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Strackharn M.,Ludwig Maximilians University of Munich | Stahl S.W.,Center for Integrated Protein Science | Puchner E.M.,University of California at San Francisco | Gaub H.E.,Ludwig Maximilians University of Munich
Nano Letters | Year: 2012

Bottom up assembly of functional molecular ensembles with novel properties emerging from composition and arrangement of its constituents is a prime goal of nanotechnology. By single-molecule cut-and-paste we assembled binding sites for malachite green in a molecule-by-molecule assembly process from the two halves of a split aptamer. We show that only a perfectly joined binding site immobilizes the fluorophore and enhances the fluorescence quantum yield by several orders of magnitude. To corroborate the robustness of this approach we produced a micrometer-sized structure consisting of more than 500 reconstituted binding sites. To the best of our knowledge, this is the first demonstration of one by one bottom up functional biomolecular assembly. © 2012 American Chemical Society.


Rojowska A.,Ludwig Maximilians University of Munich | Lammens K.,Ludwig Maximilians University of Munich | Seifert F.U.,Ludwig Maximilians University of Munich | Direnberger C.,Ludwig Maximilians University of Munich | And 4 more authors.
EMBO Journal | Year: 2014

The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50NBD (nucleotide-binding domain) in complex with Mre11HLH (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state. Synopsis The SMC protein Rad50 is a key subunit of the MRN complex, a repair factor involved in processing and tethering DNA double-strand breaks. First structural insights into its interaction with DNA show that such binding is important for some but not other functions of the MRN complex. Thermotoga maritima Rad50 in complex with dsDNA offers the first crystal structure view of DNA binding by an SMC protein. dsDNA binds to a strand-loop-helix motif on the Rad50 nucleotide-binding domain. DNA binds between the coiled-coil domains of the ATP-bound dimer of Rad50. The strand-loop-helix motif does not directly recognize DNA ends, but rather binds a DNA duplex internally. In yeast, DNA binding to the strand-loop-helix motif is important for MRN complex function in telomere maintenance but not in DNA double-strand break repair. The first structural insight into DNA binding by an SMC protein reveals the importance of Rad50-DNA interaction for some but not other functions of the MRN complex. © 2014 The Authors.


Tosi A.,Ludwig Maximilians University of Munich | Haas C.,Ludwig Maximilians University of Munich | Herzog F.,Ludwig Maximilians University of Munich | Herzog F.,ETH Zurich | And 11 more authors.
Cell | Year: 2013

INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers. © 2013 Elsevier Inc.


Hahne H.,TU Munich | Pachl F.,TU Munich | Ruprecht B.,TU Munich | Maier S.K.,TU Munich | And 8 more authors.
Nature Methods | Year: 2013

We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.


Gupta R.,Max Planck Institute of Biochemistry | Kasturi P.,Max Planck Institute of Biochemistry | Bracher A.,Max Planck Institute of Biochemistry | Loew C.,Max Planck Institute of Biochemistry | And 6 more authors.
Nature Methods | Year: 2011

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging. © 2011 Nature America, Inc. All rights reserved.


Marx H.,TU Munich | Lemeer S.,TU Munich | Schliep J.E.,TU Munich | Matheron L.,University Utrecht | And 10 more authors.
Nature Biotechnology | Year: 2013

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns. © 2013 Nature America, Inc. All rights reserved.


Schiller C.B.,Ludwig Maximilians University of Munich | Seifert F.U.,Ludwig Maximilians University of Munich | Linke-Winnebeck C.,Ludwig Maximilians University of Munich | Hopfner K.-P.,Ludwig Maximilians University of Munich | Hopfner K.-P.,Center for Integrated Protein science
Cold Spring Harbor Perspectives in Biology | Year: 2014

DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreographyof the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies. ©2014 Cold Spring Harbor Laboratory Press; all rights reserved.


Wu Z.,TU Munich | Gholami A.M.,TU Munich | Kuster B.,TU Munich | Kuster B.,Center for Integrated Protein Science
Molecular and Cellular Proteomics | Year: 2012

HSP90 is a central player in the folding and maturation of many proteins. More than two hundred HSP90 clients have been identified by classical biochemical techniques including important signaling proteins with high relevance to human cancer pathways. HSP90 inhibition has thus become an attractive therapeutic concept and multiple molecules are currently in clinical trials. It is therefore of fundamental biological and medical importance to identify, ideally, all HSP90 clients and HSP90 regulated proteins. To this end, we have taken a global and a chemical proteomic approach in geldanamycin treated cancer cell lines using stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry. We identified >6200 proteins in four different human cell lines and ∼1600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell-type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were geldanamycin treatment including >50 kinases not formerly known to be regulated by HSP90. Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture showed that protein down-regulation by HSP90 inhibition correlates with protein half-life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The proteomic responses of the HSP90 drugs geldanamycin and PU-H71 were highly similar suggesting that both drugs work by similar molecular mechanisms. Using HSP90 immunoprecipitation, we validated several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII), as novel clients of HSP90. Taken together, our study broadly defines the cellular proteome response to HSP90 inhibition and provides a rich resource for further investigation relevant for the treatment of cancer. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Gutsmiedl K.,Center for Integrated Protein Science | Fazio D.,Center for Integrated Protein Science | Carell T.,Center for Integrated Protein Science
Chemistry - A European Journal | Year: 2010

We report the regioselective Cu-free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3-Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu-free and Cu-catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high-density functionalization of DNA is simple, modular, and efficient. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


Cavlar T.,University of Bonn | Deimling T.,Ludwig Maximilians University of Munich | Ablasser A.,University of Bonn | Hopfner K.-P.,Ludwig Maximilians University of Munich | And 2 more authors.
EMBO Journal | Year: 2013

Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10-carboxymethyl-9-acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell-intrinsic type I IFN response, depending on the ER-resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C-terminal ligand-binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate (c-diGMP)-binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c-diGMP. Altogether, these results provide novel insight into ligand-sensing properties of STING and, furthermore, unravel unexpected species-specific differences of this innate sensor. © 2013 European Molecular Biology Organization.

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