Conformational assessment of proteins by hydrogen exchange-mass spectrometry

Abstract

Hydrogen exchange-mass spectrometry (HX-MS) is a key analytical tool to study protein conformation and dynamics. In this technique, backbone amide hydrogens exchange with the heavy isotope of hydrogen (deuterium) in solution to give information related to the conformational state of protein. Hydrogen exchange by a protein can be measured by mass spectrometry and the results are often presented on a structural model of the protein. This technique has been used successfully to identify changes in protein conformational information at high resolution; here I have applied this technique to study two different proteins.PsbO is an extrinsic membrane protein in photosystem II (PSII). PsbO has versatile functions in PSII. It is known that PsbO is released from PSII and contributes to the repair cycle of PSII following photodamage of PSII. The conformational states of bound and released PsbO are not the same. Previous studies found that the conformation of PsbO changes when it binds to PSII, but these studies did not reveal where exactly the structural changes were located. In this study I identified the locations of the structural changes of the released PsbO by using hydrogen exchange-mass spectrometry. Ricin toxin subunit (RTA) has been under investigation for a long time as a potential vaccine against ricin intoxication. As a part of that effort, one approach is to engineer the RTA* antigen to stimulate production of neutralizing antibodies. In this study, I analyzed an engineered version of RTA* known as RTA*-SS. This engineered version has one extra disulfide linkage which was anticipated to stabilize one of the antibody binding hotspots known as ɑ-helix B. I investigated the effect of mutation on the structure of RTA*-SS1 with special attention to the ɑ-helix B site by hydrogen exchange-mass spectrometry. While HX-MS showed that statistically significant protection and deprotection in overall conformation due to insertion of disulfide linkage, I was unable to determine the stability of ɑ-helix B in RTA*-SS1, because faster HX was observed in that region and this faster HX exchange could be due to faster, slower or unchanged structural dynamics and faster chemical exchange.