Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance sensor chips.

Chitin present in fungal cell walls has been considered as a diagnostic polymer for the detection of fungal infections. Chitin staining can be achieved with different dyes such as Calcofluor white or Congo red, but these methods have not entered into clinical routine diagnosis due to problems with sensitivity and specificity. More accurate detection can be achieved using chitin binding domains (CBDs) from a large variety of naturally occurring proteins that specifically interact with chitin. The chitin binding properties of most of these proteins have not yet been determined, because chitin is an insoluble fibrillar material rendering accurate determination of chitin binding kinetics challenging. Here we report a quartz crystal microbalance with dissipation monitoring (QCM-D) based method to determine binding constants of CBDs on chitin-coated gold surfaces. For this purpose, chitin was trimethylsilylated and coated onto the sensor chips. After desilylation, regular fibril-like structures with a typical center-to-center spacing of 85 nm were observed by atomic force microscopy. Using different experimental conditions and data evaluation methods for QCM-D measurements, we determined kon and koff and calculated the KD values for binding of a recombinant CBD from Bacillus circulans chitinase A1. Depending on the evaluation method, the KD values ranged between 0.6 and 2.5 µM. The obtained KD values were in good agreement with those measured for other bacterial CBDs usually ranging between 1 to 10 µM. Hence, we propose that the experimental approach developed in this study can be applied to determine yet unknown binding affinities of various CBDs from different origin.

Pristine DNA Hydrogels from Biotechnologically Derived Plasmid DNA.

DNA hydrogels are of great interest for a variety of biomedical applications owing to their biocompatibility and biodegradability but the advantages of DNA hydrogels have not been exploited yet because of their limited availability. Thus far, DNA hydrogels have been prepared from synthetically derived building blocks, and their production on large scale would be far too expensive. As an alternative, here the generation of DNA hydrogels from plasmid DNA is reported. Plasmid DNA can be prepared on large scale at reasonable costs by a fermentation process. The desired linear DNA building blocks are then obtained from the plasmid DNA by enzymatic digestion. Gel formation is carried out by covalent bond formation between individual building blocks via enzymatic ligation. The generation of pristine DNA hydrogels from plasmid DNA is thus presented for the first time. The viscoelastic properties of the hydrogels were studied by rheology, which confirmed that the gels have storage moduli G' of >100 Pa.

Construction of three-dimensional DNA hydrogels from linear building blocks.

A three-dimensional DNA hydrogel was generated by self-assembly of short linear double-stranded DNA (dsDNA) building blocks equipped with sticky ends. The resulting DNA hydrogel is thermoresponsive and the length of the supramolecular dsDNA structures varies with temperature. The average diffusion coefficients of the supramolecular dsDNA structures formed by self-assembly were determined by diffusion-ordered NMR spectroscopy (DOSY NMR) for temperatures higher than 60 °C. Temperature-dependent rheological measurements revealed a gel point of 42±1 °C. Below this temperature, the resulting material behaved as a true gel of high viscosity with values for the storage modulus G' being significantly larger than that for the loss modulus G''. Frequency-dependent rheological measurements at 20 °C revealed a mesh size (ξ) of 15 nm. AFM analysis of the diluted hydrogel in the dry state showed densely packed structures of entangled chains, which are also expected to contain multiple interlocked rings and catenanes.

Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology.

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.

Redox properties of LOV domains: chemical versus photochemical reduction, and influence on the photocycle.

LOV (light-oxygen-voltage-sensitive) domains comprise the light-sensitive parts of many blue light photoreceptor proteins. Photoexcitation of the chromophore flavin mononucleotide (FMN) in these LOV domains leads to formation of a covalent adduct between FMN and a cysteine residue. So far, the electronically excited singlet and triplet states of FMN have been identified as the only intermediates in the photocycles of LOV domains from several organisms. Since many flavoproteins are redox-active, however, the photocycles of LOV domains might involve other redox states of FMN, and might be controlled by the external redox potential. Here we report on the redox properties of the LOV1 domain from phototropin of the green alga Chlamydomonas reinhardtii. By equilibrium-redox spectropotentiometry a redox potential [E(fq/fhq) (flavoquinone/flavohydroquinone)] of -290 mV vs. the normal hydrogen electrode (NHE) was determined for the wild-type domain (LOV1-wt). A similar value of -280 mV was found for the mutant LOV1-C57G, in which the photoreactive cysteine is replaced by glycine. The recovery kinetics (photoadduct-->ground state) in the photocycle of LOV1-wt are not influenced by a redox potential in the range between +500 and -260 mV versus NHE. No flavosemiquinone could be generated by chemical reduction with sodium dithionite. However, photoreduction of LOV1-C57G with EDTA leads exclusively to the flavosemiquinone. This semiquinone is stable against disproportionation, and the photoreduction is not mediated by free FMN.

Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.