Fast photochemical oxidation of proteins for comparing structures of protein-ligand complexes: the calmodulin-peptide model system

Abstract

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive (•)OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not.

Figure 1

3D structure of CaM and CaM-peptide complex: Ca²⁺-free CaM structure from the average NMR structure (PDB ID: 1CFD). Ca²⁺-bound CaM structure from the x-ray crystal structure (PDB ID: 1CLL). Ca²⁺-bound CaM-M13 complex structure from the average NMR structure (PDB ID: 2BBM). M13 peptide is in pink. Ca²⁺ is displayed as yellow balls.

Figure 2

Changes of modification extent from Ca²⁺-free to Ca²⁺-bound states for various CaM tryptic peptides from different CaM complexes. (a) CaM itself, (b) CaM-Mel Complex, (c) CaM-Mas Complex, (d) CaM-M13 complex.

Figure 3

FPOP footprinting results on CaM structures. (a) Changes of oxidation are labeled on peptides with different color. M13 is black. (b) Bottom view of CaM-M13 complex. Residues in pink are several modified residues detected by LC-MS experiment. M13 is in the center of structure with ball shape.

Figure 4

Extent of modification for Ca²⁺-free (white) and Ca²⁺-bound (black) states of each modified residue for various CaM complexes. (a) CaM itself (b) CaM-Mel Complex (c) CaM-Mas Complex (d) CaM-M13 complex.