Radical approaches to probe protein structure, folding, and interactions by mass spectrometry.
This review describes mass spectrometry-based strategies and investigations to determine protein structure, folding dynamics, and protein-protein interactions in solution through the use of radical reagents. The radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources, and react with proteins on time scales that are less than those often attributed to structural reorganization 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 dynamic 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 of gelsolin to actin are among the systems presented to illustrate the approach.
Maleknia
S D
SD
Department of Physiology and Biophysics, Albert Einstein College of Medicine, New York, NY, USA.
Downard
K
K
D000818:Animals; D005609:Free Radicals; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
D005609:Free Radicals; D007202:Indicators and Reagents; D011506:Proteins