|Fig. 4 from the January 2013 Cao et al minireview in The Journal of Biological Chemistry shows the differences in deuterium exchange rates for GIVA cPLA2 upon binding to two lipophilic inhibitors. Click on the image above to see a larger version of it.
What do snake venom, sperm maturation and pancreatic fluid have in common? Phospholipase A2, a group of enzymes that release fatty acids from phospholipid components of biological membranes. The released fatty acids, especially arachidonic acid, play regulatory roles in inflammatory responses, thus making phospholipases highly valuable for therapeutic purposes.
PLA2 enzymes associate with membranes and interact with their substrates to perform their function. Unfortunately, examining the dynamics of these interactions at the molecular level has been challenging due to the insolubility of the phospholipid substrates and the large molecular size of PLA2. In a recent minireview in The Journal of Biological Chemistry, Edward Dennis and co-workers at the University of California, San Diego, describe hydrogen-deuterium exchange mass spectrometry as a useful method for examining this set of proteins, despite the technique’s shortcomings.
Hydrogen atoms that are part of the protein backbone are frequently exchanged with hydrogen atoms in the surrounding water. Deuterium is a heavier isotope of hydrogen and is interchangeable with hydrogen. Harnessing these properties, the DXMS technique has been used to examine regions of PLA2 that contact membranes, substrates and inhibitors.
The authors of the minireview describe the advantages and technical challenges of using DXMS to probe the dynamics and variety of membrane-PLA2 association modes. Highly disordered regions and inhibitor binding sites on PLA2 can be detected by DXMS. These regions are not visible in crystal structures due to high flexibility and problems with co-crystallization. On the other hand, extremes of ordered and disordered regions are beyond the detection sensitivity limits of DXMS. Also, the authors emphasize, careful experimental design is required to overcome challenges with localization of DXMS changes onto the protein as a whole.
DXMS gives moderate-resolution data; but, the authors say, in conjunction with high-resolution protein structure data, molecular models, site-directed mutagenesis and other biochemical evidence, it can serve as an efficient method to glean a more complete understanding of membrane proteins dynamics.