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MMBio - Marie Curie Training Network

Remkes Scheele

 
Remkes_Scheele_153
 

Background:

  • BSc. in Life Science & Technology, Majoring in Molecular Life Sciences
  • Msc. in Molecular Biology and Biotechnology, Specialization in Chemical Biology

My first project was during my undergrad thesis in the lab of Prof. Fraaije. Here, I engineered 5-hydroxymethylfurfural oxidase (HMFO) in order to increase formation of FDCA, a valuable biological building block. Site directed mutagenesis of residues pin pointed by docking experiments with YASARA gave a moderate boost in yields.
Excited about the prospects of biocatalysis as a clean alternative to synthetic organic chemistry, I focused my Master’s program to learn as much as possible on both the chemical and biological side of the field. As a graduate student, I worked in two different laboratories for a total of 14 months.
My first internship was in the lab of Prof. Roelfes. This project focused on the genetic incorporation of 8-hydroxyquinoline (HQala) in LmrR, a non-catalytic transcription factor using stop codon suppression technology. The side chain of HQala in LmrR showed excellent binding properties with a variety copper, rhodium and iridium metal complexes. Together, the artificial metalloenzyme showed great promise for a wide range of metal catalyzed reactions in the chiral environment of the otherwise non-catalytically active transcription factor. LmrR_HQala with copper bound in the active site was capable catalyzing both enantioselective C-C bond formations and the direct addition of water to alkenes to yield chiral alcohols.
The second internship was in the group of Prof. Arnold at Caltech. The β-subunit of TrpS, TrpB, was evolved in two consecutive rounds of directed evolution to catalyze the irreversible condensation of L-β-ethyl-Ser and L-β-propyl-Ser with indole, yielding β-substituted (2S-3S)-β-ethyl-Trp and (2S-3S)-β-propyl-Trp. Because most β-substituted serines are both expensive and not available in stereo pure form, we designed a bi-enzymatic catalytic cascade. Threonine aldolase was used to catalyze the aldol condensation of propionaldehyde or butyraldehyde with glycine to produce L-β-ethyl-Ser and L-β-propyl-Ser respectively. In combination with the engineered TrpB subunit, this facilitates formation of new tryptophan derivatives with two contiguous stereocenters from cheap achiral precursors.

Training and Transferable Skills:

- Protein engineering
- Enzymatic catalysis
- Stop codon suppression technology
- Directed evolution
- HPLC
- UV-Vis spectroscopy
- Enzymatic catalysis
- Organic synthesis

Research Projects:
During my PhD, I will use a bead-based assay to assess the factors which cause highly specific interactions between kinases in the MAPK network. This essential signal transduction pathway lays at the heart of many pathways which include cell proliferation, inflammation, and apoptosis. The multitude of MAPK proteins have evolved through gene duplication, resulting in high sequence similarity between the kinases. In physiological settings the kinases are however highly substrate specific. This is thought to originate from interactions between binding groove of MAPK and d-peptides of both its substrates (MAPKAPKs) and its activators (MAPKKs). There is ongoing debate on how the d-peptide causes specificity within the MAPK network. Using a high throughput bead based assay, we are able to assess a large portion sequence space of the d-peptide. Aside from providing fundamental information on this essential pathway of which dysregulation is associated with many forms of cancer and other diseases, this information is thought to assist in the development of more targeted drugs.

 

 

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