Influence of spectral heterogeneity of prodan and laurdan solutions on the transfer of electronic energy to octadecyl rhodamine B

Fluorescence quenching and resonance energy transfer have been studied by steady-state fluorescence spectroscopy. The experimental and theoretical values for the rate constants of the electronic energy transfer (kET) and critical radius (R0) were determined for prodan and laurdan as donors and octadecyl rhodamine B as acceptor. The spectroscopic data show, that prodan and laurdan in solution create an inhomogeneous spectroscopic medium in which multi-channel luminescence phenomena take place. This finding indicated that the modified form of the Stern-Volmer relation should be used for analyzing fluorescence quenching data. Results of performed studies point out, that dipole-dipole interaction is responsible for the resonance energy transfer from prodan and laurdan to octadecyl rhodamine B. The relative quenching efficiencies of both dyes depend on polarity of the medium and are higher for more polar solvent (AcN).     

Spectrin Organization and Dynamics: New Insights

Spectrin is the major constituent protein of the erythrocyte cytoskeleton which forms a filamentous network on the cytoplasmic face of the membrane by providing a scaffold for a variety of proteins. In this review, several aspects of spectrin organization are highlighted, particularly with respect to its ability to bind hydrophobic ligands and its interaction with membrane surfaces. The characteristic binding of the fluorescent hydrophobic probes Prodan and pyrene to spectrin, which allows an estimation of the polarity of the hydrophobic probe binding site, is illustrated. In addition, the contribution of uniquely localized and conserved tryptophan residues in the ‘spectrin repeats’ in these processes is discussed. A functional implication of the presence of hydrophobic binding sites in spectrin is its recently discovered chaperone-like activity. Interestingly, spectrin exhibits residual structural integrity even after denaturation which could be considered as a hallmark of cytoskeletal proteins. Future research could provide useful information about the possible role played by spectrin in cellular physiology in healthy and diseased states.     

Prodan Fluorescence Mimics the GroEL Folding Cycle

The non-covalent fluorescent probe 6-propionyl-2-(dimethylamino) naphthalene sulfonate (prodan) binds to hydrophobic surfaces exposed on the surface of GroEL. Under identical experimental conditions free prodan exhibits a green emission peak of intensity 390,000 cps at 520 nm. However prodan bound to GroEL, GroEL–ATP, and GroEL–ATP–GroES shows emission peaks of intensities 500,000, 540,000, and 480,000 cps at 515, 512 and 515 nm, respectively, thus mimicing the way hydrophobic surfaces on GroEL become exposed during the folding cycle. Other hydrophobic probes like bis-ANS and dansyl lysine were unable to detect the minor changes in hydrophobic exposure of GroEL after it binds to ATP, although they were able to detect hydrophobic exposure in GroEL itself.     

Specificity of Prodan for the Self-associating Domain of Spectrin: A Molecular Docking Study

Abstract

The hydrophobic fluorescent probe Prodan binds to the self-associating domain of spectrin with 1:1 stoichiometry. A model of the self-associating domain was generated based on its homology with other domains of spectrin. Prodan was then docked onto the model, and several sites with low interaction energy were identified. To verify whether the binding of Prodan is specific towards the self-associating domain of spectrin, it was docked on to several other domains of spectrin, having a known three-dimensional structure. Analysis of the docking results suggests that the binding of Prodan to the self-associating domain of spectrin will involve hydrophobic and hydrophilic groups of Prodan. The results clearly indicate the preference of Prodan for a particular binding site of the self-associating domain.     

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.     

Influence of the curvature on the water structure in the headgroup region of phospholipid bilayer studied by the solvent relaxation technique

Solvent relaxation (SR) in 1,2-dioleoyl-palmitoyl-sn-glycero-3-phosphocholine (DOPC) unilamellar vesicles of different size was probed by 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (Patman), 6-propionyl-2-dimethylaminonaphthalene (Prodan) and 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)-coumarin (DTMAC). Patman probes the amount and mobility of the bound water molecules located at the carbonyl region of the bilayer. Membrane curvature significantly accelerates the solvent relaxation process, but does not influence the total Stokes shift, showing that membrane curvature increases the mobility, without affecting the amount of water molecules present in the headgroup region. This pattern was also verified for other phosphatidylcholines. Prodan is located in the phosphate region of the bilayer and probes a more polar, mobile and heterogeneous environment than Patman. The influence of membrane curvature on SR probed by Prodan is similar, however, less pronounced compared to Patman. DTMAC (first time used in SR) shows a broad distribution of locations along the z-axis. A substantial amount of the coumarin chromophores face bulk water. No effect of curvature on SR probed by DTMAC is detectable.