Modifications generated by fast photochemical oxidation of proteins reflect the native conformations of proteins.

Autor: Chea EE; Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland, 21201., Jones LM; Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland, 21201.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2018 Jun; Vol. 27 (6), pp. 1047-1056. Date of Electronic Publication: 2018 Apr 14.
DOI: 10.1002/pro.3408
Abstrakt: Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo-dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.
(© 2018 The Protein Society.)
Databáze: MEDLINE
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