To develop and extend work on electrostatic interactions in protein with the ultimate goal of designing molecular probes capable of mapping proteins and reporting on protein dynamics and electrostatics Specifically to: 1) design and synthesize new cysteine (Cys)-specific labels, generic probes that will allow us to map static and dynamic electrostatic fields in any protein. 2) Introduce Cys residues at selective sites throughout model proteins, express proteins and label with probes for electrostatic potentials. Examples include apomyoglobin (apoMb), GB1 and T4 lysozyme as model systems. 3) Measure static protein interactions by studying shifts of vibrational transitions of CN stretch that is introduced using site-specific labeling. This vibrational label is chosen to be quantitatively using VSE. By mutating amino acid residues near to the CN, the observe vibrational bind shifts directly probe the local fields. 4) Establish whether vibrational Stark effect (VSE) spectroscopy and protein labeling can be combined to map electric fields in proteins. 5) Measure electrostatic dynamic interactions using the dynamic Strokes shift method in which the energy of a polar, fluorescent emitting state of a newly introduced probe is measured in real time. Make a parallel comparison with similar data on unnatural amino acid side chains. The proposed research includes expression and purification of mutant proteins from E. coli, synthesis of novel molecular probes, protein labeling and characterization, and, finally, an array of experimental techniques such as infrared spectroscopy and fluorescence up- conversion.
