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

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, H218O 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.

Figure: 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.

Results from: Gupta, S., D'Mello, R., Chance, M.R.
Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):14882-7. Epub 2012 Aug 27.