Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy

Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress. Here, we describe an alternative ex situ imaging method where identical molecules are recorded before and after reaction with a binding partner. Relocation of the identical molecules on the mica surface was thereby achieved by using a nanoscale scratch as marker. The method was successfully applied to study the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII), two essential haemostatic components of human blood. FVIII binding was discernible by an appearance of globular domains appended to the N-terminal large globular domains of VWF. The specificity of the approach could be demonstrated by incubating VWF with FVIII in the presence of a high salt buffer which inhibits the interaction between these two proteins. The results obtained indicate that proteins can maintain their reactivity for subsequent interactions with other molecules when gently immobilized on a solid substrate and subjected to intermittent drying steps. The technique described opens up a new analytical perspective for studying protein-protein interactions as it circumvents some of the obstacles encountered by in situ imaging and other ex situ techniques.

On the conformational flexibility of vitamin D

The conformational flexibility of vitamin D was investigated by a combination of force field calculations, LIS measurements and LIS simulations. Besides the two A-ring chair forms, A-ring twist forms were detected and were attributed to steric interactions arising from the substitution pattern of this class of seco-steroids.

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Über die konformationelle Beweglichkeit von Vitamin D

Zusammenfassung Die konformationelle Beweglichkeit von Vitamin D wurde mittels einer Kombination aus Kraftfeld Rechnungen, LIS-Messungen und LIS-Rechnungen untersucht. Neben den beiden A-Ring Sesselformen wurden auch A-Ring Twistformen aufgefunden. Die Ursache für das Vorhandensein von diesen Twistformen liegt in sterischen Wechselwirkungen, verursacht durch das Substitutionsmuster des A-Ringes in diesem seco-Steroid.

     

Analysis of Carbohydrate Mixtures by Diffusion Difference NMR Spectroscopy

Summary.  Diffusion difference NMR spectroscopy can be used as a fast and powerful tool to separate carbohydrates in a mixture by their translational diffusion properties. This method is of general interest for analyzing natural and synthetic mixtures and for monitoring and optimizing synthetic reactions. The proposed subtraction procedure applied to two varying diffusion encoded spectra can be combined with homo- and heteronuclear as well as with one- and multi-dimensional NMR experiments. Received November 26, 2001. Accepted November 30, 2001     

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE

2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.     

A core-free semicovering of the Hawaiian Earring

The connected covering spaces of a connected and locally path-connected topological space X   can be classified by the conjugacy classes of those subgroups of _π1(X,x)${\pi }_1(X,x)$ which contain an open normal subgroup of _π1(X,x)${\pi }_1(X,x)$, when endowed with the natural quotient topology of the compact-open topology on based loops. There are known examples of semicoverings (in the sense of Brazas) that correspond to open subgroups which do not contain an open normal subgroup. We present an example of a semicovering of the Hawaiian Earring H$\mathbb{H}$ with corresponding open subgroup of _π1(H)${\pi }_1(\mathbb{H})$ which does not contain any   nontrivial normal subgroup of _π1(H)${\pi }_1(\mathbb{H})$.     

Efficient option valuation using trees

An algorithm is proposed for the discrete approximation of continuous market price processes that uses trees instead of lattices. It is shown that it is convergent when used for pricing both European and American options and that it is more efficient, for some models, than the usual recombining schemes.     

Die thermische wolff-umlagerung der 1,2,3-thiadiazole

Thermal nitrogen elimination of 1,2,3-thiadiazoles leads to primary fragments, which rearrange to thioketenes. In the presence of high boiling alcohols like diglycol, the corresponding esters can be isolated in good yields. Benzo-1,2,3-thiadiazole, which does not rearrange, is an exception, because of the integrity of the aromatic nucleus.