Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy

The conformational heterogeneity
of a folded protein can affect
not only its function but also stability and folding. We recently
discovered and characterized a stabilized double mutant (L49I/I57V)
of the protein CI2 and showed that state-of-the-art prediction methods
could not predict the increased stability relative to the wild-type
protein. Here, we have examined whether changed native-state dynamics,
and resulting entropy changes, can explain the stability changes in
the double mutant protein, as well as the two single mutant forms.
We have combined NMR relaxation measurements of the ps–ns dynamics
of amide groups in the backbone and the methyl groups in the side
chains with molecular dynamics simulations to quantify the native-state
dynamics. The NMR experiments reveal that the mutations have different
effects on the conformational flexibility of CI2: a reduction in conformational
dynamics (and entropy estimated from this) of the native state of
the L49I variant correlates with its decreased stability, while increased
dynamics of the I57V and L49I/I57V variants correlates with their
increased stability. These findings suggest that explicitly accounting
for changes in native-state entropy might be needed to improve the
predictions of the effect of mutations on protein stability.