CORPORATION OF HAVERFORD COLLEGE, THE
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 C≡N 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 C≡N 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. The sensitivity of this probe vibration to burial at lipid interfaces will also be evaluated in model peptides with known membrane binding geometries. 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 C≡N 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 post-translational 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 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.