New experiments are needed to view and to understand the structure of the most dynamic and disordered proteins. Vibrational spectra of proteins can provide this specific information with very fast instrinsic time resolution. A strategy is proposed here to develop the CaN stretching vibration of the cyanylated side chain of cysteine as a site-specific probe which can be implemented in dynamic and disordered proteins of arbitrary size. The CaN stretching vibration absorbs in a transparent and uncrowded region of the aqueous biomolecular infrared spectrum, and it can be introduced via post-translational modification at free cysteine side chains. First the sensitivity of this vibration to the local structural distribution and solvent dynamics will be analyzed in the context of soluble model peptides which form predictable secondary structures. Based on preliminary results, it is expected that this probe vibration will be sensitive to formation of both local secondary structure and residue-specific hydrophobic contacts. Once its behavior is well-characterized in model peptides, the CaN vibration of cyanylated cysteine will be used to determine new structural information in two dynamic natural proteins with known binding activity but unclear bound structures. The first of these is the intrinsically disordered NTAIL protein of the measles virus. Site-directed mutagenesis to cysteine followed by posttranslational modification to introduce the probe vibration will be used to map the binding contacts of an interacting but otherwise poorly characterized piece of the NTAIL protein as it binds to its physiological partner domain XD. In a more global context, the second natural system is calmodulin. -SCN containing mutants of calmodulin will be generated via a similar mutagenesis/modification scheme in order to map both residue-specific structure formation in calmodulin and contact formation between calmodulin and its binding partner for a small group of neural peptides which bind to the dynamic host molecule in different geometries. Through these two natural systems it will be demonstrated that cyanylated cysteine is a globally useful, site-specific infrared probe of structure and contact formation as dynamic proteins undergo binding events.
Defects and dysfunction in proteins without well-defined structures are often associated with systemic human diseases. A new experimental protocol will be demonstrated in this work that allows atom-level information on binding events in disordered proteins to be determined clearly using standard analytical laboratory equipment. The generality of this approach will make it possible to understand the molecular basis of diseases associated with disordered and dynamic proteins.
