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 -