@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}
}
@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}
}