Pazos, Ileana Marquez
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Publication Spectroscopic Probes of Protein Structure, Dynamics, Hydration and Electrostatics(2015-01-01) Pazos, Ileana MarquezThe structure, dynamics and function of a protein are intimately controlled by a large number of intermolecular and intramolecular interactions. Thus, achieving a quantitative and molecular-level understanding of how proteins fold and function requires experimental techniques that can sense and differentiate various molecular forces and, in many cases, in a site-specific manner. To that end, the focus of this thesis work is to develop non-natural amino acid-based infrared and fluorescence probes that can be used to assess the local hydration status and electrostatic environment of proteins. First, we expand the utility of a well-known site-specific spectroscopic probe, p-cyano-phenylalanine (PheCN), by showing that (1) its fluorescence is sensitive to the presence of various anions and can thus be used to measure the heterogeneity of the protein conformation, (2) when placed at the N-terminal end of a peptide this non-natural amino acid can be used as a pH sensor for a wide variety of applications, and (3) its nitrile stretching vibration is a microscopic reporter of how a co-solvent, such as urea and trimethylamine N-oxide, modulates the protein-water interactions. Secondly, we demonstrate that the ester carbonyl stretching vibration of the non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, can be used to site-specifically quantify the electrostatic environment of proteins as its vibrational frequency correlates linearly with the local electrostatic field. Application of this infrared probe to amyloids allows us to gain new insight into their structure and dynamics.