Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel N-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels-Alder reaction with inverse electron demand. We demonstrate that the cell's biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3-tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels-Alder reaction with inverse electron demand and a strain-promoted azide-alkyne cycloaddition which are carried out simultaneously in a single step.

Many applications in biotechnology and molecular biology rely on modified nucleotides. Here, we present an approach for the postsynthetic labelling of enzymatically synthesized vinyl-modified DNA by Diels-Alder reaction with inverse electron demand using a tetrazine. Labelling proceeds very efficiently and supersedes several known approaches.

Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties.Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ("C12-b-M") as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors by approximately 10°C compared to C12-b-M. Another surfactant yielded a stabilization of the human Patched protein receptor by 13°C.In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b 6 f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Declaration of Interest statementThe authors report no declarations of interest. Mixed protein-surfactant micelles are often used for in vitro studies when solutions of pure, monodisperse integral membrane proteins are needed (Garavito and Ferguson-Miller, 2001). Because integral membrane proteins evolved in a lipid bilayer environment, the exchange of lipid for surfactant frequently causes a destabilization of the protein. This can result in increased activity followed by increased rate of denaturation, increased susceptibility to protease attack, accelerated heat denaturation, oxidative damage, loss of activity regulation and lack of crystallization (Tanford and Reynolds, 1976;Tate, 2010). Europe PMC Funders GroupAt the beginning of membrane protein research only surfactants developed as detergents for consumer or industrial use were available many of which were inhomogenous. In-vitro studies of solubilized membrane proteins and structural work (Rosenbusch et al., 1981;Michel and Oesterhelt, 1980) led to an increasing demand in tailored surfactants. In the micellar solution the protein should be functional and stable for extended periods of time and the micellar belt attached to the protein should be small in order to leave much of the polar protein surfaces uncovered for crystal lattice contacts (Michel, 1983).Studies of the micellar mass revealed the relation between the chemical structure of a surfactant and the size of its micelle. It was found to depend upon the ratio of the polar cross-section and the length of the extended hydrophobic chain (Israelachvili et al., 1976). In case of spherical micelles the latter matches the radius of the hydrophobic core. With alkyl chains as hydrophobic moieties of surfactants proteins require a length between an extended octyl and a dodecyl chain (Iwata, 2003).Small micelles are for...

No abstract

Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA-based material with cell-adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA-based material shows cell-repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment.