TY - JOUR A1 - Brechun, Katherine Emily A1 - Arndt, Katja Maren A1 - Woolley, G. Andrew T1 - Selection of protein-protein interactions of desired affinities with a bandpass circuit JF - Journal of molecular biology : JMB N2 - We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to beta-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for beta-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength. (C) 2018 Elsevier Ltd. All rights reserved. KW - synthetic biology KW - genetic circuit KW - biological engineering KW - protein-protein interactions KW - twin-arginine translocation KW - selection system Y1 - 2018 U6 - https://doi.org/10.1016/j.jmb.2018.11.011 SN - 0022-2836 SN - 1089-8638 VL - 431 IS - 2 SP - 391 EP - 400 PB - Elsevier CY - London ER - TY - JOUR A1 - Brechun, Katherine E. A1 - Arndt, Katja Maren A1 - Woolley, G. Andrew T1 - Strategies for the photo-control of endogenous protein activity JF - Current opinion in structural biology : review of all advances ; evaluation of key references ; comprehensive listing of papers Y1 - 2017 U6 - https://doi.org/10.1016/j.sbi.2016.11.014 SN - 0959-440X SN - 1879-033X VL - 45 SP - 53 EP - 58 PB - Elsevier CY - London ER - TY - GEN A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 983 KW - cohesive ends KW - DNA cleavage KW - genetic vectors KW - modified primers KW - molecular methods KW - polymerase chain reaction KW - recombinant Escherichia coli KW - restriction enzymes Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431085 SN - 1866-8372 IS - 983 ER - TY - JOUR A1 - Feiner, Rebecca Christine A1 - Teschner, Julian A1 - Teschner, Kathrin E. A1 - Radukic, Marco T. A1 - Baumann, Tobias A1 - Hagen, Sven A1 - Hannappel, Yvonne A1 - Biere, Niklas A1 - Anselmetti, Dario A1 - Arndt, Katja Maren A1 - Müller, Kristian Mark T1 - rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations JF - International journal of molecular sciences N2 - Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme beta-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with beta-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology. KW - adeno-associated-virus KW - beta-lactamase KW - inverted terminal repeat (ITR) KW - loop modification KW - capsid stability Y1 - 2019 U6 - https://doi.org/10.3390/ijms20225702 SN - 1422-0067 VL - 20 IS - 22 PB - MDPI CY - Basel ER - TY - JOUR A1 - Brechun, Katherine E. A1 - Zhen, Danlin A1 - Jaikaran, Anna A1 - Borisenko, Vitali A1 - Kumauchi, Masato A1 - Hoff, Wouter D. A1 - Arndt, Katja Maren A1 - Woolley, Andrew G T1 - Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo JF - Biochemistry N2 - We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts. Y1 - 2019 U6 - https://doi.org/10.1021/acs.biochem.9b00279 SN - 0006-2960 VL - 58 IS - 23 SP - 2682 EP - 2694 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Hao, Nan A1 - Shearwin, Keith E. A1 - Arndt, Katja Maren T1 - Characterizing transcriptional interference between converging genes in bacteria JF - ACS synthetic biology N2 - Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two. KW - gene regulation KW - antisense transcription KW - transcriptional interference KW - mathematical modeling KW - Escherichia coli Y1 - 2019 U6 - https://doi.org/10.1021/acssynbio.8b00477 SN - 2161-5063 VL - 8 IS - 3 SP - 466 EP - 473 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Staab, Paul R. A1 - Walossek, Jörg A1 - Nellessen, David A1 - Grünberg, Raik A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - SynBioWave : a real-time communication platform for molecular and synthetic biology N2 - Synthetic Biology is advanced by many users and relies on the assembly of genetic elements to devices, systems and finally genomes. SynBioWave is a software suite that enables multiple distributed users to analyze and construct genetic parts in real-time collaboration. It builds on Google Wave and provides an extensible robot-robot-user communication framework, a menu driven user interface, biological data handling including DAS and an internal database communication. We demonstrate its use by implementing robots for gene-data retrieval, manipulation and display. The initial development of SynBioWave demonstrates the power of the underlying Google Wave protocol for Synthetic Biology and lays the foundation for continuous and user-friendly extensions. Specialized wave-robots with a manageable set of capabilities will divide and conquer the complex task of creating a genome in silico. Y1 - 2010 UR - http://bioinformatics.oxfordjournals.org/ U6 - https://doi.org/10.1093/bioinformatics/btq518 SN - 1367-4803 ER - TY - JOUR A1 - Speck, Janina A1 - Räuber, Christina A1 - Kükenshöner, Tim A1 - Niemöller, Christoph A1 - Mueller, Katelyn J. A1 - Schleberger, Paula A1 - Dondapati, Padmarupa A1 - Hecky, Jochen A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation JF - Protein engineering design & selection N2 - The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric proteinprotein interactions enable TAT-dependent translocation of the resistance marker TEM -lactamase (L). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and L, (ii) dimeric or oligomeric proteinprotein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the L, respectively and (iii) heterotrimeric proteinprotein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split L fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections. KW - HiT selection KW - NHR2 KW - TAT selection KW - three hybrid KW - two hybrid Y1 - 2013 U6 - https://doi.org/10.1093/protein/gzs098 SN - 1741-0126 VL - 26 IS - 3 SP - 225 EP - 242 PB - Oxford Univ. Press CY - Oxford ER - TY - BOOK A1 - Arndt, Katja Maren T1 - Proteine zur Krebstherapie - Zielen, Steuern, Hemmen : Antrittsvorlesung 2010-12-08 N2 - Biotechnologie, Biologie, Protein Engineering, Therapeutische Peptide, Protein Design, Selektionssysteme / biotechnology, biology, protein enginieering, therapeutic peptides, protein design, selection systems Y1 - 2010 UR - http://info.ub.uni-potsdam.de/multimedia/show_multimediafile.php?mediafile_id=239 PB - Univ.-Bibl. CY - Potsdam ER - TY - JOUR A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V JF - BMC biotechnology N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. KW - Cohesive ends KW - DNA cleavage KW - Genetic vectors KW - Modified primers KW - Molecular methods KW - Polymerase chain reaction KW - Recombinant Escherichia coli KW - Restriction enzymes Y1 - 2013 U6 - https://doi.org/10.1186/1472-6750-13-81 SN - 1472-6750 VL - 13 IS - 10 PB - BioMed Central CY - London ER - TY - JOUR A1 - Zhang, Fuzhong Z. A1 - Timm, Katharina A. A1 - Arndt, Katja Maren A1 - Woolley, G. Andrew T1 - Photocontrol of Coiled-Coil Proteins in Living Cells N2 - Light switching of the activity of a coiled-coil protein, the AP-1 transcription factor, in living cells was made possible by the introduction of a designed azobenzene-cross-linked dominant negative peptide, XAFosW (red and yellow in the picture). In the dark, XAFosW showed decreased helical content and decreased affinity for target Jun proteins (green); irradiation at 365 nm enhanced helicity and target affinity. Y1 - 2010 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/26737/ U6 - https://doi.org/10.1002/anie.201000909 SN - 1433-7851 ER - TY - CHAP A1 - Rejiba, Soukaina A1 - Frelan, Megan A1 - Hermann, Alex A1 - Arndt, Katja Maren A1 - Hajri, Amor T1 - Interfering peptides targeting transcription factor AP1 for pancreatic cancer gene therapy T2 - Molecular therapy : the journal of the American Society of Gene Therapy Y1 - 2013 SN - 1525-0016 VL - 21 IS - 2 SP - S250 EP - S250 PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Speck, Janina A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Efficient phage display of intracellularly folded proteins mediated by the TAT pathway JF - Protein engineering design & selection N2 - Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function. KW - g3p KW - phagemid display KW - protein design KW - protein engineering KW - selection Y1 - 2011 U6 - https://doi.org/10.1093/protein/gzr001 SN - 1741-0126 VL - 24 IS - 6 SP - 473 EP - 484 PB - Oxford Univ. Press CY - Oxford ER - TY - CHAP A1 - Kükenshöner, Tim A1 - Jean-Christoph, N. A1 - Speck, J. A1 - Müller, Kristian M. A1 - Arndt, Katja Maren T1 - Targeting the microphthalmia associated transcription factor coiled coil domain with interfering peptides T2 - The FEBS journal Y1 - 2011 SN - 1742-464X VL - 278 IS - 6 SP - 159 EP - 159 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Azuma, Yusuke A1 - Kuekenshoener, Tim A1 - Ma, Guangyong A1 - Yasunaga, Jun-ichiro A1 - Imanishi, Miki A1 - Tanaka, Gen A1 - Nakase, Ikuhiko A1 - Maruno, Takahiro A1 - Kobayashi, Yuji A1 - Arndt, Katja Maren A1 - Matsuoka, Masao A1 - Futaki, Shiroh T1 - Controlling leucine-zipper partner recognition in cells through modification of a-g interactions JF - Chemical communications N2 - By focusing on the a-g interactions, successful design and selection were accomplished to obtain a leucine-zipper segment that discriminates the appropriate partner over another that provides very similar patterns of electrostatic interactions. Y1 - 2014 U6 - https://doi.org/10.1039/c4cc00555d SN - 1359-7345 SN - 1364-548X VL - 50 IS - 48 SP - 6364 EP - 6367 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Hagen, Sven A1 - Baumann, Tobias A1 - Wagner, Hanna J. A1 - Morath, Volker A1 - Kaufmann, Beate A1 - Fischer, Adrian A1 - Bergmann, Stefan A1 - Schindler, Patrick A1 - Arndt, Katja Maren A1 - Mueller, Kristian M. T1 - Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy JF - Scientific reports N2 - The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). Y1 - 2014 U6 - https://doi.org/10.1038/srep03759 SN - 2045-2322 VL - 4 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Mazumder, Mostafizur A1 - Brechun, Katherine E. A1 - Kim, Yongjoo B. A1 - Hoffmann, Stefan A. A1 - Chen, Yih Yang A1 - Keiski, Carrie-Lynn A1 - Arndt, Katja Maren A1 - McMillen, David R. A1 - Woolley, G. Andrew T1 - An Escherichia coli system for evolving improved light-controlled DNA-binding proteins JF - Protein engineering design & selection N2 - Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library. KW - directed evolution KW - fluorescent reporter KW - optogenetics Y1 - 2015 U6 - https://doi.org/10.1093/protein/gzv033 SN - 1741-0126 SN - 1741-0134 VL - 28 IS - 9 SP - 293 EP - 302 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Zuo, Zhili A1 - Gandhi, Neha S. A1 - Arndt, Katja Maren A1 - Mancera, Ricardo L. T1 - Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein JF - Biopolymers N2 - The c-Fosc-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions. KW - free energy of binding KW - coiled-coil KW - molecular dynamics KW - MM KW - GBSA KW - leucine zipper Y1 - 2012 U6 - https://doi.org/10.1002/bip.22099 SN - 0006-3525 VL - 97 IS - 11 SP - 899 EP - 909 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Hagen, Sven A1 - Mattay, Dinah A1 - Raeuber, Christina A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Characterization and inhibition of AF10-mediated interaction JF - Journal of peptide science N2 - The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd. KW - protein-protein interaction KW - protein design and selection KW - protein engineering KW - coiled coil KW - leucine zipper KW - AF10 Y1 - 2014 U6 - https://doi.org/10.1002/psc.2626 SN - 1075-2617 SN - 1099-1387 VL - 20 IS - 6 SP - 385 EP - 397 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Kuekenshoener, Tim A1 - Wohlwend, Daniel A1 - Niemoeller, Christoph A1 - Dondapati, Padmarupa A1 - Speck, Janina A1 - Adeniran, Adebola V. A1 - Nieth, Anita A1 - Gerhardt, Stefan A1 - Einsle, Oliver A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system JF - Journal of structural biology N2 - The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of alpha-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 angstrom) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach. (C) 2014 Elsevier Inc. All rights reserved. KW - Basic helix-loop-helix leucine zipper KW - Coiled coils KW - Microphthalmia associated transcription factor KW - Selection and design KW - Twin arginine translocation pathway Y1 - 2014 U6 - https://doi.org/10.1016/j.jsb.2014.03.002 SN - 1047-8477 SN - 1095-8657 VL - 186 IS - 3 SP - 335 EP - 348 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Speck, Janina A1 - Hecky, Jochen A1 - Tam, Heng-Keat A1 - Arndt, Katja Maren A1 - Einsle, Oliver A1 - Müller, Kristian M. T1 - Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution JF - Biochemistry N2 - The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM beta-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-N Delta 15C Delta 3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 degrees C in T-m, displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T-m increased by 15.3 degrees C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information. Y1 - 2012 U6 - https://doi.org/10.1021/bi2018738 SN - 0006-2960 VL - 51 IS - 24 SP - 4850 EP - 4867 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Kuekenshoener, Tim A1 - Hagemann, Urs B. A1 - Wohlwend, Daniel A1 - Raeuber, Christina A1 - Baumann, Tobias A1 - Keller, Sandro A1 - Einsle, Oliver A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - Analysis of Selected and Designed Chimeric D- and L-alpha-Helix Assemblies JF - Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences N2 - D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base. Y1 - 2014 U6 - https://doi.org/10.1021/bm5006883 SN - 1525-7797 SN - 1526-4602 VL - 15 IS - 9 SP - 3296 EP - 3305 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Kruse, Sabrina M. A1 - Arndt, Katja Maren T1 - Long-range transcriptional interference in E-coli used to construct a dual positive selection system for genetic switches JF - Nucleic acids research N2 - We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. Y1 - 2016 U6 - https://doi.org/10.1093/nar/gkw125 SN - 0305-1048 SN - 1362-4962 VL - 44 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Wohltat, Christian A1 - Müller, Kristian M. A1 - Arndt, Katja Maren T1 - A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter JF - PLoS one N2 - For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness. Y1 - 2017 U6 - https://doi.org/10.1371/JOURNAL.PONE.0181923 SN - 1932-6203 VL - 12 IS - 7 SP - 1 EP - 15 PB - PLoS CY - Lawrence, Kan. ER - TY - GEN A1 - Hoffmann, Stefan A. A1 - Wohltat, Christian A1 - Müller, Kristian M. A1 - Arndt, Katja Maren T1 - A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter N2 - For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 390 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-403406 ER - TY - GEN A1 - Brechun, Katherine E. A1 - Woolley, Andrew A1 - Arndt, Katja Maren T1 - A Bacterial Bandpass Assay for Protein-Protein Interactions T2 - Protein science : a publication of the Protein Society Y1 - 2017 SN - 0961-8368 SN - 1469-896X VL - 26 SP - 198 EP - 198 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Wohltat, Christian A1 - Mueller, Kristian M. A1 - Arndt, Katja Maren T1 - A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter JF - PLoS one N2 - For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness. Y1 - 2017 U6 - https://doi.org/10.1371/journal.pone.0181923 SN - 1932-6203 VL - 12 SP - 5944 EP - 5952 PB - PLoS CY - San Fransisco ER - TY - GEN A1 - Azuma, Yusuke A1 - Kükenshöner, Tim A1 - Ma, Guangyong A1 - Yasunaga, Jun-ichiro A1 - Imanishi, Miki A1 - Tanaka, Gen A1 - Nakase, Ikuhiko A1 - Maruno, Takahiro A1 - Kobayashi, Yuji A1 - Arndt, Katja Maren A1 - Matsuoka, Masao A1 - Futaki, Shiroh T1 - Controlling leucine-zipper partner recognition in cells through modification of a–g interactions N2 - By focusing on the a–g interactions, successful design and selection were accomplished to obtain a leucine-zipper segment that discriminates the appropriate partner over another that provides very similar patterns of electrostatic interactions. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 276 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-98758 ER -