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Phosphatidylinositides constitute only 1%–3% of plasma membranes but play vital roles in cellular signaling. In particular, phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in processes such as cytoskeleton organization and ion channel regulation. Pleckstrin homology (PH) domains are modular domains found in many proteins and are known for their strong affinity for PIP2 headgroups. The role of lipid composition in PH domain binding to PIP2, particularly the inclusion of phos phatidylserine (PS), is not well understood. This study explores the mechanisms of PH domain binding to PIP2 using fluores cence spectroscopy, Fourier transform infrared spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations. We find that anionic PIP2 and PS alter the interfacial environment compared to phosphatidylcholines. Additionally, the PH domain promotes the localization of anionic lipid domains upon binding. Our results highlight the role of PSinlipid domain formation within membranes and its potential influence on protein binding affinities and lipid geometries. Spe cifically, we discovered a strong interaction between PIP2 and PS whereby hydrogen bonding within these anionic lipids drives localization in the membrane. This interaction also regulates protein binding at the membrane interface. Our findings suggest that cooperativity between PIP2 and PS is key to the formation of localized lipid domains and the recruitment of proteins such as the PH domain of phospholipase C-d1 

Membranes serve diverse functions in biological systems. Variations in their molecular compositions impact their physical properties and lead to rich phase behavior such as switching from the gel to fluid phase and/or separation to micro- and macrodomains with different molecular compositions. We present a combined computational and experimental study of the phase behavior of a mixed membrane of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) molecules. This heterogeneous membrane changes from gel to fluid and shows separate domains as a function of temperature. Atomically detailed simulations provide microscopic information about these molecular assemblies. However, these systems are challenging for computations since approaching equilibrium necessitates exceptionally long molecular dynamics trajectories. We use the simulation method of MDAS (Molecular Dynamics with Alchemical Steps) to generate adequate statistics. Isotope-edited IR spectroscopy of the lipids was used to benchmark the simulations. Together, simulations and experiments provide insight into the structural and dynamical features of the phase diagram.