Probing Conformational Changes of Ubiquitin by Host-Guest Chemistry Using Electrospray Ionization Mass Spectrometry
JW Lee and SW Heo and SJC Lee and JY Ko and H Kim and HI Kim, JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 24, 21-29 (2013).
We report mechanistic studies of structural changes of ubiquitin (Ub) by host-guest chemistry with cucurbit6uril (CB6) using electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism spectroscopy and molecular dynamics (MD) simulation. CB6 binds selectively to lysine (Lys) residues of proteins. Low energy collision- induced dissociation (CID) of the protein-CB6 complex reveals CB6 binding sites. We previously reported (Anal. Chem. 2011, 83, 7916-7923) shifts in major charge states along with Ub-CB6 complexes in the ESI- MS spectrum with addition of CB6 to Ub from water. We also reported that CB6 is present only at Lys(6) or Lys(11) in high charge state (+13) complex. In this study, we provide additional information to explain unique conformational change mechanisms of Ub by host-guest chemistry with CB6 compared with solvent-driven conformational change of Ub. Additional CID study reveals that CB6 is bound only to Lys(48) and Lys(63) in low charge state (+7) complex. MD simulation studies reveal that the high charge state complexes are attributed to the CB6 bound to Lys(11). The complexation prohibits salt bridge formation between Lys(11) and Glu(34) and induces conformational change of Ub. This results in formation of high charge state complexes in the gas phase. Then, by utilizing stronger host-guest chemistry of CB6 with pentamethylenediamine, refolding of Ub via detaching CB6 from the protein is performed. Overall, this study gives an insight into the mechanism of denatured Ub ion formation via host-guest interactions with CB6. Furthermore, this provides a direction for designing function- controllable supramolecular system comprising proteins and synthetic host molecules.
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