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 - 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 - 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 - 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 -