Biology subjects

Hoecker, B.

Publications and source records attributed to Hoecker, B..

3 recordsLinked to original sources

bioRxiv2020

An Artificial Cofactor catalyzing the Baylis-Hillman Reaction using Designed Streptavidin as Protein Host

An artificial cofactor based on an organocatalyst embedded in a protein was used to conduct the Baylis-Hillman reaction in a buffered system. As protein host we chose streptavidin, since it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed based on high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize activity. Finally, repeated cycles of structure determination and redesign led to a system with 24 to 35-fold increased activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.

synthetic biology↗

bioRxiv2020

Ligand promiscuity in the tryptophan repressor - from structural understanding towards rational design

Receptors that promiscuously bind a range of ligands provide insights into how nature mediates affinity and biological functioning. Moreover, such receptors provide vantage points for the rational design of specific binding for biotechnological applications. Here we describe the molecular details of the ligand binding promiscuity of the well-known tryptophan repressor TrpR. We elucidated high-resolution structures of TrpR bound to the co-repressors 5-methyl-tryptophan and 5-methyl-tryptamine as well as the pseudo-repressors indole-3-propionic and indole-3-acetic acid. Furthermore, using isothermal titration calorimetry we procure the corresponding thermodynamic parameters. Together this data provides molecular explanations for the strongly varied affinities and biological effects of the ligands providing insights into how nature shapes specificity and affinity. Beyond this we use these insights to exemplarily showcase knowledge-based design of TrpR by swapping its specificity from its native ligand tryptophan to indole-3-acetic acid. Finally, we elucidate the structures of the variant bound to indole-3-acetic and indole-3-propionic acid to retrace our design rationale.

synthetic biology↗

bioRxiv2020

Design of a biosensor for direct visualisation of auxin

In plants, one of the most important regulative small molecules is indole-3-acetic acid (IAA) known as auxin. Its dynamic redistribution plays an essential role in virtually every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, the spatial and temporal distribution of auxin at cellular resolution could not be determined directly. Instead it has been inferred from visualisation of irreversible processes involving the endogenous auxin response machinery3-7. This detection system failed to record transient changes. Here we report on a genetically encoded biosensor for quantitative in vivo visualisation of auxin distributions. The sensor is based on the E. coli tryptophan repressor (TrpR)8 whose binding pocket was engineered for specific IAA binding and coupled to fluorescent proteins to employ FRET as readout. This sensor, unlike previous systems, enables direct monitoring of the fast uptake and clearance of auxin by individual cells in the plant as well as the graded spatial distribution along the root axis and its perturbation by transport inhibitors. Thus, our auxin sensor enables mapping of auxin concentrations at (sub)cellular resolution and their changes in time and space during plant life.

plant biology↗