Volodymyr Chmyrov

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aβ oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aβ, FRET-FCS detected oligomers consisting of less than 10 Aβ molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aβ oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells

Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

Efficiency enhanced colloidal Mn-doped type II core/shell ZnSe/CdS quantum dot sensitized hybrid solar cells

Colloidal Mn-doped ZnSe/CdS core/shell quantum dots (QDs) are synthesized for the first time and employed as a strategy to boost the power conversion efficiency of quantum dot sensitized solar cells. By using Mn-doping as a band gap engineering tool for core/shell QDs an effective improvement of absorption spectra could be obtained. The mid-states generated by a proper Mn content alleviate carrier separation and enhance the electron injection rate, thus facilitating electron transport to the TiO2 substrate. It is demonstrated that a device constructed with 0.25% Mn-doped ZnSe/CdS leads to an enhancement of the electron injection rate and power conversion efficiency by 4 times and 1.3, respectively.

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

Mechanisms of fluorescence decays of colloidal CdSe-CdS/ZnS quantum dots unraveled by time-resolved fluorescence measurement.

By narrowing the detection bandpass and increasing the signal-to-noise ratio in measuring the time-resolved fluorescence decay spectrum of colloidal CdSe-CdS/ZnS quantum dots (QDs), we show that directly after the photoexcitation, the fluorescence decay spectrum is characterized by a single exponential decay, which represents the energy relaxation of the photogenerated exciton from its initial high-energy state to the ground exciton state. The fluorescence decay spectrum of long decay time is in the form of β/t(2), where β is the radiative recombination time of the ground-state exciton and t is the decay time. Our findings provide us with a direct and quantitative link between fluorescence decay measurement data and fundamental photophysics of QD exciton, thereby leading to a novel way of applying colloidal QDs to study microscopic, physical and chemical processes in many fields including biomedicine.