NMR Investigations of the hyperphosphorylated IDP Osteopontin

The main goal of this proposed research project is to study how compact conformations of Intrinsically disordered proteins (IDPs) are influenced by posttranslational modification and interactions with their ligands using a workflow of recently developed methods. By using methods based on Paramagnetic Relaxation Enhancement (PRE), Cross- Correlated Relaxation (CCR) and covariance analysis we seek to describe the behavior of a phosphorylated polypeptide with preferentially extended conformation and to describe its conformational ensembles of an IDP closer to its physiological conditions. In this research project we will particularly focus on the extracellular matrix protein osteopontin (OPN), whose pathology is associated with several types of cancer and which also plays a role in formation of cancer metastases. Proteins that lack a stable tertiary structure have been in the focus of research for the past decade. Due to their conformational flexibility these polypeptides carry out important regulatory functions and play a pivotal role in many signaling pathways. IDPs constantly sample a large and heterogeneous conformational space and are capable of interacting with multiple ligands simultaneously. Since the IDPs defy the standard biochemical axiom that a protein needs to be stably folded in order to perform their function, there is a need for methods that can describe the features of an IDP ligand complex. This research project is targeted at development of such methods together with computational approach to create a conformational ensemble of a phosphorylated IDP. NMR spectroscopy has been developed into a powerful structural biology technique that offers unique opportunities for structural and dynamic studies of IDPs. Here we will investigate sophisticated experimental NMR spin relaxation methodologies to characterize structural dynamics of intrinsically disordered proteins. The applications to osteopontin will serve to illustrate the potential of the technique and will provide unprecedented insight into the conformational space of the medically highly relevant IDPs. Previous evidence suggests that although OPN possesses no stable structure in solution there are distinct structural transitions between extended and condensed conformations upon binding to a ligand. Structural characterization of such bound conformation would reveal information about the dynamics and mechanism of the complex formation.

The main goal of this proposed research project is to study how compact conformations of Intrinsically disordered proteins (IDPs) are influenced by post-translational modification and interactions with their ligands using. In this research project we particularly focused on the extracellular matrix protein osteopontin (OPN), whose pathology is associated with several types of cancer and which also plays a role in formation of cancer metastases. The starting point of the research project was the development of an efficient protein chemical method to be able to synthesize phosphorylated osteopontin in sufficient quantities in the laboratory. In a next step the influence of this post-translational modification on the structure and dynamics of the protein was investigated. By using paramagnetic relaxation enhancement (PRE) it could be shown that the phosphorylation leads to a significant structural expansion and increasing flexibility of the protein. For a more precise characterization of the biologically significant interaction between osteopontin and cellular receptors, an experimental method was developed that combines cell biological and NMR spectroscopic techniques in a new way. With the help of this new method, the interaction between osteopontin and hydroxyapatite nanoparticles could then be examined in more detail. Finally, new NMR pulse sequences for the measurement and quantitative analysis of cross-correlated relaxation measurements were developed in the project, which in the future will allow even more detailed insights into the structural dynamics of proteins and thus enable new intervention strategies for therapeutic applications. The results to be expected will not only provide new insights into the conformational space of this medically highly relevant protein, but also - based on this - enable novel possibilities for therapeutic intervention in previously inaccessible proteins.