This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The condensed-phase spectral response of a particular vibration can provide specific information on both the local structural environment and the characteristics of the medium surrounding the vibration, including solvation phenomena. A drawback of vibrational spectroscopy in complex molecular systems is spectral congestion. Several frequency windows contain most of the common molecular vibrations in organic molecules, and as the complexity of a system increases, congestion in these regions can prevent interrogation of single vibrations. In the case of polypeptides and proteins, infrared spectroscopy is widely used as a probe of global secondary (backbone) structure but rarely for single-amino-acid level study. Isotopic substitution of atoms along the peptide backbone can reveal single vibrations, but its use is limited to synthetically produced peptides. Artificial amino acids can include groups with unique spectral signatures, but with a few exceptions their incorporation into polypeptides is also limited to synthetic methods. Catalytically active, higher molecular weight species are not generally accessible by methods other than extraction from natural reservoirs or gene expression. In such cases, unique spectral probes can in principle be introduced in proteins through in situ chemical modification of amino acid side chains. The following specific aim are targeted with this collaborative project: 1) The nonlinear spectral responses of the S-H stretching vibration of cysteine and the SCaN stretching vibration of chemically modified cysteine will be characterized in different structural and solvation environments. 2) These vibrations will be used to probe site-specific structure and dynamics in proteins and protein complexes which cannot be achieved by peptide synthesis. The direct clarification of the residue-level structural distribution in domains displaying disordered structure and structural switching is of particular interest.
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