Photooxidative Crosslinking and Photoaffinity Labeling of Proteins using Naphthalene Imides and Diimides
Loading...
Links to Files
Permanent Link
Author/Creator
Author/Creator ORCID
Date
2019-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Chemistry
Citation of Original Publication
Rights
Distribution Rights granted to UMBC by the author.
Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan thorugh a local library, pending author/copyright holder's permission.
This item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.
Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan thorugh a local library, pending author/copyright holder's permission.
This item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.
Abstract
Protein structures and interactions are key to understanding their biological function. The current techniques to probe structure have limitations that can be overcome with the use of photoaffinity labeling. Traditional labeling uses benzophenone and diazarine derivatives, which require long irradiation times or photoisomerize into less reactive species, which causes non-specific crosslinking on the target protein. This study used carboxylic-acid functionalized naphthalene diimides as a new type of photoaffinity label. Nonspecific photoaffinity labeling has been achieved with these compounds. The mechanism was probed with steady-state photolysis and transient absorbance spectroscopy. The proposed labeling mechanism of these compounds involves generating a biradical upon irradiation that photodecarboxylates. This biradical can react with proteins via a hydrogen abstraction followed by radical recombination to produce a crosslink between the naphthalimide and amino acid in the protein. These naphthalene diimide compounds were then functionalized to interact with the active site of alcohol dehydrogenase. Many biological processes are governed by transient enzyme complexes, where protein interactions change throughout the catalytic cycle. Transient interactions are covalently linked with temporal control with photooxidative crosslinking. These transients are later characterized. Our group used naphthalene imides as photooxidative crosslinkers, which undergo an intermolecular photoinduced electron transfer with amino acid side chains of enzymes to generate a covalent linkage. The compounds were synthesized with phenol-functional groups to interact with the active site of mushroom tyrosinase. Site specificity was assessed with fluorescence assays and competitive enzyme kinetics. Both projects aim to use a family of compounds to facilitate efficient crosslinking with site specificity to increase biologically relevant bonds, as compared to traditional methods.