Controlled Supramolecular Polymerization via Bioinspired, Liquid-Liquid Phase Separation of Monomers.

Dynamic supramolecular assemblies, driven by noncovalent interactions, pervade the biological realm. In the synthetic domain, their counterparts, supramolecular polymers, endowed with remarkable self-repair and adaptive traits, are often realized through bioinspired designs. Recently, controlled supramolecular polymerization strategies have emerged, drawing inspiration from protein self-assembly. A burgeoning area of research involves mimicking the liquid-liquid phase separation (LLPS) observed in proteins to create coacervate droplets and recognizing their significance in cellular organization and diverse functions. Herein, we introduce a novel perspective on synthetic coacervates, extending beyond their established role in synthetic biology as dynamic, membraneless phases to enable structural control in synthetic supramolecular polymers. Drawing parallels with the cooperative growth of amyloid fibrils through LLPS, we present metastable coacervate droplets as dormant monomer phases for controlled supramolecular polymerization. This is achieved via a π-conjugated monomer design that combines structural characteristics for both coacervation through its terminal ionic groups and one-dimensional growth via a π-conjugated core. This design leads to a unique temporal LLPS, resulting in a metastable coacervate phase, which subsequently undergoes one-dimensional growth via nucleation within the droplets. In-depth spectroscopic and microscopic characterization provides insights into the temporal evolution of disordered and ordered phases. Furthermore, to modulate the kinetics of liquid-to-solid transformation and to achieve precise control over the structural characteristics of the resulting supramolecular polymers, we invoke seeding in the droplets, showcasing living growth characteristics. Our work thus opens up new avenues in the exciting field of supramolecular polymerization, offering general design principles and controlled synthesis of precision self-assembled structures in confined environments.

Fast interaction dynamics of G-quadruplex and RGG-rich peptides unveiled in zero-mode waveguides.

G-quadruplexes (GQs), a non-canonical form of DNA, are receiving a huge interest as target sites for potential applications in antiviral and anticancer drug treatments. The biological functions of GQs can be controlled by specifically binding proteins known as GQs binding proteins. Some of the GQs binding proteins contain an arginine and glycine-rich sequence known as RGG peptide. Despite the important role of RGG, the GQs-RGG interaction remains poorly understood. By single molecule measurements, the interaction dynamics can be determined in principle. However, the RGG-GQs interaction occurs at micromolar concentrations, making conventional single-molecule experiments impossible with a diffraction-limited confocal microscope. Here, we use a 120 nm zero-mode waveguide (ZMW) nanoaperture to overcome the diffraction limit. The combination of dual-color fluorescence cross-correlation spectroscopy (FCCS) with FRET is used to unveil the interaction dynamics and measure the association and dissociation rates. Our data show that the RGG-GQs interaction is predominantly driven by electrostatics but that a specific affinity between the RGG sequence and the GQs structure is preserved. The single molecule approach at micromolar concentration is the key to improve our understanding of GQs function and develop its therapeutic applications by screening a large library of GQs-targeting peptides and proteins.

Exploring the effects of cosolutes and crowding on the volumetric and kinetic profile of the conformational dynamics of a poly dA loop DNA hairpin: a single-molecule FRET study.

We investigated the volumetric and kinetic profile of the conformational landscape of a poly dA loop DNA hairpin (Hp) in the presence of salts, osmolytes and crowding media, mimicking the intracellular milieu, using single-molecule FRET methodology. Pressure modulation was applied to explore the volumetric and hydrational characteristics of the free-energy landscape of the DNA Hp, but also because pressure is a stress factor many organisms have to cope with, e.g. in the deep sea where pressures even up to the kbar level are encountered. Urea and pressure synergistically destabilize the closed conformation of the DNA Hp due to a lower molar partial volume in the unfolded state. Conversely, multivalent salts, trimethylamine-N-oxide and Ficoll strongly populate the closed state and counteract deteriorating effects of pressure. Complementary smFRET measurements under immobilized conditions at ambient pressure allowed us to dissect the equilibrium data in terms of folding and unfolding rate constants of the conformational transitions, leading to a deeper understanding of the stabilization mechanisms of the cosolutes. Our results show that the free-energy landscape of the DNA Hp is a rugged one, which is markedly affected by the ionic strength of the solution, by preferential interaction and exclusion of cosolvents as well as by pressure.

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides.

Single molecule detection provides detailed information about molecular structures and functions but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labeling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguide nanoapertures enable the observation of the UV fluorescence of single label-free ß-galactosidase proteins with increased brightness, microsecond transit times, and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient, and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. Although the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

Probing Colocalization of N-Ras and K-Ras4B Lipoproteins in Model Biomembranes.

Signaling of N-Ras and K-Ras4B proteins depends strongly on their correct localization in the cell membrane. In vivo studies suggest that intermolecular interactions foster the self-association of both N-Ras and K-Ras4B and the formation of nanoclusters in the cell membrane. As sites for effector binding, nanocluster formation is thought to be essential for effective signal transmission of both N-Ras and K-Ras4B. To shed more light on the spatial arrangement and mechanism underlying the proposed cross-talk between spatially segregated Ras proteins, the simultaneous localization of N-Ras and K-Ras4B and their effect on the lateral organization of a heterogeneous model biomembrane has been studied by using AFM and FRET methodology. It is shown that, owing to the different natures of their membrane anchor systems, N-Ras and K-Ras4B not only avoid assembly in bulk solution and do not colocalize, but rather form individual nanoclusters that diffuse independently in the fluid membrane plane.

The Deep Sea Osmolyte Trimethylamine N-Oxide and Macromolecular Crowders Rescue the Antiparallel Conformation of the Human Telomeric G-Quadruplex from Urea and Pressure Stress.

Organisms are thriving in the deep sea at pressures up to the 1 kbar level, which imposes severe stress on the conformational dynamics and stability of their biomolecules. The impact of osmolytes and macromolecular crowders, mimicking intracellular conditions, on the effect of pressure on the conformational dynamics of a human telomeric G-quadruplex (G4) DNA is explored in this study employing single-molecule Förster resonance energy transfer (FRET) experiments. In neat buffer, pressurization favors the parallel/hybrid state of the G4-DNA over the antiparallel conformation at ≈400 bar, finally leading to unfolding beyond 1000 bar. High-pressure NMR data support these findings. The folded topological conformers have different solvent accessible surface areas and cavity volumes, leading to different volumetric properties and hence pressure stabilities. The deep-sea osmolyte trimethylamine N-oxide (TMAO) and macromolecular crowding agents are able to effectively rescue the G4-DNA from unfolding in the whole pressure range encountered on Earth.

Antagonistic effects of natural osmolyte mixtures and hydrostatic pressure on the conformational dynamics of a DNA hairpin probed at the single-molecule level.

Organisms are thriving in the deep sea at pressures of up to the 1 kbar level. To withstand such harsh conditions, they accumulate particular osmolyte mixtures to counteract the pressure stress imposed. We explored the combined effects of pressure and osmolyte mixtures known from deep sea organisms on the closed-to-open conformational transition of a DNA hairpin (Hp). To this end, single-molecule Förster resonance energy transfer (smFRET) experiments were carried out in an optimized high-pressure capillary optical cell. In the absence of osmolytes, pressure shifts the conformational equilibrium of the DNA Hp towards the open, unfolded state owing to a volume decrease of about -20 cm3 mol-1. We show that the deep-sea osmolyte mixture, largely composed of TMAO, is able to rescue the DNA Hp from unfolding even up to almost 1 kbar, which is supposed to be essentially due to a distinct excluded volume effect.

Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity.

The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.

Crowders and Cosolvents-Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses.

The free energy and conformational landscape of biomolecular systems as well as biochemical reactions depend not only on temperature and pressure, but also on the particular solution conditions. Such conditions include the effects of cosolvents (for example osmolytes) and macromolecular crowding, which are crucial components to understand the energetics and kinetics of biological processes in living system. Such conditions are also important for the understanding of many debilitating diseases, such as those where misfolding and amyloid formation of proteins are involved. Moreover, understanding their effects on biomolecular processes is prerequisite for designing industrially relevant enzymatic reactions, which seldom take place under neat conditions. Here, we review and discuss experimental and theoretical studies on the characterization of cosolvent and crowding induced effects in biologically relevant systems, approaching even the complexity of living organisms. In particular, we focus on cosolvent and crowding effects on the conformational equilibrium and folding kinetics of proteins and nucleic acids as well as on enzymatic reactions, including their effects on the temperature and pressure dependence of these processes. By presenting a few representative examples, we show how such effects are unveiled and described in thermodynamic and kinetic terms.

Osmolyte Effects on the Conformational Dynamics of a DNA Hairpin at Ambient and Extreme Environmental Conditions.

The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine-N-oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single-molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single-molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co-solute urea.

Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes.

Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state-monomeric or clustered-influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners.

Probing conformational and functional substates of calmodulin by high pressure FTIR spectroscopy: influence of Ca2+ binding and the hypervariable region of K-Ras4B.

The ubiquitous Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. The conformational space of this protein needs to be explored to shed more light on the mechanism of target recognition and protein function. Here, we used pressure modulation in combination with FTIR spectroscopy to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to its ground state, which are supposed to be functionally relevant in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the hypervariable region (HVR) of the signaling peptide K-Ras4B as a binding partner. We demonstrate that the conformational dynamics of CaM is vastly affected by binding of both Ca2+ ions and the lipidated signaling peptide K-Ras4B. Moreover, we could uncover conformational substates of CaM by pressure perturbation that are partially unfolded and more solvated and conceivably facilitate target recognition by exposing the required binding surfaces.

Effect of Controlled Deposition of ZnS Shell on the Photostability of CdTe Quantum Dots as Studied by Conventional Fluorescence and FCS Techniques.

The effect of one and two monolayers of ZnS shells on the photostability of CdTe quantum dots (QDs) in aqueous and nonaqueous media has been studied by monitoring the fluorescence behavior of the QDs under ensemble and single-molecule conditions. ZnS capping of the CdTe QDs leads to significant enhancement of the fluorescence brightness of these QDs. Considerable enhancement of the photostability of the shell-protected QDs, including the suppression of photoactivation, is also observed. Fluorescence correlation spectroscopy measurements reveal an increase in the number of particles undergoing reversible fluorescent on-off transitions in the volume under observation with increasing excitation power; this effect is found to be more pronounced in the case of core-only QDs than for core-shell QDs.

Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates.

We investigated the binding between the c-MYC G-quadruplex (GQ) and berberine chloride (BCl) in an aqueous two-phase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid-liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates.

Structural transformation of bovine serum albumin induced by dimethyl sulfoxide and probed by fluorescence correlation spectroscopy and additional methods.

Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous-organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)-labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40% DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self-quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 µs in its native state to 50 µs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near-UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1-anilinonaphthalene-8-sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40% DMSO in the ensemble measurements.

Gracilaria edulis extract induces apoptosis and inhibits tumor in Ehrlich ascites tumor cells in vivo.

BACKGROUND: Marine environment is inestimable for their chemical and biological diversity and therefore is an extraordinary resource for the discovery of new anticancer drugs. Recent development in elucidation of the mechanism and therapeutic action of natural products helped to evaluate for their potential activity. METHODS: We evaluated Gracilaria edulis J. Ag (Brown algae), for its antitumor potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Cytotoxicity evaluation of Ethanol Extract of Gracilaria edulis (EEGE) using EAT cells showed significant activity. In vitro studies indicated that EEGE cytotoxicity to EAT cells is mediated through its ability to produce reactive oxygen species (ROS) and therefore decreasing intracellular glutathione (GSH) levels may be attributed to oxidative stress. RESULTS: Apoptotic parameters including Annexin-V positive cells, increased levels of DNA fragmentation and increased caspase-2, caspase-3 and caspase-9 activities indicated the mechanism might be by inducing apoptosis. Intraperitoneally administration of EEGE to EAT-bearing mice helped to increase the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. Extensive hematology, biochemistry and histopathological analysis of liver and kidney indicated that daily doses of EEGE up to 300 mg/kg for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity. CONCLUSION: Comprehensive antitumor analysis in animal model and in Ehrlich Ascites Tumor cells was done including biochemical, and pathological evaluations indicate antitumor activity of the extract and non toxic in vivo. It was evident that the mechanism explains the apoptotic activity of the algae extract.

Antitumor activity of ethanol extract of Gracilaria edulis (Gmelin) Silva on Ehrlich ascites carcinoma-bearing mice.

OBJECTIVE: To evaluate antitumor activity of Gracilaria edulis in Swiss albino mice with Ehrlich ascites carcinoma (EAC). METHODS: Tumors were induced in mice by intraperitoneal injection of EAC cells. Ethanol extract of Gracilaria edulis (EEGE) was administered to the experimental animals in different doses after 24 h of tumor inoculation. The antitumor effect of the EEGE was evaluated by assessing in vitro cytotoxicity, survival time, biochemical parameters and hepatic enzyme levels. RESULTS: EEGE increased the life span of EAC-bearing mice compared with that of the model control mice (P<0.05 or P<0.01). EEGE treatment also converted the changes of biochemical parameters and hepatic enzyme levels in the EAC-bearing mice (P<0.05 or P<0.01). EEGE induced inhibition of tumor formation in EAC-bearing mice compared with that of the model control group (P<0.05 or P<0.01). CONCLUSION: The present study scientifically proved the antitumor activity of marine algae G. edulis and the effect can be correlated with doses.

Novel mutations in the human MCCA and MCCB gene causing methylcrotonylglycinuria.

Methylcrotonylglycinuria (MCG) is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The clinical phenotypes are highly variable ranging from neonatal onset with severe neurological involvement to asymptomatic adults. Here we identified two novel MCCA (exon 3: c.137G>A; p.46G>E), (IVS7-1G>A splice site mutation), and four novel MCCB (exon 11: c.1065A>T; p.355L>F), (exon 15: c.1430A>G; p.477Q>R), (exon 16: c.1549G>A; p.517G>R), (exon 16: c.1559A>C; p.520Y>S) mutant alleles from five MCC-deficient patients.

Fluorescence quenching of CdS quantum dots by 4-azetidinyl-7-nitrobenz-2-oxa-1,3-diazole: a mechanistic study.

Fluorescence quenching of CdS quantum dots (QDs) by 4-azetidinyl-7-nitrobenz-2-oxa-1,3-diazole (NBD), where the two quenching partners satisfy the spectral overlap criterion necessary for Förster resonance energy transfer (FRET), is studied by steady-state and time-resolved fluorescence techniques. The fluorescence quenching of the QDs is accompanied by an enhancement of the acceptor fluorescence and a reduction of the average fluorescence lifetime of the donor. Even though these observations are suggestive of a dynamic energy transfer process, it is shown that the quenching actually proceeds through a static interaction between the quenching partners and is probably mediated by charge-transfer interactions. The bimolecular quenching rate constant estimated from the Stern-Volmer plot of the fluorescence intensities, is found to be exceptionally high and unrealistic for the dynamic quenching process. Hence, a kinetic model is employed for the estimation of actual quencher/QD ratio dependent exciton quenching rate constants of the fluorescence quenching of CdS by NBD. The present results point to the need for a deeper analysis of the experimental quenching data to avoid erroneous conclusions.