The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

• Keywords
• MD simulation, environment-sensitive probes, lipid saturation, model membranes, spectral imaging, time-resolved emission shift,
• MeSH
• Cell Membrane chemistry   MeSH
• Cholesterol MeSH
• Fluorescent Dyes * analysis   chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Fluorescent Dyes * MeSH

Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.

• Keywords
• DFT, Laurdan, TDDFT, classical molecular dynamics, fluorescence,
• MeSH
• 1,2-Dipalmitoylphosphatidylcholine chemistry   MeSH
• 2-Naphthylamine analogs & derivatives   chemistry   MeSH
• Models, Chemical * MeSH
• Fluorescence MeSH
• Phosphatidylcholines chemistry   MeSH
• Quantum Theory MeSH
• Laurates chemistry   MeSH
• Lipid Bilayers chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1,2-Dipalmitoylphosphatidylcholine MeSH
• 1,2-oleoylphosphatidylcholine MeSH Browser
• 2-Naphthylamine MeSH
• Phosphatidylcholines MeSH
• Laurates MeSH
• laurdan MeSH Browser
• Lipid Bilayers MeSH

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

• MeSH
• Cell Membrane metabolism   MeSH
• Phospholipids chemistry   metabolism   MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Liposomes chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Calcium Signaling MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Phospholipids MeSH
• Lipid Bilayers MeSH
• Liposomes MeSH
• Calcium MeSH

Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• Kinetics MeSH
• Lipid Bilayers chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Lipid Bilayers MeSH