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 -