Capabilities of MS for analytical quantitative determination of the ratio of α- and ßAsp7 isoforms of the amyloid-ß peptide in binary mixtures.

There is strong evidence that the amyloid-ß peptide (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), a lethal neurodegenerative disorder of the elderly. During pathology development, the peptide as well as its various chemically modified isoforms is accumulated in specific brain tissues as characteristic proteinaceous deposits, the so-called amyloid plaques, which are the pathomorphological mark of AD, although the level of Αß in the blood is the same for healthy individuals and for AD patients. Earlier, it has been shown that isomerization of aspartate 7, the most abundant post-translational modification of the Αß peptide, is tightly involved in a set of molecular processes associated with AD progression. Therefore, the isoAsp 7-containing Αß isomer (isoAß) is assumed to be a potential biomarker of AD that can be identified in the blood. Here, we present an analytical mass spectrometric method for quantitative determination of the ratio of normal and isomerized Αß fragments 1-16 in their binary mixtures, and all analytical capabilities, such as accuracy, detection limits, and sensitivity of the presented method, are determined and thoroughly discussed. On the basis of this method, an analytical approach for quantitative determination of this modification in the blood will be developed in further studies.

Cation binding mode of fully oxidised calmodulin explained by the unfolding of the apostate.

Calmodulin is the most ubiquitous calcium binding protein. The protein is very sensitive to oxidation and this modification has pronounced effects on calmodulin function. In this work, we decided to fully oxidise calmodulin in order to study the consequences on cation binding, domain stability, and alpha helicity. Oxidation of methionines unfolds completely the apostate of the protein, which upon calcium binding recovers the major part of its secondary and tertiary structure. However, the unstructuring of the apostate results in a protein that binds calcium to any site in an independent manner, does not bind magnesium and does not possess auxiliary sites anymore.

DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.

DNA gyrase is a major bacterial protein that is involved in replication and transcription and catalyzes the negative supercoiling of bacterial circular DNA. DNA gyrase is a known target for antibacterial agents since its blocking induces bacterial death. Quinolones, coumarins, and cyclothialidines have been designed to inhibit gyrase. Significant improvements can still be envisioned for a better coumarin-gyrase interaction. In this work, we obtained the crystal costructures of the natural coumarin clorobiocin and a synthetic analogue with the 24 kDa gyrase fragment. We used isothermal titration microcalorimetry and differential scanning calorimetry to obtain the thermodynamic parameters representative of the molecular interactions occurring during the binding process between coumarins and the 24 kDa gyrase fragment. We provide the first experimental evidence that clorobiocin binds gyrase with a stronger affinity than novobiocin. We also demonstrate the crucial role of both the hydroxybenzoate isopentenyl moiety and the 5'-alkyl group on the noviose of the coumarins in the binding affinity for gyrase.