The factor that makes the greatest contribution to stabilization of the
unfolded state is its conformational entropy. It has been proposed that
decreasing the conformational flexibility of the unfolded chain (by substitution
with proline, or by replacement of glycine) should lead to an increase in
the stability of the folded relative to the unfolded protein (Matthews
et al, 1987) (See also disulphide
bonds, below.) Two such substitutions (A82P and G77A) in T4 lysozyme
gave rise to only a small amount of stabilization (0.8 and 0.4 kcal/mol),
possibly due to the counterbalancing removal of favourable interactions
upon mutation.(Matthews et al,
1987) A similar mutation that also eliminated a hydrogen bond and some
hydrophobic interactions was destabilizing, suggesting that these factors
outweighed the decrease in conformational stability (Dixon
et al, 1992).
Watanabe et al (1994)
have found a correlation between the number of proline residues in oligo-1,6-glucosidases
from a number of bacterial species and their thermostability. Structural
analysis suggests that the optimal placement is at the N-cap of alpha helices
and at the second position beta type I and beta type II turns.
PPS link to beta turns.
When they substituted prolines at these positions in the homologous mesophilic enzymes they observed an increase in thermostability. However, they made their substitutions based on recruitment from the more stable enzyme. When I tried this approach in the thermostable alpha amylase from Bacillus lichinoformis, based on structural parameters alone, the mutants were either equal in stability to wild-type or destabilized (A. Day, unpublished). Structural analysis of at least one destabilized mutant (A. Day & A. Shaw unpublished) revealed the creation of a hydrophobic surface cavity upon mutation, which presumably counterbalanced any stabilizing effect of the conformational rigidity. This suggests that, in the absence of any hints from nature in terms of homology, proline substitution for stabilization should be used with some care.
Watanabe et al ascribe
their increase in stability to a decrease in conformational entropy of the
unfolded state (c.f. the T4 lysozyme case described above by Matthews
et al, 1987). As their enzyme (and ours) is irreversibly inactivated
upon heating, it is equally likely that any observed increase in stability
is due, not to an overall stabilization of the protein, but to a slowing
of the rate of unfolding. (See Kinetic Stability
Hydrogen Bonds. Other Factors Affecting Protein Stability Beginning