lipid composition and total lipid abundance. These different types of asymmetries play a major role in determining the biophysical properties of the membrane; however, they have proven challenging to assay experimentally even in simpler model systems. Molecular dynamics simulations offer the means for detailed computational investigation of systematically varied interleaflet lipid distributions, but opportunities for critical validation with wet lab experiments are scarce. To help address this problem, here we use atomistic simulations of asymmetric bilayers to generate synthetic experimental data and thus investigate the sensitivity of various approaches to changes in relative lipid composition, number, and cholesterol distribution. Contrary to trends in symmetric bilayers, the simulations showed a decrease in lipid packing with increasing cholesterol in differentially stressed asymmetric bilayers, with more pronounced changes in the more loosely packed leaflet. Representative experimental data computed from the simulation trajectories indicated that the detection of asymmetry-induced changes in leaflet properties should be possible with environment-sensitive fluorescent probes and NMR observables, but may require optimization of sample preparation conditions. On the other hand, small-angle scattering data are already experimentally accessible and can reveal differential leaflet packing densities through a model-free analysis. We further show that computationally generated cryo-EM intensity profiles are highly sensitive to phospholipid imbalance between membrane leaflets. Together, these findings provide a roadmap for developing targeted applications of the techniques and obtaining experimental data critical for validating computationally derived principles related to membrane asymmetry.
生物膜有两个小叶，其脂质组成和总脂质丰度都可能不同。这些不同类型的不对称性在决定膜的生物物理性质方面起着主要作用；然而，即使在更简单的模型系统中，它们在实验测定中也被证明具有挑战性。分子动力学模拟为系统地改变小叶间脂质分布的详细计算研究提供了手段，但通过湿实验室实验进行关键验证的机会却很少。为了帮助解决这个问题，我们在这里使用不对称双层的原子模拟来生成合成实验数据，从而研究各种方法对相对脂质组成、数量和胆固醇分布变化的敏感性。与对称双层的趋势相反，模拟结果表明，在差异应力不对称双层中，随着胆固醇含量的增加，脂质堆积减少，在堆积较松散的小叶中变化更为明显。从模拟轨迹计算出的代表性实验数据表明，使用环境敏感荧光探针和核磁共振可观测量应该能够检测到小叶性质中不对称诱导的变化，但可能需要优化样品制备条件。另一方面，小角散射数据已经可以通过实验获得，并且可以通过无模型分析揭示不同的小叶堆积密度。我们进一步表明，计算生成的冷冻电镜强度分布对膜小叶之间的磷脂失衡高度敏感。总之，这些发现为开发这些技术的靶向应用以及获得对验证与膜不对称性相关的计算推导原理至关重要的实验数据提供了路线图。
