Many scientists out there want to know about the dynamics of a protein or how a protein binds to small molecules. But sometimes that information is hard to get using classical techniques such as X-ray crystallography or NMR.
Maybe your protein is too large (NMR won’t work) or maybe it just won’t crystallize. A mass spec technique called hydrogen/deuterium exchange mass spectrometry (HDX-MS) can be useful when you have almost given up and can’t think of anything else to try!
The basis behind HDX is simple: one exchanges the hydrogens in your protein sample with deuteriums by soaking the protein in D2O for specific amounts of time. Because deuterium weighs one dalton more than hydrogen (and it mimics the function of hydrogen) you can accurately measure the exchange rate with mass spec.
So what will the information tell you? Well, it won’t give you the structure of a protein (you still need X-ray crystallography or NMR information), but it can tell you how dynamic certain regions in your protein are. An example of such a study was published by Griffin and colleagues (Zhang et al., 2010) using β2 adrenergic receptor (β2AR) bound to the inverse agonist carazolol.
β2AR is part of the G-protein signaling cascade and is involved in regulation of muscle relaxation, glycogenolysis, etc. β2AR is activated through binding of endogenous or synthetic ligands that modulate the receptor-G protein interaction. In the resting state, the receptor associates with G-protein via its intracellular 3rd loop and C-terminus. When a ligand binds, conformational changes occur that facilitate the release of the G-protein. This, in turn, initiates the downstream signaling cascade. Carazolol, which is used to treat asthma, is known to bind to β2AR, but the precise mechanism of action is still unknown.
The crystal structure of carazolol-bound β2AR had been previously determined; however, it was obtained by replacing the highly dynamic third intracellular loop with the T4-lysozyme domain (which is more rigid), so no information on the flexible loop was determined. This is where HDX-MS can help. The group was able to get dynamic information about the loop by examining the changes in deuterium exchange over time; the more flexible a region is, the more readily it will exchange hydrogens for deuteriums. Hydrogens are not exchanged when they are protected in some way – such as a region involved in hydrogen binding. The exchange information can then be overlaid onto the crystal structure (see picture below).
Peptides in the 3rd intracellular loop demonstrated the most rapid exchange kinetics, meaning the loop can move around very easily. The rapid movement could be important when G-protein binds or is released from this region. After an exchange time of 30s, 300s, and 15h, the HDX results were mapped onto the 3-D crystal structure of β2AR bound to carazolol (see picture below).
In addition to the data for the 3rd intracellular loop, the results also showed that the 2nd extracellular domain (the region at the top of the structure) contains a loop that may act as a lid, capable of moving away and over the ligand binding site. So, while it might seem obvious that the loops of membrane proteins are more flexible, the dynamic secondary elements indicate functional importance of these regions for β2AR. These conclusions had not been determined by X-ray crystallography. Pretty cool, huh?
Another hot area in HDX-MS is getting “single amide resolution.” One limitation with most HDX-MS experiments is that one can only see changes in a region of the protein, but not shifts in single amino acids.
A recent paper in JASMS (Journal of the American Society for Mass Spectrometry) describes an HDX-MS technique to study the PPARγ receptor when it’s bound to different drugs. This receptor is important because it plays a role in controlling genes involved in carbohydrate and lipid metabolism. Drugs targeted for this receptor are shown to improve insulin sensitivity in diabetic patients, but cause a long list of side effects.
So, it’s important to understand how different drugs bind to PPARγ to determine if there is a correlation between the binding interactions and the side effects. In the past, researchers believed that ligand-mediated activation of PPARγ occurred through a stabilization of helix 12 of the ligand binding domain. A recent study, however, has shown that several drugs can bind in a helix 12-independent manner. Griffin and colleagues (Landgraf et al., 2011) determined that another region of the protein, helix 3, is actually stabilized by drug binding instead of helix 12. So, by using mass spectrometry they determined exactly where binding occurs for two drugs, MRL24 (a partial agonist) and rosiglitazone (a full agonist), as well as the effects on the protein.
In addition to using an Orbitrap (a high resolution mass spec instrument), Griffin and colleagues used a gas phase fragmentation method different from what’s normally used. Instead of collision induced dissociation (CID), they used electron transfer dissociation (ETD), which minimizes the chances of “scrambling.”
Blah blah blah… basically this means that not only will they have a good chance of getting amide resolution because they used a high resolution instrument, the Orbitrap, but they will also be confident in the information they obtain because they are using ETD to minimize the chance of protons jumping around in the gas phase.
In the end, they were able to tell that MRL24 makes more hydrophobic contacts within helix 3 of PPARγ compared to rosiglitazone. The results could explain the differences in the magnitudes of the agonist response between MRL24 and rosiglitazone.
So basically, this is yet another reason why I think mass spectrometry is a wonderful tool that can help all scientists!
Zhang, X., Chien, E., Chalmers, M., Pascal, B., Gatchalian, J., Stevens, R., & Griffin, P. (2010). Dynamics of the β-Adrenergic G-Protein Coupled Receptor Revealed by Hydrogen−Deuterium Exchange. Analytical Chemistry, 82 (3), 1100-1108 DOI: 10.1021/ac902484p
Landgraf, R., Chalmers, M., & Griffin, P. (2011). Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution. Journal of The American Society for Mass Spectrometry, 23 (2), 301-309 DOI: 10.1007/s13361-011-0298-2
Wales, T., & Engen, J. (2006). Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrometry Reviews, 25 (1), 158-170 DOI: 10.1002/mas.20064