Abiotic Stress Adaptation: Protein Folding Stability and Dynamics - Abiotic Stress: Plant Responses and Applications in Agriculture - page 15

sample k f (s -1 ) k u (s -1 ) p E (%)

none 574,4 16,7 2,8

myo- Inositol 871,5 12,3 1,4

pinitol 935,9 12,7 1,3

quebrachitol 653,8 10,8 1,6

quercitol 661,8 13,0 1,9

Table 1. Two-site conformational exchange parameters of KIX. The response of R 2.eff to v CPMG can be fitted to extract
exchange parameters. A two-site exchange model (G↔E) was fit to 15 N relaxation dispersion data, yielding site-
specific values of G→E and E→G rate constants (k u and k f ). k f and k u are the first order rate constants for folding and
unfolding transitions, calculated from global fits of 15 N backbone relaxation experiments.

The two-site conformational exchange of KIX between its natively folded ground state and a
partially unfolded high-energy state, that represents the equilibrium analog of a folding in‐
termediate [54], was shown to be highly sensitive to the addition of osmolytes. NMR data
showed that the composition of these two states differed between the protein in buffer alone
and the osmolyte containing sample. Addition of 0.4M pinitol led to a decrease of more than
50% in the population of the partially unfolded state (p E ). Accordingly, the first order rate
constant for folding (k f ) increased from 574.4s -1 to 935.9s -1 in the presence of pinitol, while
the rate constant for unfolding (k u ) decreased (from 16.7s -1 to 12.7s -1 ). These data provide
evidence that even under native conditions osmolytes shift the folding equilibrium towards
the folded state. NMR relaxation experiments revealed that osmolytes play an important
role on the structure of the folding intermediate, which is the main determinant for protein
folding and dynamics. Even though intermediate states are extremely short-lived (in the or‐
der of ms), osmolytes greatly influence these states. A decrease in the population of the par‐
tially folded state is associated with a destabilization of this state relative to the folded state
in the osmolyte containing sample. The interaction of the osmolyte with the protein surface
is not favorable and therefore osmolytes are preferentially excluded from the protein sur‐
face. Osmolytes indirectly act by changing the properties of water surrounding the protein
and hence modify protein-solvent interactions by altering the specific arrangement of the
hydrophobic and hydrophilic residues. Folded states are relatively favored over (partially)
unfolded states due to their compact structure and smaller surface exposed (solvent accessi‐
ble) area, leading to a net stabilization of the folded state even under native conditions. Ac‐
cumulation of high amounts of osmolytes does not seem to be useful under non-stress
conditions as they influence protein conformation and dynamics, but they confer great ad‐
vances to enhance protein stability under stress conditions by counteracting the forces driv‐
ing protein unfolding. Compact folded conformations are generally less prone to unfolding,
misfolding and aggregation that lead to loss of protein function. Increased conformational
stability through osmolytes on the other hand allows for greater protein flexibility under
elevated temperatures, since thermal motion decreases rigidity and enhances flexibility. This
mechanism greatly contributes to preserve protein function under stress conditions in
plants.