NIH Research Festival
Slow proline cis-trans isomerization can serve as a rate-limiting step in protein folding by
creating energy barriers that must be overcome to achieve the native structure. Several studies
have reported that the cis isomer of proline can act as a kinetic trap during protein folding,
thereby altering the folding pathway, or preventing folding, while the
trans isomer is usually preferred in folded proteins. Therefore, the presence of proline residues
within protein domains can significantly impact their function, and ability to fold
correctly. In this study, we utilize NMR
spectrometer-controlled hardware that performs rapid and repeatable pressure switching within
a sample cell from 1bar (folding condition) to 2.5kbar (unfolding condition) to alter the
population of cis proline isomers. Hydrostatic pressure can reversibly shift the thermodynamic
equilibrium between folded and unfolded states, enabling the experimental control over folding
and unfolding under physiologically relevant conditions. Single and double pressure-jump
techniques enable us to measure cis-trans isomerization time constants ranging from seconds to
many minutes and to record specific rates for each proline.
Ubiquitin L50A, used as a model system in this study, contains three proline residues with
cis fractions of 9.5%, 6.5% and 3% for P19, P37, and P38, respectively, under unfolding conditions.
The rates and energy barriers for proline cis-to-trans isomerization in ubiquitin differ between
conditions that favor the folded and unfolded states, with 2-8 times faster rates observed under
folding conditions at ambient pressure, suggesting that cis Pro residues may form less of a kinetic
barrier to folding than often assumed.
Scientific Focus Area: Structural Biology
This page was last updated on Monday, September 25, 2023