@article{ZuoGandhiArndtetal.2012, author = {Zuo, Zhili and Gandhi, Neha S. and Arndt, Katja Maren and Mancera, Ricardo L.}, title = {Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein}, series = {Biopolymers}, volume = {97}, journal = {Biopolymers}, number = {11}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0006-3525}, doi = {10.1002/bip.22099}, pages = {899 -- 909}, year = {2012}, abstract = {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.}, language = {en} } @article{ZhangTimmArndtetal.2010, author = {Zhang, Fuzhong Z. and Timm, Katharina A. and Arndt, Katja Maren and Woolley, G. Andrew}, title = {Photocontrol of Coiled-Coil Proteins in Living Cells}, issn = {1433-7851}, doi = {10.1002/anie.201000909}, year = {2010}, abstract = {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.}, language = {en} } @article{StaabWalossekNellessenetal.2010, author = {Staab, Paul R. and Walossek, J{\"o}rg and Nellessen, David and Gr{\"u}nberg, Raik and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {SynBioWave : a real-time communication platform for molecular and synthetic biology}, issn = {1367-4803}, doi = {10.1093/bioinformatics/btq518}, year = {2010}, abstract = {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.}, language = {en} } @article{SpeckRaeuberKuekenshoeneretal.2013, author = {Speck, Janina and R{\"a}uber, Christina and K{\"u}kensh{\"o}ner, Tim and Niem{\"o}ller, Christoph and Mueller, Katelyn J. and Schleberger, Paula and Dondapati, Padmarupa and Hecky, Jochen and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation}, series = {Protein engineering design \& selection}, volume = {26}, journal = {Protein engineering design \& selection}, number = {3}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1741-0126}, doi = {10.1093/protein/gzs098}, pages = {225 -- 242}, year = {2013}, abstract = {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.}, language = {en} } @article{SpeckHeckyTametal.2012, author = {Speck, Janina and Hecky, Jochen and Tam, Heng-Keat and Arndt, Katja Maren and Einsle, Oliver and M{\"u}ller, Kristian M.}, title = {Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution}, series = {Biochemistry}, volume = {51}, journal = {Biochemistry}, number = {24}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/bi2018738}, pages = {4850 -- 4867}, year = {2012}, abstract = {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.}, language = {en} } @article{SpeckArndtMueller2011, author = {Speck, Janina and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {Efficient phage display of intracellularly folded proteins mediated by the TAT pathway}, series = {Protein engineering design \& selection}, volume = {24}, journal = {Protein engineering design \& selection}, number = {6}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1741-0126}, doi = {10.1093/protein/gzr001}, pages = {473 -- 484}, year = {2011}, abstract = {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.}, language = {en} } @inproceedings{RejibaFrelanHermannetal.2013, author = {Rejiba, Soukaina and Frelan, Megan and Hermann, Alex and Arndt, Katja Maren and Hajri, Amor}, title = {Interfering peptides targeting transcription factor AP1 for pancreatic cancer gene therapy}, series = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {21}, booktitle = {Molecular therapy : the journal of the American Society of Gene Therapy}, number = {2}, publisher = {Nature Publ. Group}, address = {New York}, issn = {1525-0016}, pages = {S250 -- S250}, year = {2013}, language = {en} } @article{MazumderBrechunKimetal.2015, author = {Mazumder, Mostafizur and Brechun, Katherine E. and Kim, Yongjoo B. and Hoffmann, Stefan A. and Chen, Yih Yang and Keiski, Carrie-Lynn and Arndt, Katja Maren and McMillen, David R. and Woolley, G. Andrew}, title = {An Escherichia coli system for evolving improved light-controlled DNA-binding proteins}, series = {Protein engineering design \& selection}, volume = {28}, journal = {Protein engineering design \& selection}, number = {9}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1741-0126}, doi = {10.1093/protein/gzv033}, pages = {293 -- 302}, year = {2015}, abstract = {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.}, language = {en} } @inproceedings{KuekenshoenerJeanChristophSpecketal.2011, author = {K{\"u}kensh{\"o}ner, Tim and Jean-Christoph, N. and Speck, J. and M{\"u}ller, Kristian M. and Arndt, Katja Maren}, title = {Targeting the microphthalmia associated transcription factor coiled coil domain with interfering peptides}, series = {The FEBS journal}, volume = {278}, booktitle = {The FEBS journal}, number = {6}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1742-464X}, pages = {159 -- 159}, year = {2011}, language = {en} } @article{KuekenshoenerWohlwendNiemoelleretal.2014, author = {Kuekenshoener, Tim and Wohlwend, Daniel and Niemoeller, Christoph and Dondapati, Padmarupa and Speck, Janina and Adeniran, Adebola V. and Nieth, Anita and Gerhardt, Stefan and Einsle, Oliver and Mueller, Kristian M. and Arndt, Katja Maren}, title = {Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system}, series = {Journal of structural biology}, volume = {186}, journal = {Journal of structural biology}, number = {3}, publisher = {Elsevier}, address = {San Diego}, issn = {1047-8477}, doi = {10.1016/j.jsb.2014.03.002}, pages = {335 -- 348}, year = {2014}, abstract = {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.}, language = {en} }