Heyne, BelindaValencia-Perez, Adriana Zulema2017-12-182017-12-182012Valencia-Perez, A. Z. (2012). How to blow out a molecular lantern: interaction of aequorea victoria green fluorescent protein and its variants with singlet oxygen (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/4590http://hdl.handle.net/1880/105591Bibliography: p. 147-152Herein, we studied the interaction between Aequorea victoria Green Fluorescent Proetein and some of its mutants with exogenous single oxygen using spectroscopic and electrophoretic techniques. According to our results, exogenous 02 mainly oxidizes the ??-barrel of the proteins, resulting in formation of a covalent protein dimer. Further characterization of the underlying mechanism utilizing monomeric variants, suggested the interface of the natural occurring dimer as the initial area of oxidation, specifically the histidine in position 148 (H148). By mutating this residue to a less reactive side chain, its involvement in the 02 mediated mechanism was corroborated. The reactivity of endogenous 02 towards fluorescent proteins, was also analyzed by direct excitation of the proteins chromophores. Spectroscopy data suggested a variety of complex mechanisms, including generation of 02 and photodecarboxylation, occurring upon irradiation of the protein. Taken together, these results indicate that production of exogenous and/or endogenous 02 during standard imaging techniques influences fluorescent proteins, which could deteriorate the data obtained from the studied system.xxiii, 154 leaves : ill. ; 30 cm.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.master thesis10.11575/PRISM/4590