Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1

Abstract

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

Figure 1

Peptide-1 before (A) and after oxidation (B), indicating oxidation of phenylalanine (m/z 799) and histidine (m/z 761 and 773).

Figure 2

MS spectrum of tryptic digestion of galectin-1 before oxidation (A), and after oxidation (B). Oxidation leads to more facile proteolysis, formation of peaks from oxidation products, and a decrease in the abundance of non-oxidized peptides (see peak indicated by arrows).

Figure 3

Tandem mass spectra of a tryptic peptide from galectin-1 in non-oxidized (A) and oxidized (B) forms indicating peaks associated with a 22 Da mass loss, typical of histidine oxidation.

Figure 4

Per-residue oxidation levels plotted on the solvent accessible surface of dimeric galectin-1 (A). Galectin-1 dimer interface in detail. Reporter residues are indicated in shades of red according to the measured level of side chain oxidation. From left: ribbon structure of the homodimer, solvent accessible surface structure of the homodimer, and monomeric subunit illustrating interfacial residues (B).

Figure 5

Solvent accessible surface area () for each of the 134 side chains in galectin-1. Values for monomer (pink) and dimer (black) domains are reported. Shaded regions indicate the dimer interface (blue). Other regions of low exposure include the sheets associated with the β-sandwich structure (yellow).

Figure 6

Percentage of oxidation versus side chain for selected amino acids. Highly reactive side chains (A, B) are characterized by steep slopes (%Ox/Ų) and low minimal exposure values (Ų). Moderately reactive side chains (C, D) require significant exposure before oxidization may occur. Side chains that are inert under these experimental conditions (E, F) show no sensitivity to level of exposure.