Macromolecular crowding effects on two homologs of ribosomal protein s16: protein-dependent structural changes and local interactions.

Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent.

Synthesis of fluorescent ring-fused 2-pyridone peptidomimetics.

Thiazolino fused 2-pyridone peptidomimetics are of significant biological importance due to their ability to interfere with adhesive fiber formation in uropathogenic Escherichia coli and oligomerization of amyloid fibers. We have developed an efficient synthetic route to fluorescent BODIPY analogues, with structural diversification from a key intermediate enabling introduction of C-2 substituents and late incorporation of the BODIPY moiety. A mild lithium halide mediated hydrolysis enabled preparation of peptidomimetic fluorophores with useful photophysical properties for further chemical biology applications.

Direct observation of protein unfolded state compaction in the presence of macromolecular crowding.

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system.

Förster resonance energy transfer (FRET) between heterogeneously distributed probes: application to lipid nanodomains and pores.

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer.

Dynamics and size of cross-linking-induced lipid nanodomains in model membranes.

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics.

Design and synthesis of fluorescent pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms.

Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores.

Limitations of electronic energy transfer in the determination of lipid nanodomain sizes.

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area.

On the analyses of fluorescence depolarisation data in the presence of electronic energy migration. Part II: Applying and evaluating two-photon excited fluorescence.

Electronic energy migration within a bifluorophoric molecule has been studied by time-resolved two-photon excited (TPE) fluorescence depolarisation experiments. Data were analysed by using a recently developed quantitative approach [O. Opanasyuk and L.

On the analyses of fluorescence depolarisation data in the presence of electronic energy migration. Part I: Theory and general description.

A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D(1)-R-D(2)) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R).

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures.

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.

A versatile standard for bathochromic fluorescence based on intramolecular FRET.

A perylene and a terrylene tetracarboxylic bisimide dyad was prepared in which an efficient energy transfer from the former to the latter is observed. The absorption spectrum of this compound covers a broad range. Bathochromic fluorescence with a high quantum yield was obtained independent of excitation wavelengths (λ < 655 nm).

Locations and reorientations of multi-ring-fused 2-pyridones in ganglioside G(M1) micelles.

Fluorescent multi-ring-fused 2-pyridones, with chemical resemblance to other biologically active 2-pyridone systems, were solubilized in spherical micelles formed by the ganglioside G(M1) and studied with respect to their spatial localization and rotational mobility. For this, electronic energy transfer between the multi-ring-fused 2-pyridone (donor) and BODIPY-FL-labeled G(M1) was determined, as well as their fluorescence depolarization. From the obtained efficiency of energy transfer to the acceptor group (BODIPY-FL), either localized in the polar or in the nonpolar part of the ganglioside, it has been possible to estimate the most likely localization of the multi-ring-fused 2-pyridones.

Luminescence enhancement from silica-coated gold nanoparticle agglomerates following multi-photon excitation.

Multi-photon absorption induced luminescence (MAIL) from bare gold nanoparticles, silica-coated particles, as well as silica-coated agglomerated gold nanoparticles suspended in aqueous solution was studied by using time-resolved and steady-state luminescence spectroscopy. The nanoparticles were excited by femtosecond pulses of wavelengths ranging from 630 nm to 900 nm. The luminescence from the particles exhibits a broad spectrum in the UV and VIS region.

Extended Förster theory: a quantitative approach to the determination of inter-chromophore distances in biomacromolecules.

This review highlights recent theoretical and experimental advances in the study of biomacromolecular structure by using electronic transfer. The considered electronic transport in the extended Förster theory occurs within donor-acceptor pairs, donor-donor pairs, as well as within regular arrangements of many donors which may undergo reorienting and translational dynamics. The classical and the extended Förster theory are compared.

Unusual light spectroscopic properties of a 2-pyridone-based multi-ring-fused fluorescent scaffold.

UV-VIS absorption and fluorescence spectroscopic properties of six related polyaromatic 2-pyridones have been studied. Excitation of the lowest and rather weak and structure-less transition [ε (max) (430 nm) ≈ 3,000 mol(-1)dm(3)cm(-1)] gives rise to a broad fluorescence band in the visible region, for these compounds. These S (0) ↔ S (1) transitions are compatible with symmetrically forbidden transitions, promoted by intensity borrowing, as is revealed by fluorescence depolarisation data.

Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers.

A series of sn-2 acyl-labelled phosphatidyl-cholines (PC), bearing 4,4-difluoro-1-3-5-7-tetra-methyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY) at the end of the C(n)-acyl chains were solubilised in unilamellar vesicles and studied with respect to the order and location of the Me(4)-BODIPY (denoted: B) group. The obtained results are based on time-resolved electronic energy transfer from donors (2-(9-anthroyloxy)-stearic acid) localised in the lipid-water interface to acceptors BnPC (n = 3, 5, 7, 9, 11, 13, 15), as well as the energy migration among the Me(4)-BODIPY groups of BnPC:s. The donor-acceptor and the donor-donor experiments strongly suggest that the Me(4)-BODIPY group in BnPC tends to loop back close to the lipid-water interface.

A characterisation study on the application of inverted lyotropic phases for subcutaneous drug release.

An experimental characterisation of lipid mixtures consisting of inverted hexagonal and inverted cubic phases composed of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) was performed. The release of five chromophores of varying lipophilicity, used as model drugs, was investigated. Two experimental setups were applied: one based on maintaining sink condition, while a constant volume release medium was employed for the other.

Two-photon excited fluorescence depolarisation and electronic energy migration within donor-donor pairs.

A unified theoretical description is presented for one- and two-photon excited fluorescence depolarisation and electronic energy migration within pairs of chromophores. Two weakly coupled donor groups are linked via a rigid macromolecule with the ability to undergo restricted reorienting motions. Describing these reorienting motions as well as their influence on the coupling is rather complex, but can be accounted for by using the extended Förster theory.

A comparative study on ganglioside micelles using electronic energy transfer, fluorescence correlation spectroscopy and light scattering techniques.

Ganglioside (G(M1)) micelles have been studied by means of three different techniques: fluorescence correlation spectroscopy (FCS), electronic energy transfer, as monitored by time-resolved fluorescence spectroscopy, as well as static and dynamic light scattering. The aggregation numbers obtained, 168 +/- 4, remain constant over a wide range of G(M1) concentrations (0.764-156 muM), are very consistent when using different donor-acceptor energy transfer pairs and have served as reference values in tests of the FCS method.

Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions.

New fluorophore-labelled G(M1) gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G(M1).

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.

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state.

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16(meso)) and hyperthermophilic (S16(thermo)) bacterium by using a variety of biophysical methods. As basis for the study, the 2.

The symmetry of two-photon excited states as determined by time-resolved fluorescence depolarization experiments.

A new experimental and theoretical approach is presented for the quantitative determination and assignment of the two-photon absorption tensor of fluorophores dissolved in liquid solutions. Two linearly independent time-resolved fluorescence anisotropies and the two-photon polarization ratio were determined from experiments based on using the time-correlated single photon counting technique. The data were analyzed in a global manner under the assumption of prevailing diffusive molecular reorientations and when accounting for the influence of rapid unresolved reorientations.

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.

Extended Förster theory for determining intraprotein distances: 2. an accurate analysis of fluorescence depolarisation experiments.

The extended Förster theory (EFT) is for the first time applied to the quantitative determination of the intramolecular distances in proteins. It is shown how the EFT (J. Chem.