No abstract

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the structure of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions using synchrotron light modify amino acid side-chains on millisecond timescales. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side-chains to the bulk solvent. The aromatic and sulphur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for a number of proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates correlate closely with a theoretical measure of the accessibility of residue side-chains to the solvent in the native protein structure. This approach can distinguish the relative accessibility of the tryptophan residue side-chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues, and phenylalanine at position 34 from phenylalanine residues at positions 3 and 38 based upon their rates of oxidation.

Highlights d SMAPs bind at an intersubunit pocket defined by all three PP2A subunits d DT-061 (SMAP) binding results in selective stabilization of PP2A-B56a heterotrimers d Stabilization of B56a heterotrimers biases PP2A toward substrates such as c-Myc d Accumulation of methylated, B56a heterotrimers, is a potential clinical biomarker

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca 2؉ -dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca 2؉ concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca 2؉ sensitive. Ca 2؉ titration isotherms monitoring the oxidation within residues 49 -72 (within subdomain S1), 121-135 (S1), 162-166 (S2), and 722-748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca 2؉ concentration of 1-5 M that is competent for capping and severing activity. A second structural transition with a midpoint of Ϸ60 -100 M, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical ''latch'' of S6 (including Pro-745) that contact an F-actinbinding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca 2؉ transition. However, residues within S4 that are part of an extended ␤-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca 2؉ concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.