Fluorescence Depolarization Studies Research Articles

Tuning effect of local environment to control mechanism of fluorescence depolarization: Rotational diffusion and resonance energy transfer within homo-aggregates of xanthenes

Controlling excitation energy transfer within self-assembled molecular aggregates has been a major challenge since such systems represent as prototypes for highly efficient light-harvesting pigment assemblies within photosynthetic organisms. Using time-resolved fluorescence depolarization studies, here we report complete reversal of trends in anisotropy decay traces up on increasing fluorophore concentration while traversing a series of solvent mixtures (water and glycerol) with increasing viscosity. For glycerol, in dilute solution depolarization results from rotational diffusion whereas Förster resonance energy transfer (FRET) is found to be the key mechanism in concentrated solution; in contrast, for water, rotational diffusion is the dominant reason for fluorescence depolarization at all concentrations. We further investigate how viscosity, rather than dielectric constant of the solvent, controls contribution from FRET towards depolarization by partially immobilizing the fluorophores. The results are explained within the framework of analytical models for fluorescence depolarization due to either rotational diffusion or FRET. We further examine the unusual ‘dip-and-rise’ behavior in anisotropy traces for intermediate range of concentration which is suggestive of presence of heterogeneity within homochromophoric system. These results show how one can fine-tune the explicit nature of solvent to facilitate FRET over other competing dynamical processes.

Effect of fibrillation on the excited state dynamics of tryptophan in serum protein – A time-resolved fluorescence study

Abstract The aggregation of proteins into the amyloid fibrils is mainly responsible for several neurological diseases. Knowledge of dynamics in amyloid fibril is very essential to understand its biological activity. Although, the effects of environment like pH, ionic strength, temperature, etc. on the fibril have been studied extensively, studies on the dynamics of amyloid fibril and the role of water in fibril are scarce. In this article, we have reported the results on the excited state dynamics of amyloid fibrils formed by a well-known blood plasma protein, human serum albumin (HSA), at neutral pH. The sole tryptophan residue, W214, has been used as the intrinsic fluorescent probe to monitor its excited state dynamics. Steady-state and time-resolved fluorescence data suggests that the W214 becomes more closer to the quencher amino acid residues in the fibrillar phase than in the native protein. From detailed time-resolved emission measurements, it is shown that despite having a more ordered structure, the water molecules around W214 in amyloid fibril is more labile than that in the native protein. Fluorescence depolarization studies also indicate that the W214 residue is located in a relatively more flexible region of the ordered amyloid fibril than that in the native protein.

Island versus Archipelago Architecture for Asphaltenes: Polycyclic Aromatic Hydrocarbon Dimer Theoretical Studies

To shed some light on the problem of the most likely architecture, island or archipelago, of the polycyclic aromatic hydrocarbon (PAH) core in asphaltenes, molecular orbital (MO) calculations have been carried out considering two PAHs per asphaltene molecule cross-linked (archipelago model) and the same two PAHs aggregated in a parallel fashion and not cross-linked (island model of interacting PAHs). The calculated highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gaps (H―L gaps) are compared to the 0―0 band experimental fluorescence emission data of asphaltenes. The calculations of the H―L gap were performed at the semi-empirical ZINDO approach using density functional theory (DFT) dimer-optimized structures (relaxed structures). This methodology was validated against experimental data and it was proven to have a good predictive power. A total of 48 PAHs with 5―10 fused aromatic rings (5FAR―10FAR) were calculated, as well as all of their possible combinations in dimers formed by large core (5FAR―10FAR)―large core (5FAR― 10FAR) systems (large―large core dimers). In total, 1176 systems in the archipelago architecture, crossed linked with a C 5 alkane chain, and 1176 systems in the island-stacked interaction were calculated. Also, island and archipelago models were constructed with the 48 PAHs, interacting in both architectures, with 10 PAHs that contain 1―4 fused aromatic rings in the structure (1FAR―4FAR), thus comprising 960 calculations of large core (5FAR―10FAR)―small core (1FAR―4FAR) dimers (large-small core dimers). Therefore, a total of 3312 systems are calculated in this work. For both cases, large-large and large―small dimer systems, and for both architectures, island and archipelago, statistical analysis of the results was carried out. It is found that the calculated H―L gap of the dimers with the archipelago architecture is similar to the H―L gap of the monomers. For the case of the island dimers (stacked dimers), there is a dependency of the H―L gap upon both the size of the monomers, in terms of the number of fused aromatic rings, and the type of interlayer geometries or stacking arrangement between the monomers composing the island (stacked) dimer. The stacked dimers are more stable in most of the cases than the archipelago dimers. The higher stability in turn produces larger H―L gaps. The remarkably broad variation of the energy gap, of 0.9 eV, between the island and archipelago architectures for the large―large dimers strongly depends upon the dimer size and the stacking arrangement. In the later, the π―π interactions play a role but have a minor influence. The stacked island systems that fall inside the experimental range of asphaltenes are, in general, dimers composed with the same monomer in hexagonal (H) arrangement and 6FAR―10FAR combinations with parallel displacement (PD) arrangement, mainly without fjords or coves in the structure. Of all of the PAH systems that fall inside the experimental range of asphaltenes when interacting with any other PAH, to form a dimer, in either island or archipelago architecture, the 7FAR PAHs are more abundant in agreement with the former literature. When the percentage of all the calculated dimers, large-large and large―small, is compared, in both architectures, and whose H― L gap falls inside the experimental asphaltene fluorescence range, the large―large dimers are more abundant than the large―small dimers. The large-large dimers are more abundant by 20.6% in island architecture, and by 17.9% in archipelago architecture. It is concluded that indeed, if two asphaltene core PAHs are directly bound via a single bond, then optical methods, such as time-resolved fluorescence depolarization (TRFD) studies, would identify this as a single chromophore, thereby blurring the distinction between island versus archipelago. Some decomposition studies might find this single-bonded pair of PAHs as a single entity as well.

Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

Interaction Between Zwitterionic and Anionic Surfactants: Spontaneous Formation of Zwitanionic Vesicles

The physicochemical properties, such as critical micelle concentration (cmc), surface tension at cmc (γ(cmc)), and surface activity parameters of the mixtures of a new amino acid-based zwitterionic surfactant, N-(n-dodecyl-2-aminoethanoyl)-glycine (C(12)Gly) and an anionic surfactant, sodium dodecyl sulfate (SDS) at different molar fractions, X(1) (= [C(12)Gly]/([C(12)Gly] + [SDS])) of C(12)Gly were studied. A synergistic interaction was observed between the surfactants in mixtures of different X(1). The self-organization of the mixtures at different molar fractions, concentrations, and pH was investigated. Fluorescence depolarization studies in combination with dynamic light scattering, and transmission electron microscopic and confocal fluorescence microscopic images suggested the formation of bilayer vesicles in dilute solutions of SDS rich mixtures with X(1) ≤ 0.17 in the pH range 7.0 to 9.0. However, the electronic micrographs showed structures with fingerprint-like texture in moderately dilute to concentrated C(12)Gly/SDS mixture at X(1) = 0.50. The vesicles were observed to transform into small micelles upon lowering the solution pH and upon increase of total surfactant concentration in mixtures with X(1) ≤ 0.17. However, decrease of SDS content transformed vesicles into wormlike micelles. The structural transitions were correlated with bulk viscosity of the binary mixtures.

Temperature Response of Self-Assembled Micelles of Telechelic Hydrophobically Modified Poly(2-alkyl-2-oxazoline)s in Water

Hydrophobically end-modified (HM) poly(2-ethyl-2-oxazolines) (PEtOx) and poly(2-isopropyl-2-oxazolines) (PiPrOx) bearing an n-octadecyl chain on both termini or on one chain end only were prepared by cationic ring-opening polymerization of 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, respectively, and subsequent end-group modification. The polymers had a molar mass (Mn) ranging from 7000 to 13 000 g mol−1, a size distribution Mw/Mn < 1.20, and end-group functionality > 0.97. All polymers, except the semitelechelic sample C18-PiPrOx-OH 13K (Mn = 13 000 g mol−1), formed core−shell micelles in cold water with a hydrodynamic radius (RH), measured by dynamic light scattering, between 7 and 12 nm and a core of radius (Rc), determined by analysis of small-angle X-ray scattering (SAXS) data, of ∼1.3 nm. Aqueous solutions of all polymers underwent a heat-induced phase transition detected by an increase in solution turbidity at a temperature (Tcp, cloud point) ranging from 32 to 62 °C, depending on polymer structure and size. Temperature-dependent light scattering (LS) measurements and fluorescence depolarization studies with the probe diphenylhexatriene (DPH) revealed that extensive intermicellar bridging takes place in solutions heated in the vicinity of Tcp leading to large assemblies (RH ≥ 1 μm). Further heating caused these assemblies to shrink into objects with RH ∼ 300−700 nm, depending on the size and structure of the polymer. The formation of H-bonds between water molecules and the main-chain amide nitrogen atoms imparts distinct features to the flower/star micelles formed by telechelic/semitelechelic PiPrOx and PEtOx, compared to the micelles formed by other hydrophobically end-modified water-soluble polymers, such as poly(ethylene oxide) or poly(N-isopropylacrylamide).

Excited-State Symmetry and Reorientation Dynamics of Perylenes in Liquid Solutions: Time-Resolved Fluorescence Depolarization Studies Using One- and Two-Photon Excitation

The excited-state symmetry and molecular reorientation of perylene, 1,7-diazaperylene, and 2,5,8,11-tetra- tert-butylperylene have been studied by different fluorescence depolarization experiments. The first excited electronic singlet state was reached through one-photon excitation (OPE) and two-photon excitation (TPE). A 400 and 800 nm femtosecond laser pulse was used for this purpose, and data were collected by means of the time-correlated single-photon counting technique. It is found that the rotational correlation times for each perylene derivative are very similar in the OPE and TPE depolarization experiments. For the determination of the two-photon absorption tensor, a recently described theoretical model has been applied (Ryderfors et al. J. Phys. Chem. A 2007, 111, 11531). It was found that the two-photon process can be described by a 2 x 2 absorption tensor for which the components are solvent dependent and exhibit mixed vibronic character. In the dipole approximation this is compatible with a parity-forbidden two-photon absorption into the first excited singlet state.

Fluorescence Depolarization Studies of Filamentous Actin Analyzed with a Genetic Algorithm

A new method, in which a genetic algorithm was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyze fluorescence depolarization data collected by the time-correlated single photon-counting technique. It was applied to studies of BODIPY-labeled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by electronic energy migration within the actin polymers. Analyses of F-actin samples composed of different fractions of labeled actin molecules revealed the known helical organization of F-actin, demonstrating the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerization experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.

Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences

We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.

Thermal Unfolding Process of Dihydrolipoamide Dehydrogenase Studied by Fluorescence Spectroscopy

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.

Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine

Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] plays a key role in the modulation of actin polymerization and vesicle trafficking. These processes seem to depend on the enrichment of PI(4,5)P(2) in plasma membrane domains. Here, we show that PI(4,5)P(2) does not form domains when in a fluid phosphatidylcholine matrix in the pH range of 4.8-8.4. This finding is at variance with the spontaneous segregation of PI(4,5)P(2) to domains as a mechanism for the compartmentalization of PI(4,5)P(2) in the plasma membrane. Water/bilayer partition of PI(4,5)P(2) is also shown to be dependent on the protonation state of the lipid.

Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences†

We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.

Domain Motions and the Open-to-Closed Conformational Transition of an Enzyme: A Normal Mode Analysis ofS-Adenosyl-l-homocysteine Hydrolase

The structure and fluctuations of the enzyme S-adenosyl-L-homocysteine hydrolase (SAHH) are analyzed in an effort to explain its biological function. Besides the previously identified open structure, characteristic of the substrate-free enzyme, we find two distinct structures in enzyme-inhibitor complexes, the closed and closed-twisted conformers. Both closed conformers differ from the open form by a hinge bending motion of two large domains within each subunit, which isolate the inhibitor bound in the active site from the bulk solvent. The closed-twisted form further differs from the closed form by a rigid body twist of the two-subunit dimers. The local structural fluctuations of SAHH are analyzed by performing block normal mode analysis of the tetrameric enzyme in its three forms. For the open form, we find that the four lowest-frequency normal modes, corresponding to the collective motions of the protein with the largest amplitudes, are essentially combinations of the hinge bending deformations of the individual subunits. Thus, the mechanical properties of the open structure of SAHH lead to the presence of structural fluctuations in the direction of the open-to-closed conformational transition. A candidate for such a motion has been observed in previous fluorescence depolarization studies of the enzyme. Both structural and normal mode analyses indicate that residues 180-190 and 350-356 form hinge regions, connecting large domains which tend to move as rigid bodies in response to interactions with substrate, intermediates, and the product of the enzymatic reactions. We propose that these hinge regions play a crucial role in the enzymatic mechanism of SAHH. In contrast to the open form, normal mode calculations for the closed conformations show strong coupling of the hinge bending motions of the individual subunits to each other and to other low-frequency vibrations. Thus, information about structural changes related to reaction progress in one active site may be mechanically transmitted to other subunits of the protein, explaining the cooperativity found in the enzyme kinetics.

Morphology‐dependent luminescence properties of poly(benzyl ether) dendrimers

AbstractPoly(benzyl ether) dendrimers with o‐, m‐, and p‐isomers of dialkoxybenzene at their focal points [o‐, m‐, and p‐(Gn)2Ar], having generation numbers (n) of 0–3, were synthesized. 1H NMR pulse relaxation times (T1) of the exterior MeO groups of o‐ and m‐(Gn)2Ar (n = 0–3) all remained in the range of 0.92–1.43 s. In sharp contrast, an exceptionally short T1 value (0.23 s) was observed for p‐(G3)2Ar. Although their absorption spectral profiles were slightly different from one another, an essential difference was observed for their fluorescence properties. When the generation number was increased, the fluorescence efficiency of o‐(Gn)2Ar increased, but that of p‐(Gn)2Ar decreased, whereas m‐(Gn)2Ar exhibited a relatively small change in the fluorescence efficiency. Fluorescence depolarization studies showed a highly efficient intramolecular energy migration in p‐(G3)2Ar as compared with o‐(G3)2Ar and m‐(G3)2Ar. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3524–3530, 2003

Elucidation of the Nature of the Conformational Changes of the EF-interhelical Loop in Bacteriorhodopsin and of the Helix VIII on the Cytoplasmic Surface of Bovine Rhodopsin: A Time-resolved Fluorescence Depolarization Study

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150 mM KCl and 20 °C requires a surface potential change that corresponds to Δσ≈−1.0 e −/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.

An EPR and Fluorescence Depolarization Study of Intermolecular Interactions of Dendrimers at Medium and Highly Concentrated Aqueous Solutions

The intermolecular dendrimer interactions between poly (amidoamine) dendrimers (SBDs) of different generations ( G=2, 4, and 6) were studied in aqueous solution by EPR and fluorescence depolarization. For the EPR studies a spin probe (TEMPO) was covalently linked to the SBDs, and for the fluorescence depolarization studies a fluorophor (fluorescein) was covalentely linked to the SBDs. The mobility of the TEMPO labeled SBDs was determined by EPR over a range of the concentration of added SBDs (unlabeled SBD of the same generation) from 0.01 to 75% (w/w) in water solutions. The mobility of the TEMPO label was not influenced by added SBD below a concentration of approximately 5%, suggesting that no significant interdendrimer interactions, such as aggregation, occurred. Above a concentration of 30% SBD in water (w/w), the mobility of the TEMPO label decreased dramatically; the effect is mostly attributed to the viscosity increase at such high concentrations. Fluorescence depolarization studies, employing fluorescein-labeled dendrimers are in agreement with the EPR studies.

Dendritic multiporphyrin arrays as light-harvesting antennae: effects of generation number and morphology on intramolecular energy transfer.

A series of star- and cone-shaped dendritic multiporphyrin arrays, (nPZn)4PFB and (nPZn)1PFB, respectively, that contain energy-donating dendritic zinc porphyrin (PZn) wedges of different numbers (n = 1, 3, and 7) of the PZn units, attached to an energy-accepting free-base porphyrin (PFB) core, were synthesized by a convergent growth approach. For the cone-shaped series ((nPZn)1PFB), the efficiency of energy transfer (phi ENT) from the photoexcited PZn units to the focal PFB core, as evaluated from the fluorescence lifetimes of the PZn units, considerably decreased as the generation number increased: (1PZn)1PFB (86%), (3PZn)1PFB (66%), and (7PZn)1PFB (19%). In sharp contrast, the star-shaped series ((nPZn)4PFB) all showed high phi ENT values: (1PZn)4PFB (87%), (3PZn)4PFB (80%), and (7PZn)4PFB (71%). Energy transfer efficiencies of (3PZn)4-ester-PFB, (1PZn)4-ester-PFB, and (3PZn)1-ester-PFB, whose dendritic PZn wedges are connected by an ester linkage to the PFB core, were almost comparable to those of the corresponding ether-linked versions. Fluorescence depolarization (P) studies showed much lower P values for star-shaped (7PZn)4PFB and (3PZn)4PFB than cone-shaped (7PZn)1PFB and (3PZn)1PFB, respectively, indicating a highly efficient energy migration among the PZn units in the star-shaped series. Such a morphology-assisted photochemical event is probably responsible for the excellent light-harvesting activity of large (7PZn)4PFB molecules.

Mobility of Poly(Ethylene Oxide) Segments on Surface of Lecithin Liposomes

To better understand the dynamics of PEO-lipid-modified phosphatidylcholine liposomes (“fusogenic liposomes”), the mobility of the PEO segment on the liposomal surface and the fluidity of the bilayer were investigated by measuring the spin-lattice relaxation time (T1by 13C-NMR spectroscopy and by fluorescence depolarization. 13C-NMR studies revealed that the PEO-lipids in this work exhibit similar behavior on liposomal surfaces irrespective of the PEO chain length. Furthermore, with regard to the relationship between morphology and dynamics of the PEO-lipid and phosphatidylcholine mixture, it was found that the mobility of PEO segments are altered by the morphology; liposome < aqueous micelle < monomeric dispersion in organic solution. Fluorescence depolarization studies revealed that liposomal membranes were tighter with the addition of PEO-lipid(12,a31) than with the addition of PEO-lipid(12,a13). In contrast, the reconstitution of PEO-lipid(12,a5) into liposomes increased membrane fluidity.

Effect of Adriamycin on the boundary lipid structure of cytochrome c oxidase: pico-second time-resolved fluorescence depolarization studies

The fluorescence dynamics of the dye 3,3′-diethyloxadicarbocyanine iodide (DODCI) was used to probe the microenvironment of cytochrome c oxidase (C cO) and cardiolipin. The dye was partitioned between an aqueous and a hydrophobic phase. The ‘bound’ and ‘free’ populations of DODCI could be separated by analysis of the time-resolved fluorescence decay of the dye. The anisotropy decay of the DODCI bound to C cO showed a unique ‘dip and rise’ shape that was analyzed by a combination of rotational correlation times with time-dependent weight factors for each lifetime component. Rotational dynamics studies revealed the existence of a restricted motion of the dye bound at the enzyme surface. Adriamycin, an anticancer, albeit cardiotoxic drug, was previously proposed to affect the surface structure of C cO, most likely by causing a disorder to the surface lipid arrangement. A drastic change in the rotational correlation time of the dye bound to the enzyme surface was observed, which suggested a depletion of cardiolipin layer due to complexation with the drug. The effect of Adriamycin on cardiolipin was drastic, leading to its phase separation. The present study suggests that the effect of Adriamycin on C cO is primarily a segregation of the cardiolipins.

Molecular dynamics simulation of local motion of polystyrene chain end—comparison with the fluorescence depolarization study

Molecular dynamics (MD) simulation of the local motion of a polystyrene (PS) chain with anthryl group at the chain end surrounded by benzene molecules was performed and the results were compared with those obtained experimentally by the fluorescence depolarization method. The molecular weight dependence of the relaxation time of the probe obtained by the MD simulation was qualitatively in agreement with the results obtained by the fluorescence depolarization method. We also estimated the molecular weight dependence of the relaxation time for the end-to-end vector. Below the degree of polymerization (DP)≤3, the mean relaxation time Tm for the end-to-end vector was similar to that for the vector corresponding to the transition moment of the probe. With the increase of DP, the Tm for the probe tended to reach an asymptotic value unlike that for the end-to-end vector, which monotonically increased with DP. This indicates that the entire motion of a polymer coil contributes to the local motion to a lesser extent as the molecular weight increases. The MD simulations using artificial restraints showed that the rotational relaxation of the probe at the chain end for a dynamically stiff PS chain is realized by the cooperative rotation of the main chain bonds. The internal modes which takes place below 5 monomer units mainly led to the rotational relaxation of the probe at the PS chain end. Finally, the change of Tm with the position along the PS main chain was examined.