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Herein we present an efficient synthesis of a biomimetic probe with modular construction that can be specifically bound by the mannose binding FimH protein – a surface adhesion protein of E. coli bacteria. The synthesis combines the new and interesting DBD dye with the carbohydrate ligand mannose via a Click reaction. We demonstrate the binding to E. coli bacteria over a large concentration range and also present some special characteristics of those molecules that are of particular interest for the application as a biosensor. In particular, the mix-and-measure ability and the very good photo-stability should be highlighted here.
Hepcidin-25 (Hep-25) plays a crucial role in the control of iron homeostasis. Since the dysfunction of the hepcidin pathway leads to multiple diseases as a result of iron imbalance, hepcidin represents a potential target for the diagnosis and treatment of disorders of iron metabolism. Despite intense research in the last decade targeted at developing a selective immunoassay for iron disorder diagnosis and treatment and better understanding the ferroportin-hepcidin interaction, questions remain. The key to resolving these underlying questions is acquiring exact knowledge of the 3D structure of native Hep-25. Since it was determined that the N-terminus, which is responsible for the bioactivity of Hep-25, contains a small Cu(II)-binding site known as the ATCUN motif, it was assumed that the Hep-25-Cu(II) complex is the native, bioactive form of the hepcidin. This structure has thus far not been elucidated in detail. Owing to the lack of structural information on metal-bound Hep-25, little is known about its possible biological role in iron metabolism. Therefore, this work is focused on structurally characterizing the metal-bound Hep-25 by NMR spectroscopy and molecular dynamics simulations. For the present work, a protocol was developed to prepare and purify properly folded Hep-25 in high quantities. In order to overcome the low solubility of Hep-25 at neutral pH, we introduced the C-terminal DEDEDE solubility tag. The metal binding was investigated through a series of NMR spectroscopic experiments to identify the most affected amino acids that mediate metal coordination. Based on the obtained NMR data, a structural calculation was performed in order to generate a model structure of the Hep-25-Ni(II) complex. The DEDEDE tag was excluded from the structural calculation due to a lack of NMR restraints. The dynamic nature and fast exchange of some of the amide protons with solvent reduced the overall number of NMR restraints needed for a high-quality structure. The NMR data revealed that the 20 Cterminal Hep-25 amino acids experienced no significant conformational changes, compared to published results, as a result of a pH change from pH 3 to pH 7 and metal binding. A 3D model of the Hep-25-Ni(II) complex was constructed from NMR data recorded for the hexapeptideNi(II) complex and Hep-25-DEDEDE-Ni(II) complex in combination with the fixed conformation of 19 C-terminal amino acids. The NMR data of the Hep-25-DEDEDE-Ni(II) complex indicates that the ATCUN motif moves independently from the rest of the structure. The 3D model structure of the metal-bound Hep-25 allows for future works to elucidate hepcidin’s interaction with its receptor ferroportin and should serve as a starting point for the development of antibodies with improved selectivity.
The goal of this work was to study the binding of ions to polymers and lipid bilayer membranes in aqueous solutions. In the first part of this work, the influence of various inorganic salts and polyelectrolytes on the structure of water was studied using Isothermal Titration Calorimetry (ITC). The heat of dilution of the salts was used as a scale of water structure making and breaking of the ions. The heats of dilution could be attributed to the Hofmeister Series. Following this, the binding of Ca2+ to poly(sodium acrylate) (NaPAA) was studied. ITC and a Ca2+ Ion Selective Electrode were used to measure the reaction enthalpy and binding isotherm. Binding of Ca2+ ions to PAA, was found to be highly endothermic and therefore solely driven by entropy. We then compared the binding of ions to the one-dimensional PAA polymer chain to the binding to lipid vesicles with the same functional groups. As for the polymer, Ca2+ binding was found to be endothermic. Binding of calcium to the lipid bilayer was found to be weaker than to the polymer. In the context of these experiments, it was shown that Ca2+ not only binds to charged but also to zwitterionic lipid vesicles. Finally, we studied the interaction of two salts, KCl and NaCl, to a neutral polymer gel, PNIPAAM, and to the ionic polymer PAA. Combining calorimetry and a potassium selective electrode we observed that the ions interact with both polymers, whether containing charges or not.
A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein
(2013)
We present two thermoresponsive water soluble copolymers prepared via free radical statistical copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates (OEGMAs), respectively, with a solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)- functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic changes in its fluorescence profile in response to a change in solution temperature as well as to the presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its emission colour when heated above its cloud point. Such drastic changes in the fluorescence and absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the molecular recognition event. On the other hand, heating of the polymer–antibody complexes releases a fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture, the released antibody competitively binds to the monomer and the antibody-free chains of the polymer undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular design of responsive polymers aimed at specific applications, such as biosensing.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.