Search

Eine neue Pulssequenz: Die Abstände zwischen Kupferzentren der homotrimeren Nitrit ‐ Reduktase wurden mit EPR ‐ Spektroskopie bestimmt (siehe Bild). Die Differenzen in der Elektronenspinrelaxation der Kupferionen ermöglichen das selektive Entfernen von Abständen aus der komplexen Abstandsverteilung. Diese Filtertechnik in Kombination mit PELDOR ‐ Experimenten dürfte bei Abstandsbestimmungen mittels EPR ‐ Spektroskopie hilfreich sein. [ABSTRACT FROM AUTHOR]

A new pulse sequence: The distances between copper sites of the homotrimeric Cu‐containing nitrite reductase were determined by EPR spectroscopy (see picture). By exploiting the differences in the electron spin relaxation of the Cu ions, a filtering technique allows the selective removal of distances from a complex distance distribution. This filter technique combined with the PELDOR experiment promises to be useful for distance mapping by EPR spectroscopy. [ABSTRACT FROM AUTHOR]

Proliferation of cerebellar granular neuronal precursors (CGNPs) is mediated by Sonic Hedgehog (Shh), which activates the Patched and Smoothened (Smo) receptor complex. Although its protein sequence suggests that Smo is a G protein coupled receptor (GPCR), the evidence that this receptor utilizes heterotrimeric G proteins as downstream effectors is controversial. In Drosophila, Gαi is required for Hedgehog (Hh) activity, but the involvement of heterotrimeric G proteins in vertebrate Shh signaling has not yet been established. Here, we show that Shh-induced proliferation of rat CGNPs is enhanced strongly by the expression of the active forms of Gαi/o proteins (Gαi1, Gαi2, Gαi3, and Gαo) but not by members of another class (Gα12) of heterotrimeric G proteins. Additionally, the mRNAs of these different Gαi members display specific expression patterns in the developing cerebellum; only Gαi2 and Gαi3 are substantially expressed in the outer external granular layer, where CGNPs proliferate. Consistent with this, Shh-induced proliferation of CGNPs is reduced significantly by knockdowns of Gαi2 and Gαi3 but not by silencing of other members of the Gαi/o class. Finally, our results demonstrate that Gαi2 and Gαi3 locate to the primary cilium when expressed in CGNP cultures. In summary, we conclude that the proliferative effects of Shh on CGNPs are mediated by the combined activity of Gαi2 and Gαi3 proteins. [ABSTRACT FROM AUTHOR]

Over the past few decades, single-molecule manipulation has been widely applied to the real-time analysis of biomolecular interactions. It has enabled researchers to decipher structure-function relationships for polymers, enzymes, and larger-scale molecular machines, in particular by harnessing force to probe both chemical and mechanical stabilities. Nucleic acids have played a central role in this effort because, in addition to their biological significance, they exhibit unique polymeric properties which have recast them as key components participating in numerous experimental designs. In this review, we introduce recent developments highlighting this dual nature of nucleic acids in biophysics, as objects of study but also as tools allowing novel approaches. More specifically, we present molecular scaffolds as an emerging concept and describe their use in single-molecule force spectroscopy. Aspects related to folding and noncovalent interactions will be presented in parallel to research in enzymology, with a focus on the acquisition of thermodynamic and kinetic data. [ABSTRACT FROM AUTHOR]

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow. for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism. [ABSTRACT FROM AUTHOR]

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C- terminal copper-binding ioop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self- assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrange- ments accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short- range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein∔SAM interaction. [ABSTRACT FROM AUTHOR]

Abstract: The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at ∼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins. [Copyright &y& Elsevier]