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

August 27, 2012

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: Example of LC-MS. (A) Selected ion chromatogram (SIC) shows the abundance of doubly protonated (z = 2) unmodified (748.33 m/z) and modified (+16 Da, 756.33 m/z) peptide 61.72 of 10 μM cyt c irradiated for 30 ms at room temperature (RT, 25 °C) and frozen state (-35 °C). Site of modifications are confirmed by MS/MS (Fig. S2). The abundance of modified M65/Y67 are nearly the same at RT and the frozen state, while that for modified E61-62 and P71 are remarkably different. In this example the modified peptide fragments were eluted as a separate peak. The ability to chromatographically isolate the different species is critical for the analysis. (B) The average abundance of unmodified and modified peptide fragment extracted from 37.7-42.1 min. The various fractions of modifications plotted in Figs. S3 and S4A are calculated from each of the SICs for all the detected peptides.

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.