Application of FPOP to Apomyoglobin

We tested FPOP by applying it to apomyoglobin [30], a protein that is well characterized in the holo form [127] and is often used as a model in the field of protein folding [128-130]. Radical footprinting by FPOP in the presence of 20 mM phenylalanine, a concentration that is 2000 times greater than that of the protein, should be complete in @70 ns [30]. Indeed, virtually no protein oxidation occurs with this high concentration of scavenger (Fig. 11.8A). In the presence of 20 mM glutamine, a less reactive scavenger, the reaction duration lengthens from 70 ns to 1 ms, and oxidation occurs (Fig. 11.8B). In the absence of scavenger, the reaction duration lengthens to >100 ms, and now considerable oxidation occurs (Fig. 11.8C). These trends suggest that the kinetics of oxidation can be followed, at least roughly, by varying the nature and concentration of the scavenger.

The next step is to digest the protein and analyze the peptides for sites of oxidation. If the reactions indeed modify residues at the surface of the protein, one should find a correlation between reactive sites and those that are predicted to

Protein Oxidation

Fig. 11.9 Deconvolved mass spectra [deconvolution by a maximum entropy algorithm (MaxEnt) supplied by instrument manufacturer] for S-peptide and S-protein, showing differences upon oxidation with 15 mM H2O2 using 15 mM Gln as scavenger.

(A) S-peptide oxidized in absence of protein.

(B) S-peptide oxidized while bound to RNase S protein, showing less oxidation. (C) RNase S protein oxidized in absence of peptide. (D) RNase S-protein oxidized while bound to S-peptide.

Fig. 11.9 Deconvolved mass spectra [deconvolution by a maximum entropy algorithm (MaxEnt) supplied by instrument manufacturer] for S-peptide and S-protein, showing differences upon oxidation with 15 mM H2O2 using 15 mM Gln as scavenger.

(A) S-peptide oxidized in absence of protein.

(B) S-peptide oxidized while bound to RNase S protein, showing less oxidation. (C) RNase S protein oxidized in absence of peptide. (D) RNase S-protein oxidized while bound to S-peptide.

have significant solvent exposure. To test the hypothesis, one can calculate side-chain solvent accessibility using the X-ray structure and a 1.1-A probe in the program GetArea 1.1, available on the web [131]. For apomyoglobin, the only protein tested thus far, the correlations are good [30].

The ability to measure the change in oxidation as a protein is titrated with its ligand may, like for PLIMSTEX, enable the characterization of the binding affinity. We know, for example, that hydroxyl radicals are suitable reagents for following the denaturation-induced unfolding of apomyoglobin [132]. Although fast radical footprinting has not yet been extended to affinity measurements, Fig. 11.9 shows one example where we can see large changes in the extent of oxidation of a peptide and protein when the protein is unligated and when it is interacting with the ligand (peptide). For S-peptide, Fig. 11.9A shows the extent of oxidation for the peptide in the absence of its binding partner, RNase S protein. In Fig. 11.9B, we see that the extent of oxidation of the peptide is attenuated because the peptide is now complexed with the S-protein. Similar changes are observed when the order of addition is reversed; that is, when S-protein is in solution in the absence of S-peptide, there is considerably more oxidation of the protein (Fig. 11.9C) than when the peptide is added to form the complex (Fig. 11.9D). Following the extent of oxidation as the protein is titrated with the peptide may afford the binding affinity of the complex, S-peptide/S-protein, as well as simultaneously reveal the residues involved in complex formation.

366 | 11 Quantification of Protein-Ligand Interactions in Solution 11.5.6

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