Steady-state Fluorescence Anisotropy Research Articles

Study on intramolecular charge transfer processes, solvation dynamics and rotational relaxation of coumarin 490 in reverse micelles of cationic gemini surfactant

The steady-state fluorescence properties, steady-state fluorescence anisotropy, solvation dynamics and rotational relaxation of coumarin 490 (C-490) have been studied in cationic gemini surfactant 1,4-bis(dodecyl-N,N-dimethylammoniumbromide)butane (12-4-12)–cyclohexane–n-pentanol–water reverse micelles (RMs). C-490 molecule resides in the water pool of RMs. A lesser extent of hydrogen bonding interactions occurs between the –OH group of the alcohol and the C-490 molecule at the interface of the present RMs as compared to the RMs of reported conventional cationic surfactants. The effect of the more hydrophobic nature of coumarin 480 (C-480) used to study the RMs of conventional cationic surfactants cannot be ruled out. C-490 migrates to the water pool with increasing content of water molecules. Therefore, the results of steady-state fluorescence properties, rotational relaxation and solvation dynamics depend on the water loading of RMs, unlike the RMs of conventional cationic surfactants. Thus, the solvation dynamics in gemini surfactant RMs are one order of magnitude faster as compared to the RMs of conventional cationic surfactants. The faster solvation dynamics in gemini surfactant RMs as compared with the reported AOT RMs are due to the absence of hydrogen bonding interactions between the water molecules and quaternary ammonium headgroups of gemini surfactants. The polarity of the surrounding microenvironment is found to have very little effect on the rates of non-radiative processes in the water pool of gemini surfactant RMs. The rates of non-radiative processes in the present RMs are found to be higher than those in reported AOT RMs.

Molecular Mechanism of Dream -Presenilin-1 Interactions

Downstream Regulatory Element Antagonist Modulator (DREAM), also known as calsenilin or potassium channel interacting protein 3 (KChIP3), interacts with presenilin 1 (PS1) and presenilin 2 (PS2) to promote accumulation of β-amyloid peptide in brain. To understand the molecular mechanism by which DREAM mediates β-amyloid formation, the interactions between calsenilin and presenilin were characterized using steady-state and time-resolved fluorescence anisotropy techniques. In addition, the structural changes in DREAM oligomerization state triggered by presenilin binding were studied. Here, we report the interaction between DREAM and a short segment of PS1 C-terminus (helix-9). The binding of DREAM to helix-9 is calcium dependent. The dissociation constant for Ca2+DREAM:helix-9 complexes was determined to be 0.77 ± 0.03 μM, whereas the Mg2+DREAM:complex helix-9 exhibits lower affinity, Kd = 19.3 ± 0.5 μM. Moreover, association of Ca2+DREAM to helix 9 results in slower depolarization with the rotational correlation time of Ф = 22.5 ± 0.1 ns, which matches w the estimated rotational correlation time for DREAM dimer bound to a helix-9. These results suggest that DREAM binds to presenilin in its dimeric form and the formation of the inter-protein complex in regulated by Ca2+.View Large Image | View Hi-Res Image | Download PowerPoint Slide

An Investigation into the Effect of the Structure of Bile Salt Aggregates on the Binding Interactions and ESIHT Dynamics of Curcumin: A Photophysical Approach To Probe Bile Salt Aggregates as a Potential Drug Carrier

This work demonstrates the utilization of bile salt aggregates as a potential biological host system for studying the binding interactions and dynamics of the poorly-water-soluble drug curcumin by means of photophysical techniques. We found that the level of degradation of curcumin is greatly suppressed upon encapsulation into the nanocavities of three different bile salt aggregates. However, NaTC aggregates are more effective to suppress the level of degradation of curcumin than NaCh and NaDC aggregates. We also report the modulation of the photophysical and dynamical properties of curcumin into the nanocavities of bile salt aggregates using steady-state and time-resolved fluorescence spectroscopy. The reduced level of interaction of curcumin with water upon incorporation into the different binding sites of bile salt aggregates results in an enhanced fluorescence intensity along with the blue shift in the emission maxima of curcumin. However, the observation of higher fluorescence quantum yield as well as longer fluorescence lifetime in NaTC aggregates compared to that in NaCh and NaDC aggregates clearly indicates a more effective decrease in the excited-state intramolecular hydrogen atom transfer (ESIHT) mediated nonradiative deactivation of curcumin by the interaction with the anionic headgroup of NaTC. The binding and location of curcumin into the bile salt aggregates has been further confirmed from the steady-state fluorescence anisotropy measurements. In addition, we have shown the effect of addition of salt on the photophysical properties of curcumin in the confined environments of bile salt aggregates. Our results indicate that on addition of salt the time scale of ESIHT process of curcumin in bile salt aggregates is markedly increased.

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.

Photophysical Behavior of 8-Anilino-1-Naphthalenesulfonate in Vesicles of Pulmonary Surfactant Dipalmitoylphosphatidylcholine (DPPC) and Its Sensitivity toward the Bile Salt–Vesicle Interaction

The photophysical behavior of 8-anilino-1-naphthalenesulphonate (ANS) in vesicles of dipalmitoylphosphatidylcholine (DPPC), a pulmonary surfactant, has been carried out in a detailed manner. ANS shows notable variations in fluorescence intensity, lifetime, and anisotropy parameters as it gets into the vesicle. It was found that ANS partitions well into the DPPC bilayer membrane with an estimated partition coefficient of ~2.0 × 10(5). Among the various fluorescence parameters of ANS, fluorescence anisotropy was found to be most responsive to the temperature induced phase change of the bilayer membrane. These interesting fluorescence parameters of ANS were then used to study the hydration of lipid bilayer membrane by submicellar concentration of bile salts. From the steady-state fluorescence intensity and dynamic fluorescence lifetime analyses it is clear that ANS is able to probe the submicellar concentration (≤1 mM) of bile salt induced hydration of lipid bilayer membrane that accompanies expulsion of ANS from the bilayer to the aqueous bulk phase. Lower-temperature shift in the phase transition of DPPC bilayer indicates that fluorescence anisotropy of ANS is sensitive enough to the bile salt induced perturbation in the packed acyl chains of DPPC bilayer and modification in the membrane fluidity. In presence of sodium deoxycholate (NaDC) and sodium cholate (NaC) in DPPC vesicles, ANS experiences restriction in rotational mobility which is evident from the variation in steady-state fluorescence anisotropy and fluorescence anisotropy decay parameters.

Possible mechanism of structural transformations induced by StAsp-PSI in lipid membranes

In the present work we have analyzed the effect of StAsp-PSI (plant-specific insert of potato aspartic protease) on the structural and thermotropic properties of the major phospholipid types of bacterial and animal cells. Results obtained suggest that StAsp-PSI induces a destabilization of the membrane bilayers, depending on the time of interaction between the protein and the bilayers, rather than on its concentration. This temporal delay would be consistent with a lateral diffusion of StAsp-PSI monomers to assemble into aggregates to form pores. Like with the results previously reported for the StAsp-PSI circular dichroism, data obtained here from IR spectroscopy show that there are slight changes in the StAsp-PSI secondary structure in the presence of lipid membranes; suggesting that these changes could be related with the StAsp-PSI self-association. Results obtained from steady-state fluorescence anisotropy and differential scanning calorimetry assays suggest that StAsp-PSI interacts with both uncharged and negatively charged phospholipids, modulates the phase polymorphic behavior of model membranes and partitions and buries differentially in the membrane depending on the presence of negatively charged phospholipids.

New Fluorene-Based Fluorescent Probe with Efficient Two-Photon Absorption

The synthesis, linear photophysical characterization, and two-photon absorption (2PA) properties of new fluorene derivative 3,30-(pyridine-2,6-diyl)bis(1-(7-(diphenylamino)-9,9-dihexyl-9H-fluoren-2-yl)propane-1,3-dione) (1) have been presented. The steady-state absorption, fluorescence and excitation anisotropy spectra along with the fluorescence decay kinetics of 1 are obtained in the solvents of different polarities at room temperature with respect to its potential application in bioimaging. The analysis of linear photophysical properties revealed a complicated nature of the main one-photon absorption band of 1, and the strong solvatochromic effect in steady-state fluorescence spectra is observed. The degenerate 2PA spectrum of 1 is measured in the spectral range 570–970 nm with the use of the open aperture Z-scan method under the 1-kHz femtosecond excitation, and the maximum values of two-photon action cross sections ~(100–130) GM are obtained. The nature of the linear absorption and the 2PA bands is analyzed by quantum chemical methods using a Gaussian program package.

Competitive binding of nile red between lipids and β-cyclodextrin

Fluorometric measurements are exploited to explore the binding interactions of nile red (NR) with anionic lipid dimyristoyl-L-α-phosphatidylglycerol (DMPG), zwitterionic lipid dimyristoyl-L-α-phosphatidylcholine (DMPC) as well as neutral cyclic oligosaccharide β-cyclodextrin (β-CD) solutions. The binding constants are found to be quite high and comparable (within a factor of five). Series of spectral techniques like steady state fluorescence and fluorescence anisotropy study, micropolarity study, quenching study and time resolved experiments reveal that the addition of β-CD to the probe–lipid complexes results in weakening of the lipid–probe interaction and formation of probe–β-CD inclusion complexes leading to the removal of some of the probe (NR) molecules from the lipid environments. The extent of removal of NR is, however, more from DMPG than DMPC lipid. The phenomena are explained from the concept of competitive binding of the probe between the lipids and β-CD. Since lipids are the principal constituents of the cell walls, the work might make a foundation for the possible removal of excess of molecules like nile red adsorbed on the cell walls.

Photophysical Properties and Ultrafast Excited-State Dynamics of a New Two-Photon Absorbing Thiopyranyl Probe

Comprehensive linear photophysical and photochemical characterization, two-photon absorption (2PA) properties, and femtosecond excited-state dynamics of a symmetrical fluorene derivative 2-(2,6-bis((E)-2-(7-(diphenylamino)-9,9-dihexyl-9H-fluoren-7-yl)vinyl)-4H-thiopyran-4-ylidene)malononitrile (1) are reported. The linear one-photon absorption (1PA), steady-state fluorescence, excitation, and excitation anisotropy spectra of 1 were investigated in organic solvents of different polarities at room temperature, exhibiting rather complex absorption and emission behavior. The relatively strong 2PA of thiopyranyl 1 was investigated by the open aperture femtosecond Z-scan technique in the main long wavelength 1PA contour with maxima cross sections up to 600–700 GM. Femtosecond dynamics of the excited-state absorption (ESA) and gain processes in 1 exhibited fast complicated relaxation phenomena with a strong dependence on solvent polarity and a weak dependence on excitation wavelength. The nature of the observed transient absorption kinetics was explained based on the short-lived ESA bands of 1 and solvate relaxation phenomena. Quantum chemical calculations, based on density functional theory, were employed for additional analysis of the 1PA and 2PA properties of 1.

Analysis of high-affinity assembly for AMPA receptor amino-terminal domains

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.

Influence of surfactants on the photophysics of 4,4-diaminodiphenyl sulfone encapsulated in self-assembled nanocage of diblock copolymer

• Influence of cationic and non-ionic micelles drastically reduces the critical aggregation constant but not in the case of anionic micelle. • Addition of anionic micelle causes the probe to move toward the edge of the aggregation due to a possible disruption. • Further addition of polymer in polymer-anionic micelle gains rigidness with probe position unchanged. • Wobbling motion of the whole polymer-micelle assembly is fastest in non-ionic micelle with minimum semi-cone angle. This paper reports the influence of anionic, cationic and nonionic surfactant on the spherical nonionic polymeric micelles composed of block copolymers polyethylene- b -polyethylene glycol (PE- b -PEG) and the modulation of excited state emission characteristics of 4,4-diaminodiphenyl sulfone (Dapsone) in the mixed polymer-micelle environments. The critical micellar concentration (CMC) of diblock polymer was determined as ~0.12 mM from the variation in fluorescence intensity and solvatochromic shift of the probe with polymer concentration. Addition of surfactant in polymer solution of Dapsone causes a decrease in the value of critical aggregation concentration (CAC) to roughly one tenth the values of CMC of the individual surfactants (cationic or non-ionic) except in the case of anionic surfactant. The huge enhancement in fluorescence intensity and large blue shift of the emission maximum of the probe incorporated inside the nanocages formed by the diblock copolymer and cationic or nonionic micelle indicate an entirely different environment than that in neat water, polymer and micellar system separately. The steady state fluorescence anisotropy and dynamic light scattering (DLS) results on addition of micelle to the polymeric solution show two classes of events; (1) addition of anionic micelle causes lesser rotational restriction and (2) addition of cationic and non-ionic micelles cause to form more rigid environment to the probe. The rotational motion of the probe analyzed in terms of “wobbling–in-cone” model with the fluorescence anisotropy decay data shows that the wobbling is fastest in non-ionic micelle with minimum semi-cone angle. The increased rotational time and the value order parameter obtained from the time-resolved anisotropy decay confirm a very restricted environment experienced by the probe inside the self assembly system. The translational and the wobbling motions are also restricted inside these aggregations.

Biopolymer-based hydrogels as injectable materials for tissue repair scaffolds

The progress in tissue regeneration is strongly dependent on the development of biocompatible materials with properties resembling those of a native tissue. Also, the application of noninvasive methods of delivering the scaffold into the tissue defect is of great importance. In this study we present a group of biopolymer-based materials as potential injectable scaffolds. In contrast to other studies involving collagen neutralization or additional incubation of gel in genipin solution, we propose collagen and collagen–chitosan gels crosslinked in situ with genipin. Since some parameters of the cells should be considered in the microscale, the steady-state fluorescence anisotropy was applied to study the microenvironment of the gels. To our knowledge we are the first to report on microrheological properties, such as gel time and microviscosity, for this group of hydrogels. Rapid gelation at physiological temperatures found makes these materials of special interest in applications requiring gel injectability. Physico-chemical investigation showed the influence of the crosslinking agent concentration and chitosan addition on the crosslinking degree, swelling ratio, gel microviscosity, and the degradation rate. Strong correlation was revealed between the surface wettability and the viability of cultured mesenchymal stem cells. Cytotoxicity studies indicated that the collagen–chitosan hydrogels showed the best biocompatibility.

Study on Mixed Micelles of Cationic Gemini Surfactants Having Hydroxyl Groups in the Spacers with Conventional Cationic Surfactants: Effects of Spacer Group and Hydrocarbon Tail Length

Gemini surfactants, being more surface-active than their conventional counterparts, have potential applications in various industries. The properties of mixed surfactant systems are far better than those of neat surfactants in many cases, and as a result, mixed surfactants are used in many industrial applications. In the present work, the micellar properties of binary mixtures of the monomeric cationic surfactants hexadecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), and dodecyltrimethylammonium bromide (DTAB) with the cationic gemini surfactants 1,3-bis(dodecyl-N,N-dimethylammonium bromide)-2-propanol and 1,4-bis(dodecyl-N,N-dimethylammonium bromide)-2,3-butanediol were studied in aqueous solution at 303.15 K by means of conductivity, steady-state fluorescence, and fluorescence anisotropy techniques. The presence of a small amount of gemini surfactant was found to improve the physicochemical properties of the conventional surfactant. For example, the cmc value of DTAB was reduced to one-sixth of its original value in the presence of 0.1 mole fraction of a gemini surfactant. The spacer group of the gemini surfactant and the hydrocarbon chain of the monomeric surfactant play a significant role in the interactions between the surfactants in mixed micelles. These interactions are greatest when there are similarities in the structures of their hydrocarbon chains; however, the micellization process is favored by increasing hydrophobicity of the monomeric surfactant. The microenvironments of mixed micelles were studied using fluorescence techniques.

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Electronic energy transfer in tetracene-doped p-terphenyl nanoparticles: Extraordinarily high fluorescence enhancement and quenching efficiency

A series of tetracene (Tc)-doped p-terphenyl (pTP) nanoparticles (Tc/pTP-NPs) were produced at various doping ratios by reprecipitation in water. The Tc/pTP-NPs are disk-like with a mean diameter of 75nm and height of 7nm, which were determined by scanning electron microscopy and atomic force microscopy, and exhibited electronic delocalization through H-type aggregation of the pTP molecules. Electronic energy transfer in the Tc/pTP-NPs was examined using steady-state and time-resolved fluorescence spectroscopy and fluorescence anisotropy experiments: pTP-NPs serve as an excellent light-harvesting nano-matrix with a large absorption coefficient that exceeds 109M−1cm−1. Furthermore, Stern–Volmer analysis of the donor emission was performed by changing the dopant concentration; this showed that a single Tc acceptor quenched more than 104pTP donors. Comparison of the experimental and theoretical energy transfer efficiencies indicated that the efficient energy transfer can be attributed to two-dimensional exciton diffusion in the host nanoparticles.

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.

How Different Lipidic Mimetic Systems of E. COLI Membranes Affect Membrane Protein Reconstitution: A Steady-State Fluorescence Anisotropy Study

Outer membrane proteins (Omp) in bacteria, porins, have an important role on antibiotic translocation trough bacterial membranes. OmpF is one of the most important E. coli porins in the permeation of beta-lactam and quinolone antibiotics. Moreover, these antibiotics might penetrate the bacterial outer membrane through porins or through the lipid/protein interface. We focused on the characterization of E. coli membranes, once this knowledge is crucial for studies of antibiotic translocation and useful to understand and counteract bacterial antibiotic resistance, one of the biggest concerns of nowadays. Steady-state fluorescence anisotropy is a good technique to detect changes in lipid environments through the use of fluorescent probes. We used two fluorescent probes, DPH and TMA-DPH (which provide information from the core and from the interface regions of the bilayer, respectively), and determined the temperature dependence of these two anisotropy probes, incubated or incorporated into POPE/POPG (0.75:0.25), POPE/POPG/Cardiolipin (0.67:0.23:0.10) and E. coli total lipid extract liposomes (LUVs) and OmpF proteoliposomes. POPE/POPG and POPE/POPG/Cardiolipin mixtures are conventionally used to mimic the E. coli membranes, while the E. coli total lipid extract is a natural system. In this study we determine the phase transition temperatures characteristics of each membrane mimetic system and still prove the correct insertion of the protein in the membrane. When comparing the transition temperatures obtained for each mimetic system, our study suggest that the ternary lipid mixture is a better system to mimic the E. coli membranes than the binary system.

Cardiolipin's Impact on Model Membranes Thermodynamic: Drug-Lipid Interactions and Protein Conformation Implication

Steady-state fluorescence anisotropy and dynamic light scattering (DLS) were used to determine the thermotropic properties of lipid model systems of bacterial membranes. Diferent lipid proportions of PE:PG:CL were used in order to mimic Yersinia kristensenii, Proteus mirabilis and Escherichia Coli membranes. Cardiolipin's inclusion as a third lipid component of any PE:PG mixture considerably changes the system's properties. The results obtained by these two techniques were shown to be reproducible and were the same within experimental errors, suggesting that either technique can be used to obtain accurate values to characterize the thermotropic behavior of such systems. Moreover they show that the main transition temperatures obtained are undoubtedly cardiolipin dependent. Additionally AFM experiments were performed and these results show that even at small concentration cardiolipin produces important changes not only in the membrane thermotropic properties, but also in the bilayer structure. Moxifloxacin and enrofloxacin's interaction studies with these model systems were also performed and show that cardiolipin's absence greatly influence the conclusions obtained. Preliminary circular dichroism results of an E. coli membrane protein, OmpF, reconstituted in different model system membranes, with and without cardiolipin, also point out for its influence on protein's conformation.Taken all together these results show that cardiolipin's incorporation in model system membranes can have a significant impact on the membrane's properties and its inclusion should be considered when the aim is to construct model system of bacterial membranes.

Conformational dynamics and excitation wavelength dependent photoluminescence of decameric organic nanoparticles

We report a new type of fluorescent organic nanoparticles (16-EDFONP) which are composed of ten bis(3,4,5-tris(16-alkyloxy) monobenzoylglyceride) bisphenol A molecules. The nanoparticles are only found in high concentration solution and exhibit the excitation-dependent fluorescence (EDF). The 16-EDFONP shows clear spectral relaxation on the nanosecond time scale. We have observed similar spectral shifts in cyclohexane, and thus attribute the time-dependent Stokes shift to the emission from multiple conformations of 16-EDFONP. With the employment of steady state and time-resolved fluorescence anisotropy measurements, the hydrodynamic radius of 16-EDFONP is estimated to be 3.13 nm, which is consistent with the size measured using the dynamic light scattering and high-resolution transmission electron microscopy techniques. The time-resolved anisotropy reveals the change in fundamental anisotropy upon different excitation wavelengths, arising from the structural heterogeneity of hydrogen-bonded monoacylglycerol clusters of the 16-EDFONP. Our findings indicate that incomplete spectral relaxation and the size distribution of nanoparticles are not the source of the observed EDF. The EDF comes from the selective excitation of the 16-EDFONP with different monoacylglycerol hydrogen-bond conformations.

Exploring the Kinetics of Protein Birth

Fidelity and efficiency in protein folding are essential to sustain cell life. Overall however, very little is known about the way proteins are able to attain their native structure within the context of the cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes may have strong influence on the early stages of protein folding in the cell and may be crucial for the production of folded unaggregated proteins. The conformational changes that occur within a nascent protein during its release from the ribosome have yet to be elucidated. Here, we present a kinetic study on the release time-course of ribosome bound model proteins upon addition of the antibiotic puromycin. By time-resolved gel electrophoresis, we are able to discern that puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3' end of tRNA occurs quickly. Steady-state fluorescence anisotropy reveals the presence of two additional slower kinetic phases. Finally, time decay fluorescence anisotropy analysis complements the above results by providing insights into the local motions experienced by the nascent protein during different stages of the protein birth process.