Analysis of the oxidative damage-induced conformational changes of apo- and holocalmodulin by dose-dependent protein oxidative surface mapping

Abstract

Calmodulin (CaM) is known to undergo conformational and functional changes on oxidation, allowing CaM to function as an oxidative stress sensor. We report the use of a novel mass spectrometry-based methodology to monitor the structure of apo- and holo-CaM as it undergoes conformational changes as a result of increasing amounts of oxidative damage. The kinetics of oxidation for eight peptides are followed by mass spectrometry, and 12 sites of oxidation are determined by MS/MS. Changes in the pseudo-first-order rate constant of oxidation for a peptide after increasing radiation exposure reveal changes in the accessibility of the peptide to the diffusing hydroxyl radical, indicating conformational changes as a function of increased oxidative damage. For holo-CaM, most sites rapidly become less exposed to hydroxyl radicals as the protein accumulates oxidative damage, indicating a closing of the hydrophobic pockets in the N- and C-terminal lobes. For apo-CaM, many of the sites rapidly become more exposed until they resemble the solvent accessibility of holo-CaM in the native structure and then rapidly become more buried, mimicking the conformational changes of holo-CaM. At the most heavily damaged points measured, the rates of oxidation for both apo- and holo-CaM are essentially identical, suggesting the two assume similar structures.

FIGURE 1

High-resolution structures of human CaM. Helix A, red; helix B, pink; helix C, aqua; helix D, yellow; helix E, green; helix F, violet; helix G, blue; helix H, cyan. Methionine side chains are shown in orange, and calcium is shown as green spacefill models. (Top) X-ray crystal structure of holo-CaM (1CLL). (Bottom) Average NMR structure of apo-CaM (1CFD).

FIGURE 2

Sequence of bovine CaM. Peptides detected by mass spectrometry are shown as underlined portions, with solid lines indicating peptides with detected oxidation, and dashed lines indicating peptides with no detected oxidation. Oxidation targets are shown in bold. Lys¹¹⁵ (italicized) is trimethylated in bovine CaM, and the N-terminus is acetylated.

FIGURE 3

Quadrupole ion trap MS/MS spectrum of the +3 charge state of peptide 127–148 with the addition of one oxygen (m/z 836.7). Y-type ions contain the C-terminus and are counted from C-terminus to N-terminus. B-type ions contain the N-terminus and are counted from N-terminus to C-terminus. All oxidation occurred on Met¹⁴⁴; no oxidation was detected on Met¹⁴⁵. (Inset) A detailed view of the y4 ion (the C-terminal MTAK). Only the unmodified fragment is detected; no signal for the modified fragment is detected.

FIGURE 4

(A) Mass spectrum of the peptides from apo-CaM after 0-Gy and 1398-Gy radiation exposure. (B) Mass spectra of peptide 31–37 from apo-CaM and its major oxidation product as a function of radiation dosage. The triply charged ion found at lower radiation dosages is from an unidentified source.

FIGURE 5

Kinetics of oxidation for the eight oxidized peptides detected, corrected for preirradiation oxidation caused by protein purification and storage. The abscissa is the radiation dosage, and the ordinate is the apparent pseudo-first order rate constant. If there are pseudo-zero-order kinetics and no alteration in solvent accessibility, the data points should form a line with a slope of 0. An increase in the rate reflects an increase in the solvent accessibility of the oxidation target, whereas a decrease in the rate reflects either a decrease in the solvent accessibility or saturation of the target. ▪, holo-CaM; ♦, apo-CaM. (A) Peptide 1–13; (B) peptide 14–22; (C) peptide 31–37; (D) peptide 38–74; (E) peptide 75–77; (F) peptide 95–106; (G) peptide 107–126; (H) peptide 127–148.