@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}
}