Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry

Abstract

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. 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 sulfur-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 each of the 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 have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to 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 based on their rates of oxidation.

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

“…Bovine calmodulin (Swiss-Prot ID: P62157) is comprised°of°148°amino°acid°residues° [30]°of°which°30°are potentially oxidizable under the conditions applied in these°limited°oxidation°experiments° [21][22][23][24][25][26][27][28][29].°Solutions of calmodulin alone and an equimolar mixture of calmodulin and melittin, both at pH 7 in the presence of 300 M calcium, were separately oxidized within the electrical discharge source. The products were analyzed directly by mass spectrometry.…”

“…Collectively, these segments span over 42% of the protein sequence. The six oxidizable tryptic peptides each contain sulphur (within methionine) and aromatic (within phenylalanine and tyrosine) containing amino acid residues that all have been shown to oxidize°in°our°previous°studies° [21][22][23][24][25][26][27][28][29].°Selected°ion chromatograms were generated for each of the tryptic peptide ions from the digest of calmodulin oxidized alone and in the presence of melittin. The levels of oxidation within the peptides were quantified based upon the areas under the selected chromatogram for each peptide in its unoxidized and mono-oxidized form.…”

“…To explore further the interaction between this im-portant protein and the peptide melittin, we have applied an approach developed over a number of years to probe protein structure and interactions using oxygen-based radicals [21][22][23][24][25][26][27][28]. Briefly, proteins or their complexes are treated with a high flux of radicals on millisecond timescales that results in the limited (less than a few percent) oxidation of individual amino acid side chains.…”

“…Briefly, proteins or their complexes are treated with a high flux of radicals on millisecond timescales that results in the limited (less than a few percent) oxidation of individual amino acid side chains. It has been found that where the reaction timescales are short (less than 10 -30 ms), the extent of oxidation at reactive amino acid residue side chains is highly influenced by the accessibility of these sites to the bulk solvent [21][22][23][24][25][26][27][28]. This would not be the case were the structure of the protein or complex to be perturbed.…”

“…This radical based approach coupled to mass spectrometric analysis [21][22][23][24][25][26][27][28][29] requires minimal (nanogram) levels of protein, can be applied to protein mixtures without their purification, and is capable of providing protein structure information with single amino acid resolution (at reactive residues) with relatively high sample throughput. The method has been successfully applied to follow the dynamics of protein folding at both the global and local level [23] and subsequently to several protein complexes [25,27,28].°In°the°case°of protein-protein complexes, the interaction can be mapped at either or both of the binding partners based upon the reduced levels of oxidation measured at reactive amino acid residue side chains within the interface.…”