Collaborative projects with groups from Bielefeld University

Expression of L-amino acid oxidases for biocatalytic applications (collaboration with Harald Gröger, Thomas Dierks, Thomas Hellweg, Tilman Kottke, Norbert Sewald)

l-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate l-amino acids and produce the corresponding Α-keto acids, ammonia and hydrogen peroxide. LAAOs with broad substrate spectra are of interest for biotechnological applications, but could not be expressed in sufficient amounts so far. We succeded in expressing an LAAO from the fungus Rhizoctonia solani (details). The recombinant enzyme could be activated by the detergent SDS and undergoes a conformational change (details). The L-AAO4 from the fungus Hebeloma cylindrosporum was also expressed and characterized. It is activated by exposure to acidic pH, SDS and freezing. hcLAAO4 has a broader substrate spectrum and higher specific activity than rsLAAO1. An aldehyde tag was added to site-specifically immobilize the enzyme (details).

 

Cytotoxic dinuclear Cu₂ Complex (collaboration with Thorsten Glaser and Dario Anselmetti)

A dinuclear Cu₂complex was designed and synthesized that targets two neighboring phosphates of DNA. We could demonstrate this complex binds DNA, blocks DNA synthesis in PCR reactions and is cytotoxic for human cancer cells (details).

 

Expression of Channelrhodopsin-2 in Pichia pastoris (collaboration with Joachim Heberle, now Freie Universität Berlin)

The light-gated cation channel Channelrhodopsin-2 from the algae Chlamydomonas reinhardtii became an important optogenetic tool. By expression of this channel in neurons, light can be used to trigger neurophysiological responses and thus revolutionizing studies of such processes with high spatial and temporal resolution. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown.

We expressed functional tagged Channelrhodopsin-2 and mutants with single amino acid exchanges in the yeast Pichia pastoris. The group of Joachim Heberle used time-resolved FTIR spectroscopy to trace structural changes in these proteins upon light absorption. They found that a crucial hydrogen-bonding interaction between D156 and C128 may be involved in gating (details). In addition, they identifies transient protonation changes (details).

 

 

Investigating RET signaling using inhibitor affinity purification (collaboration with Norbert Sewald and Bernhard Küster (TU München))

RET is a receptor tyrosine kinase which interacts with a co-receptor of the GFRa family to respond to growth factors of the GDNF family. These growth factors are important in neuronal survival. A mutated form of RET has been observed in several tumors. Upon activation, RET phosphorylates and activates several kinases. A kinase inhibitor, which can distinguish between the inactive and active conformation of many kinases was immobilized. This tool was used for inhibitor affinity purification (IAP)-based activity-profiling of cells stimulated with GDNF compared to unstimulated cells. By mass spectrometry known RET target proteins as well as additional kinases were identified (details).