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Volker Dötsch is at the Institute of Biophysical Chemistry, Goethe University and Centre for Biomolecular Magnetic Resonance (BMRZ) and the Cluster of Excellence Frankfurt (Macromolecular Complexes), Frankfurt ...doi:10.1038/embor.2009.190 pmid:19721457 pmcid:PMC2750066 fatcat:wvdgmfapojeblo7im7o3natayu
The crystal structure of a nucleotide exchange factor in white blood cells reveals an autoinhibitory mechanism that reinforces the switch-like behaviour of the signalling protein Ras.doi:10.7554/elife.01159 pmid:23908771 pmcid:PMC3728619 fatcat:j42qkz7bnzaifodcynzwolnafa
Volker Dötsch is at the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt (Main), Germany. email: Ivan.Dikic@biochem2.de ubiquitin linkages ... make a difference Ivan Dikic & Volker Dötsch ubiquitin chains have critical roles in activating the nf-κB pathway and mediating immune responses. recent structural work on distinct ubiquitin chains in ...doi:10.1038/nsmb1209-1209 pmid:19956206 fatcat:p33daju27zdtvleexk5ev4ru2e
The production of haploid gametes through meiosis is central to the principle of sexual reproduction. The genetic diversity is further enhanced by exchange of genetic material between homologous chromosomes by the crossover mechanism. This mechanism not only requires correct pairing of homologous chromosomes but also efficient repair of the induced DNA double-strand breaks. Oocytes have evolved a unique quality control system that eliminates cells if chromosomes do not correctly align or if DNAdoi:10.3390/molecules25235714 pmid:33287328 fatcat:7vro7zvvyjcmnergp74civ3dwm
more »... repair is not possible. Central to this monitoring system that is conserved from nematodes and fruit fly to humans is the p53 protein family, and in vertebrates in particular p63. In mammals, oocytes are stored for a long time in the prophase of meiosis I which, in humans, can last more than 50 years. During the entire time of this arrest phase, the DNA damage checkpoint remains active. The treatment of female cancer patients with DNA damaging irradiation or chemotherapeutics activates this checkpoint and results in elimination of the oocyte pool causing premature menopause and infertility. Here, we review the molecular mechanisms of this quality control system and discuss potential therapeutic intervention for the preservation of the oocyte pool during chemotherapy.
Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membranedoi:10.1016/j.febslet.2015.04.045 pmid:25937121 fatcat:fhzmtejdjnhtvjmcparjfaxi5u
more »... otein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.
Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribution of this technology. Membrane protein synthesis by cell-free expression does not appear to be restricted by origin, size or topology of the target, and its global application is therefore a highly valuabledoi:10.1007/978-1-60761-344-2_11 pmid:20099146 fatcat:slknm2ibcze7thb4v55lsjooau
more »... The technology is relatively fast to establish in standard biochemical labs, and it does not require expensive equipment. Moreover, it enables the production of membrane proteins in completely new modes, like the direct translation into detergent micelles, which is not possible with any other expression system. In this protocol, we focus on the currently most efficient cell-free expression system for membrane proteins based on Escherichia coli extracts.
Krauskopf, K.; Gebel, J.; Kazemi, S.; Tuppi, M.; Lohr, F.; Schafer, B.; Koch, J.; Guntert, P.; Dotsch, V.; Kehrloesser, S. ... .; Dotsch, V.; De Felici, M.; et al. The p63 C-terminus is essential for murine oocyte integrity. Nat. Commun. 2021, 12, 383. [CrossRef] 51. ...doi:10.3390/cancers13030536 pmid:33572532 fatcat:neguszp4qfcfjd6a2a5gt3e72i
In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent thedoi:10.3390/ijms21155369 pmid:32731622 pmcid:PMC7432864 fatcat:o3svxst4xfbfxkhg4oxaliosta
more »... ion of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.
Simple peak-picking algorithms, such as those based on lineshape fitting, perform well when peaks are completely resolved in multidimensional NMR spectra, but often produce wrong intensities and frequencies for overlapping peak clusters. For example, NOESY-type spectra have considerable overlaps leading to significant peak-picking intensity errors, which can result in erroneous structural restraints. Precise frequencies are critical for unambiguous resonance assignments. Results: To alleviatedoi:10.1186/1471-2105-15-46 pmid:24511909 pmcid:PMC3931316 fatcat:k6m5h4wydzcn7cealyojfcwnr4
more »... is problem, a more sophisticated peaks decomposition algorithm, based on non-negative matrix factorization (NMF), was developed. We produce peak shapes from Fourier-transformed NMR spectra. Apart from its main goal of deriving components from spectra and producing peak lists automatically, the NMF approach can also be applied if the positions of some peaks are known a priori, e.g. from consistently referenced spectral dimensions of other experiments. Conclusions: Application of the NMF algorithm to a three-dimensional peak list of the 23 kDa bi-domain section of the RcsD protein (RcsD-ABL-HPt, residues 688-890) as well as to synthetic HSQC data shows that peaks can be picked accurately also in spectral regions with strong overlap.
doi:10.1038/s41418-018-0107-6 pmid:29666471 fatcat:j2a7kmslqncppm7qw4gve7s5fy
The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they bothdoi:10.1038/sj.emboj.7601764 pmid:17581633 pmcid:PMC1933395 fatcat:3dydp5sk2jebdbmuskfwyxd4gu
more »... iffer significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal b-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.
Nuclear magnetic resonance (NMR) structure calculations of the a-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary fordoi:10.1016/j.str.2012.03.010 pmid:22560730 fatcat:4vyj6xa4brfldpqwz4llr4ivni
more »... ing enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables a-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.
doi:10.1038/s41418-021-00818-8 pmid:34131311 pmcid:PMC8257794 fatcat:mlapbi4jgvgoncicf6oodirer4
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