Long-Lived States to Monitor Protein Unfolding by Proton NMR

1. Aurélien Bornet, 
2. Puneet Ahuja, 
3. Riddhiman Sarkar, 
4. Laetitia Fernandes, 
5. Sonia Hadji, 
6. Shirley Y. Lee, 
7. Aydin Haririnia,
8.  David Fushman, 
9. Geoffrey Bodenhausen, and 
10. Paul R. Vasos

1. The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T₁ and T₂ relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.

1. DOI

1. Journal: ChemPhysChem