Coexisting liquid ordered (Lo) and liquid disordered (Ld) lipid phases in synthetic and plasma membrane-derived vesicles are commonly used to model the heterogeneity of biological membranes, including their putative ordered rafts. However, raft-associated proteins exclusively partition to the Ld and not the Lo phase in these model systems. We believe that the difference stems from the different microscopic structures of the lipid rafts at physiological temperature and the Lo phase studied at room temperature. To probe this structural diversity across temperatures, we performed atomistic molecular dynamics simulations, differential scanning calorimetry, and fluorescence spectroscopy on Lo phase membranes. Our results suggest that raft-associated proteins are excluded from the Lo phase at room temperature due to the presence of a stiff, hexagonally packed lipid structure. This structure melts upon heating, which could lead to the preferential solvation of proteins by order-preferring lipids. This structural transition is manifested as a subtle crossover in membrane properties; yet, both temperature regimes still fulfill the definition of the Lo phase. We postulate that in the compositionally complex plasma membrane and in vesicles derived therefrom, both molecular structures can be present depending on the local lipid composition. These structural differences must be taken into account when using synthetic or plasma membrane-derived vesicles as a model for cellular membrane heterogeneity below the physiological temperature.

• Publication type
• Journal Article MeSH

The organization of biomolecules and bioassemblies is highly governed by the nature and extent of their interactions with water. These interactions are of high intricacy and a broad range of methods based on various principles have been introduced to characterize them. As these methods view the hydration phenomena differently (e.g., in terms of time and length scales), a detailed insight in each particular technique is to promote the overall understanding of the stunning "hydration world." In this prospective mini-review we therefore critically examine time-dependent fluorescence shift (TDFS)-an experimental method with a high potential for studying the hydration in the biological systems. We demonstrate that TDFS is very useful especially for phospholipid bilayers for mapping the interfacial region formed by the hydrated lipid headgroups. TDFS, when properly applied, reports on the degree of hydration and mobility of the hydrated phospholipid segments in the close vicinity of the fluorophore embedded in the bilayer. Here, the interpretation of the recorded TDFS parameters are thoroughly discussed, also in the context of the findings obtained by other experimental techniques addressing the hydration phenomena (e.g., molecular dynamics simulations, NMR spectroscopy, scattering techniques, etc.). The differences in the interpretations of TDFS outputs between phospholipid biomembranes and proteins are also addressed. Additionally, prerequisites for the successful TDFS application are presented (i.e., the proper choice of fluorescence dye for TDFS studies, and TDFS instrumentation). Finally, the effects of ions and oxidized phospholipids on the bilayer organization and headgroup packing viewed from TDFS perspective are presented as application examples.

• Keywords
• biomembranes, calcium, cholesterol, hydration, lipid headgroups, membrane dynamics, oxidized phosholipids, time-dependent fluorescence shift,
• Publication type
• Journal Article MeSH
• Review 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

A nucleoside bearing a solvatochromic push-pull fluorene fluorophore (dCFL ) was designed and synthesized by the Sonogashira coupling of alkyne-linked fluorene 8 with 5-iodo-2'-deoxycytidine. The fluorene building block 8 and labeled nucleoside dCFL exerted bright fluorescence with significant solvatochromic effect providing emission maxima ranging from 421 to 544 nm and high quantum yields even in highly polar solvents, including water. The solvatochromism of 8 was studied by DFT and ADC(2) calculations to show that, depending on the polarity of the solvent, emission either from the planar or the twisted conformation of the excited state can occur. The nucleoside was converted to its triphosphate variant dCFLTP which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes by primer extension or PCR. The fluorene-linked DNA can be used as fluorescent probes for DNA-protein (p53) or DNA-lipid interactions, exerting significant color changes visible even to the naked eye. They also appear to be suitable for time-dependent fluorescence shift studies on DNA, yielding information on DNA hydration and dynamics.

• Publication type
• Journal Article 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

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

• MeSH
• Hydrocarbons, Brominated chemistry   MeSH
• Spectrometry, Fluorescence MeSH
• Hydrolases chemistry   genetics   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering * MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Stereoisomerism MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hydrocarbons, Brominated MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Water MeSH

The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-Gi1α (Cys(351)-Ile(351)) fusion protein. Agonist [(3)H]DADLE binding was decreased in the order Na(+) ≫ Li(+) > K(+) > (+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na(+)-responsive sites (r (2) = 0.99). High-affinity Na(+)-sites were characterized by Kd = 7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (Kd = 463 mM). Inhibition of [(3)H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [(35)S]GTPγS binding was increased in a reverse order: Na(+) < K(+) < Li(+). This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to Gi1α(Cys(351)-Ile(351)) within the δ-OR-Gi1α fusion protein, but is also valid for stimulation of endogenous G proteins of Gi/Go family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs(+) < K(+) < Na(+) < Li(+). The results are discussed in terms of interaction of Na(+), K(+) and Li(+) with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl(-), Br(-), and I(-) counter anions in inhibition of both [(3)H]DADLE and [(35)S]GTPγS binding.

• MeSH
• Cell Membrane metabolism   MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• HEK293 Cells MeSH
• Enkephalin, Leucine-2-Alanine metabolism   MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Lithium pharmacology   MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go metabolism   MeSH
• Receptors, Opioid, delta metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Sodium metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Guanosine 5'-O-(3-Thiotriphosphate) MeSH
• Enkephalin, Leucine-2-Alanine MeSH
• Lipid Bilayers MeSH
• Lithium MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go MeSH
• Receptors, Opioid, delta MeSH
• Recombinant Proteins MeSH
• Sodium MeSH