Proline Peptide Bond Isomerization in Ubiquitin under Folding and Denaturing Conditions by Pressure-Jump NMR

J Mol Biol. 2024 Apr 23:168587. doi: 10.1016/j.jmb.2024.168587. Online ahead of print.ABSTRACTProline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kCal/mole for denatured polypeptides but showed a substantial drop to 12.7 kCal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensit...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Source Type: research