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Kaufenstein M.,Albert Ludwigs University of Freiburg | Van Der Laan M.,Institute For Biochemie Und Molekularbiologie | Graumann P.L.,Albert Ludwigs University of Freiburg
Journal of Bacteriology | Year: 2011

Many bacteria possess the ability to actively take up DNA from the environment and incorporate it into the chromosome. RecA protein is the key protein achieving homologous recombination. Several of the proteins involved in the transport of DNA across the cell envelope assemble at a single or both cell poles in competent Bacillus subtilis cells. We show that the presumed structure that transports DNA across the cell wall, the pseudopilus, also assembles at a single or both cell poles, while the membrane receptor, ComEA, forms a mobile layer throughout the cell membrane. All other known Com proteins, including the membrane permease, localize again to the cell pole, revealing that the uptake machinery has three distinct layers. In cells having two uptake machineries, one complex is occasionally mobile, with pairs of proteins moving together, suggesting that a complete complex may lose anchoring and become mobile. Overall, the cell pole provides stable anchoring. Only one of two uptake machineries assembles RecA protein, suggesting that only one is competent for DNA transfer. FRAP (fluorescence recovery after photobleaching) analyses show that in contrast to known multiprotein complexes, the DNA uptake machinery forms a highly stable complex, showing little or no exchange with unbound molecules. When cells are converted into round spheroplasts, the structure persists, revealing that the assembly is highly stable and does not require the cell pole for its maintenance. High stability may be important to fulfill the mechanical function in pulling DNA across two cell layers. Copyright © 2011, American Society for Microbiology. All Rights Reserved. Source

Opalinska M.,Institute For Biochemie Und Molekularbiologie | Meisinger C.,Institute For Biochemie Und Molekularbiologie | Meisinger C.,Center for Biological Signalling Studies
Current Opinion in Cell Biology | Year: 2015

Mitochondria have to import most of their proteins in order to fulfill a multitude of metabolic functions. Sophisticated import machineries mediate targeting and translocation of preproteins from the cytosol and subsequent sorting into their suborganellar destination. The mode of action of these machineries has been considered for long time as a static and constitutively active process. However, recent studies revealed that the mitochondrial protein import machinery is subject to intense regulatory mechanisms that include direct control of protein flux by metabolites and metabolic signalling cascades. © 2014 Elsevier Ltd. Source

Anbazhagan V.,Institute For Biochemie Und Molekularbiologie | Cymer F.,Institute For Biochemie Und Molekularbiologie | Cymer F.,Albert Ludwigs University of Freiburg | Schneider D.,Institute For Biochemie Und Molekularbiologie | Schneider D.,Johannes Gutenberg University Mainz
Archives of Biochemistry and Biophysics | Year: 2010

The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions albeit without modifying the helicity of the peptides. The energetics and kinetics of the dimer dissociation in mixed micelles is analyzed and discussed, and the experimental data demonstrate that mixed micelles can be used as a general method to investigate unfolding of α-helical membrane proteins. © 2010 Elsevier Inc. All rights reserved. Source

Venne A.S.,Leibniz Institute for Analytical Sciences | Vogtle F.-N.,Institute For Biochemie Und Molekularbiologie | Meisinger C.,Institute For Biochemie Und Molekularbiologie | Meisinger C.,Albert Ludwigs University of Freiburg | And 3 more authors.
Journal of Proteome Research | Year: 2013

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities. © 2013 American Chemical Society. Source

Cymer F.,Institute For Biochemie Und Molekularbiologie | Cymer F.,Albert Ludwigs University of Freiburg | Schneider D.,Institute For Biochemie Und Molekularbiologie
Biochemistry | Year: 2010

Like many other α-helical membrane proteins, the monomeric Escherichia coli aquaglyceroporin GlpF associates within cellular membranes and forms higher-order oligomeric structures. A potential impact of the oligomeric state on the protein function remains enigmatic. We have analyzed the role of residues W42 and E43 in the oligomerization of the E. coli GlpF protein in vitro and in vivo. In contrast to W42, the polar glutamate residue at position 43 appears to be critical for oligomerization. While other polar residues can substitute for the function of E43, replacement of E43 with alanine results in a greatly reduced GlpF oligomerization propensity. The reduced interaction propensity of GlpF E43A correlates with an impaired in vivo function as well as a decreased in vivo stability. Therefore, E43 is critical for the proper oligomerization of GlpF, and protein oligomerization appears to be crucial for the channel function as well as for the in vivo stability of the protein. ©2009 American Chenical Society. Source

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