This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recently, p-cyanophenylalanine (PheCN) has emerged as a useful spectroscopic probe of protein structure and dynamics. For example, its CN stretching frequency is sensitive to environment and thus has been used to probe the orientation and hydration status of individual sidechains of a peptide that is bound to model membranes , as well as the structure of amyloid fibers. In addition, the fluorescence quantum yield of PheCN in water is about five times larger than that of phenylalanine and decreases upon dehydration, making it an attractive fluorescent probe of protein conformational changes. What is more, its utility as a spectroscopic probe is further increased by its ability to excite tryptophan (Trp) fluorescence via the mechanism of fluorescence energy transfer (FRET). Finally, and perhaps most importantly, the major advantage of using PheCN in protein conformational studies is that as a non-natural amino acid, it minimally perturbs the physical properties of the native protein in question because of its small size, especially when it replaces either a Phe or Tyr (tyrosine) residue in the native sequence. While previous studies have established the utility of PheCN as a valuable fluorescent probe, many details of its photophysics are not known, which, in many cases, limits its application to a qualitative context. Herein, we propose to investigate the fluorescence lifetime and quantum yield of PheCN in a variety of solvents, attempting to understand how environmental factors, such as hydrogen bonding and polarity, determine its fluorescence properties
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