Local and global conformational changes in signaling proteins studied by solution NMR spectroscopy

The proteins involved in cell signaling must be tightly regulated in order for the highly orchestrated process to continue in a harmonious manner. One common method of regulating activity is through conformational changes of the protein. Because regions involved in these conformational changes are often intrinsically unstructured, solution NMR spectroscopy provides a powerful tool-box from which to study these processes, on both a residue specific and global level. The first aim of this research was to explore the conformation of the carboxyl tail of Protein Kinase C. This region of PKC is key to the maturation and regulation of this ubiquitous kinase. It was discovered that regardless of phosphorylation state the last 48 residues of PKC (PKC-CT) demonstrate a helical propensity, but overall this region is intrinsically unstructured, and the functional importance of this property is addressed. Additionally, by investigating time dependent changes which were repeatedly observed in the spectra, an intrinsic proline isomerization in PKC-CT was revealed. More importantly, it was discovered that this isomerization can be catalyzed by the peptidyl-prolyl isomerase Pin1. A new model for the maturation and regulation of PKC, addressing some of the previously unknown aspects of the process is proposed. The second aim of this research was to implement additional NMR methods for studying conformational changes in proteins. PFG-Diffusion experiments are an elegant means for studying the global structure of a protein under a variety of solution conditions. However, the precision to which one can determine the hydrodynamic radius via this method is not well established. There are many conformations involved in the biology of the pleiotropic cytokine Interleukin 1-β and thus provides a well behaved yet biologically relevant system on which to implement these experiments. Because all known sources of error were addressed and the measurements were acquired in such replicate, it was possible to determine the lower limit to conformational changes which can be detected. It was also shown that an external sample can be used for a viscosity control. Finally, methods for simplifying spectra through specific labeling with 19F were explored, and experimental protocols for acquiring artifact free spectra were implemente