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Issue 8, 2004
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Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins

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Abstract

The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein–protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin–melittin protein–peptide complexes are presented to illustrate the approach.

Graphical abstract: Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins

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Article information


Submitted
08 Dec 2003
Accepted
10 May 2004
First published
28 May 2004

Photochem. Photobiol. Sci., 2004,3, 741-748
Article type
Perspective

Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins

S. D. Maleknia, J. W. H. Wong and K. M. Downard, Photochem. Photobiol. Sci., 2004, 3, 741
DOI: 10.1039/B315904C

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