In cell NMR
The Folkers group has a strong expertise in protein expression and purification of mammalian proteins, membrane proteins and designer proteins both in E.coli and in mammalian cells This expertise is applied for in-cell NMR studies on membrane proteins, the role of Ubiquitin in cellular stress and Sumo in cell cycle regulation The research builds on the experience acquired over the years in collaboration with the Baldus group on using in-cell NMR for structural analysis of proteins and nucleic acid within the cell using solid state NMR.
High Throughput methods for protein production
We have implemented within many European projects a strong experience in cloning, expression and purification of proteins for structural biology. We use both bacterial expression systems as well as mammalian expression systems. Through transient transfection or electroporation we can introduce proteins in eukaryotic cells and study these proteins in their native environment, see below. We have used these high throughput approach in the past to determine the solution structures of numerous protein domains using NMR spectroscopy. We recently started to use the infrastructure to design binder proteins using BindCraft and biochemically and biophysically characterize the binding affinities. In collaboration with the Bonvin group we would like to integrate the HADDOCK docking approach to rank putative designer-target complexes and to improve the protein-protein interfaces.
An integrated In-cell NMR approach to study proteins in their native environment
The ability to obtain structural information on proteins and protein complexes is increased tremendously due to for example CryoEM and Alfa-fold. Presently most structural information is obtained or based on protein structures in isolation. It is getting increasingly clear that proteins can adopt multiple conformations and that these conformations could play a crucial role in the activation or inactivation of proteins. Most structural approaches lack dynamic information and the native environment the proteins act in. It is our approach to obtain structural and dynamical information on proteins in their native environment and try to combine the dynamical properties with the function of the protein. We therefore use in-cell solid state NMR to study proteins at near native concentrations within the native environment. In order to be able to study these proteins in vivo we . introduce isotope enriched protein samples through electroporation or transient transfection into the cell leading to correctly localized expression within the cells These samples of whole cells, nuclei, or membrane fractions can subsequently be analyzed by solution state or solid state NMR. We use both in house as well as the UU center for cell imaging facility to characterize expression and to evaluate the functional consequence of the expression of these proteins and mutants thereof in the cell. This integrated structural biology approach allows us to study relevant proteins in their native environment and correlate structural changes with functional adaptations of the cell
We apply these methods to study, a short description of these projects and opportunities for internships is provided in the hyperlink