Project description:Thermal Proteome Profiling (TPP) allows for identification of drug (off-)targets by evaluating shifts in apparent melting temperature for proteins. In this study, we use TPP to identify the targets for a novel antifolate (C1).
Project description:To carry out population genetics analyses of the Arctic gregion we carried out Illumina Bead-Array-based enotyping on 18 samples from Greenland.
Project description:The organohalide-respiring Sulfurospirillum multivorans uses chlorinated ethenes as electron acceptors for growth under anoxic conditions. However, little is known about the interaction of these substrates with proteins. Here, we apply thermal proteome profiling (TPP) to analyze enzyme-trichloroethene interactions. TPP is commonly used to investigate protein-ligand binding through protein melting curve shifts. Several modifications in the protocol, e.g. performing the incubation under anaerobic conditions and increasing the temperature range up to 97°C, improved the detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of the tetrachloroethene reductive dehalogenase PceA with trichloroethene and confirms the enzyme’s specificity for this substrate. Another 19 proteins showed significant melting curve shifts with trichloroethene, pointing to other proteins directly or indirectly interacting with trichloroethene. Interestingly, a putative response regulator reacted similarly towards trichloroethene, which is potentially in line with its proposed role in regulating trichloroethene respiration. The TPP approach is here proven to facilitate the identification of substrate-enzyme interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and potential signal-sensing proteins in other difficult to study bacteria.
Project description:Background biology: Global warming has accelerated in recent decades, with the Arctic warming 2–3 times faster than the global average. As a result boreal species are expanding into the Arctic, at a pace reflecting environmental warming. Nevertheless, the poleward expansion of boreal marine species is restricted by their ability to tolerate low water temperatures, and in the case of intertidal species, sub-zero air temperatures during winter. In Greenland, however, the number of days with extreme sub-zero air temperatures has decreased by more than 50% since the 1950’s, suggesting that the low air temperature constraint is weakening. Although boreal intertidal species could potentially benefit from this warmer climate to establish populations in the Arctic, recent work has shown that local intertidal summer air temperatures in Greenland can exceed 36°C. This temperature is above the thermoregulatory capacity of many boreal intertidal species, including the highly abundant blue mussel Mytilus edulis. Therefore will further colonisation of M. edulis in Greenland be inhibited by the increasingly warm summer temperatures. Aim of experiment: Intertidal animals (Greenland blue mussel M. edulis) were sampled in situ on the first warm days of the year from the inner (warmer) and outer (cooler) regions of the Godthåbsfjorden around Nuuk (64°N) to examine the fjord temperature gradient effect. In addition, subtidal M. edulis were also collected and subjected to two acute temperature shocks of 22 and 32°C, which represented common and extreme summer air temperatures for intertidal habitats near Nuuk.
Project description:To carry out population genetics analyses of the Arctic gregion we carried out Illumina Bead-Array-based enotyping on 18 samples from Greenland. 19 samples were analysed with the Illumina platform Human660W-Quad v1.0 Genotyping BeadChip and are described herein.
Project description:This study aimed to model formamide-based melting for the optimization of the sensitivity and specifcity of oligonucleotide probes in dignostic high-density microarrays. Formamide melting profiles of DNA oligonucleotides were obtained with a high-density microarray targeting 16S rRNA genes of Escherichia coli and Rhodobacter sphaeroides. One or two mismatched versions of perfect match probes were included on the array to systematically analyze the effect of formamide on mismatch stability and mismatch discrimination. A thermodynamics-based mathematical model of formamide denaturation was developed to predict the formamide melting profiles with sufficient accuracy to help with oligonucleotide design in microbial ecology applications.