Nuclear receptors (NRs) are important pharmaceutical targets. The access to these proteins for biochemical, biophysical and structural studies is generally however limited to one specific domain only, the ligand binding domain (LBD). This severely limits the molecular insights that can be obtained with the aforementioned studies on nuclear receptors. We have previously shown that nuclear receptor semi-synthesis is a powerful means to generate Estrogen Receptors (ERs) featuring molecular elements beyond the ligand binding domain, such as a phosphorylated tyrosine and the flexible F-domain. The thus generated proteins allowed for important structural and biophysical insights, such as the first crystal structure of a phosphorylated nuclear receptor ligand binding domain and the molecular understanding of the effect of the protein F-domain on ER – coactivator interaction and transcriptional activity. Nevertheless there are still many outstanding questions in relation to the regul
ation of nuclear receptors via post-translational modifications and via interdomain interactions. Molecular answers to these questions are critically dependent on the availability of sufficient quantities of nuclear receptor constructs featuring post-translational modifications (PTMs) and multiple domains for biophysical and structural studies. Since these constructs are as such not available via techniques of molecular biology alone, chemical biology approaches are needed for the generation of specifically modified and labelled nuclear receptors.
This project aims to generate Nuclear Receptor constructs featuring the Ligand Binding Domain and the hinge region, including receptor specific PTMs. The objective is to apply protein semi-synthesis for generating these extended ER and AR constructs and subsequently use these constructs in biophysical and structural studies to investigate the effects of the hinge region and of PTMs on relevant pharmacological effects such as ligand binding, cofactor interactions and receptor dynamics. The results from these molecular studies will subsequently be correlated with the described cellular studies on these NRs and the sites of post-translational modification. In addition cellular studies will be performed to correlate the observed molecular effects with the transcriptional activity or protein localization of constructs of analogous length or with specific point mutations in cell based studies. This will allow linking the observed molecular effects of specific PTMs and of the length
of hinge region length with their cellular characteristics.
The protein semi-synthesis approach will be based on the application of expressed protein ligation to nuclear receptors, as previously established by us. The larger fragments (the LBDs) will be expressed in E. coli to feature an N-terminal cysteine, and the smaller fragments (the hinge regions featuring the PTMs) will be made accessible as C-terminal thioesters via peptide chemistry. These fragments will then be ligated to assemble the post-translational modified multi-domain constructs. Specific protein constructs will be generated for protein crystallography studies, whereas other constructs will be made for biophysical studies such as coactivator and ligand binding studies and protein dynamic studies. As an example the introduction of an N-terminal fluorophore will allow to probe how the hinge region dynamics depends on ligand and coactivator binding and on PTMs. Finally, the introduction of specific PTMs will allow to probe their effects on the interactions of NRs with co
factor proteins, responsible for the transcriptional activity of the NRs.
Starting date is a guidline.
Methoden:
Peptide synthesis
Protein engineering (cloning, expression)
Fluorescence spectroscopy
ITC
X-Ray
NMR
CD-spectrocospy
Anfangsdatum: 1. Januar 2012
geschätzte Dauer: 4 years
Bezahlung: Starting from around 1500 euro netto to around 1800 euro net
We are seeking candidates with a strong interest in chemical biology, to enforce our group and work on the theme described above. The candidate should for example have a background in one of the following themes: protein engineering, molecular biology, biochemistry, peptide chemistry, chemical biology, organic synthesis or a related field, with,
• Knowledge and experience in the molecular sciences,
• Strong affinity for multidisciplinary research and good communicative skills,
• Capacity to write and communicate in English,
• Enthusiasm and talent.