Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry

Abstract

Water is critical for the structure, stability, and functions of macromolecules. Diffraction and NMR studies have revealed structure and dynamics of bound waters at atomic resolution. However, localizing the sites and measuring the dynamics of bound waters, particularly on timescales relevant to catalysis and macromolecular assembly, is quite challenging. Here we demonstrate two techniques: first, temperature-dependent radiolytic hydroxyl radical labeling with a mass spectrometry (MS)-based readout to identify sites of bulk and bound water interactions with surface and internal residue side chains, and second, H(2)(18)O radiolytic exchange coupled MS to measure the millisecond dynamics of bound water interactions with various internal residue side chains. Through an application of the methods to cytochrome c and ubiquitin, we identify sites of water binding and measure the millisecond dynamics of bound waters in protein crevices. As these MS-based techniques are very sensitive and not protein size limited, they promise to provide unique insights into protein-water interactions and water dynamics for both small and large proteins and their complexes.

Fig. 1.

Pictorial representation of modification sites on cyt c (A and B) and ubiquitin (C and D) in two orientations. Colored surface representation based on the X-ray crystal structure of cyt c (1HRC) (27) and ubiquitin (1UBQ) (28) indicate the side-chain residues that are consistently modified after irradiation under RT and/or frozen conditions. Blue indicates the residues that show 13- to 200-fold decreases in modification upon freezing, violet indicates 3- to 10-fold decreases, and red indicates minimal to no change (< 2-fold) in the modification rate when sample is frozen compared to RT. Y67 in cyt c is completely buried inside the heme cavity and not visible in these orientations.

Fig. 2.

Identification and quantification of ¹⁸O labeled residues. MS analysis in zoom scan mode of cyt c samples in 97% and 50% H₂¹⁸O provide a quantitative analysis of isotopologues of singly protonated ¹⁸O and ¹⁶O labeled peptides 9–22, 28–38, 40–53, 56–60, 61–72, and 80–86 of cyt c with side-chain modifications on the residues C14/C17, F36, F46/Y48, W59, M65/Y67, and M80, respectively. The first labeled peak indicates the position of the monoisotopic mass of the ¹⁶O-adduct; the peaks shifted by 2 m/z units from this mass contain a mixture of the ¹⁸O monoisotopic mass and the (two) C¹³ containing ¹⁶O-isotope. The decreased abundance at this m/z value relative to the monotisotopic mass, for 50% vs. 97% H₂¹⁸O, is indicated by an arrow and represents the potential signal for an exchange experiment for the respective peptide. For peptide 9–22, the largest signal is the increase in the 9–16 monoisotopic peak.

Fig. 3.

Time-resolved radiolytic ¹⁸O labeling and water exchange in cyt c. (A) Rapid mixing combined with ¹⁸O-mediated hydroxyl radical labeling monitored the time course of exchange of water in cyt c. LC-ESI-MS is used to identify and isolate the modified peptides, targeted MS/MS is used to identify the sites of ¹⁸O labeling, and zoom scan is used quantify the ratio of ¹⁸O vs. ¹⁶O labeling at various mixing delays. (B) Zoom scans for singly protonated peptide 61–72 showing decease in the abundance of the 2 m/z shifted ¹⁸O monoisotopic mass (arrow) that corresponds to the water exchange at M65 and Y67 with increase in the mixing delays. (C) Progress curves (circles and error bars) of water exchange for the ¹⁸O labeled side-chain residues. The solid line represents the fit to single exponential function. Residues W59 and F36 have exchange that is complete at the first measurement, while the rates of exchange of C14/C17, F46/Y48, M65/Y67, and M80 are discretely measured.

Fig. 4.

Cyt c ¹⁸O-labeling map. The sites of ¹⁸O-modifications are visualized from the crystal structure 1HRC (27) using PyMOL. The ¹⁸O-labeled residues (light blue) in and around the heme (light pink) crevices, and the position of residue T78 (gray) and conserved waters (cyan spheres) HOH112, HOH139 are shown in two orientations of the cyt c molecule.