Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry

Abstract

The synchrotron X-ray protein radiolysis technique is based on a quantitative determination of the extent and the site of millisecond radiolytic oxidation of amino-acid side chains by mass spectrometry. The amino acids most susceptible to radiolytic oxidation are cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, and leucine. These residues serve as reactive markers within a protein structure that can be used to monitor changes in solvent accessibility during folding or as part of macromolecular interactions. To monitor the unfolding, the extent of radiolytic products of side chains of reactive amino acids is quantitatively measured by mass spectrometry as a function of the denaturant concentration following proteolysis. This approach provides site-specific unfolding isotherms for various segments of a protein without the use of mutation or labeling techniques. Application of this technique to the equilibrium urea unfolding of apomyoglobin at pH 7.8 has demonstrated the cooperative unfolding of helices A to C consistent with midpoints, DG, and m values derived from fluorescence data. The G helix, in contrast, showed a local unfolding behavior. The similarity of the thermodynamic data derived by this synchrotron-based method for helix A (containing two oxidizable tryptophan residues) to that of the fluorescence data indicates that the limited oxidation of proteins by exposure to X-rays on millisecond timescales does not alter the structure of apomyglobin. This supports the viability of the method for the study of protein folding and the mapping of protein interaction sites.Keywords: protein folding; X-ray radiolysis; synchrotron; mass spectrometry; apomyoglobin.The interactions between amino-acid side chains play an important role in influencing protein structure and are significant for the stability of the hydrophobic core of the protein. Amino-acid side chains have well known propensities for secondary structure formation and influence the formation of helices and sheets [1 -6]. The specific nature and thermodynamic stability of these tertiary contacts are very important in understanding protein folding, protein structure, and formation of macromolecular complexes [4,[7][8][9][10][11], and have been shown to be useful in predicting misfolding or aggregation events in relation to disease [12][13][14][15][16][17].A common method for probing the nature and energetic contributions of these tertiary contacts has been the use of site-directed mutagenesis combined with chemical denaturation, where CD or fluorescence spectroscopy is commonly used as a probe, to generate isotherms allowing the global free energy change to be measured [4,7,8,18]. The small, single domain proteins usually analyzed by this method have a highly cooperative, two-state unfolding transition. Thus, for each mutation, the change in DG value (analyzed as a DDG ) can be interpreted as the specific sidechain contribution to the overall global stability. This analysis must be carefully applied because any particular terti...

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.…”

“…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.…”

“…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.…”