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alone. Retention of secondary structure in osmolyte solution was monitored even at temper‐
atures where proteins were fully unfolded when heated without additives.
Studies on RnaseA previously showed that increases in ΔH m by the addition of trehalose re‐
sulted in a lower ΔCp-vlaue (heat capacity change). [41]. The heat capacity change, ΔCp, is a
very sensitive thermodynamic parameter that correlates with the amount of the protein sur‐
face that is exposed to the solvent [44]. A decrease in ΔCp upon the addition of osmolytes
reflects a lower surface exposed area and/or decreased exposure of hydrophobic groups to
the solvent. Decreases in ΔCp may also result in flattening of thermal unfolding curves,
leading to conformational stability over a wider range of temperature. This has shown to be
an effective strategy for many mesophilic proteins.
The thermal stability of a protein is determined by the response to thermal energy, concern‐
ing globally and locally unfolding and the ability to refold into its active conformation. Ther‐
mal unfolding was shown to be highly reversible for thermostable proteins of
hyperthermophilic organisms. The far-UV CD spectrum of the native protein was identical
to that after heat denaturing and re-cooling [45]. Many mesophilic proteins, however, aggre‐
gate or precipitate after thermal unfolding making the unfolding process irreversible. Find‐
ing co-solvent conditions that facilitate refolding is as important as increasing the melting
temperature. Facilitated refolding was observed for ribonuclease that undergoes a reversible
denaturation in the presence of trehalose [46].
Taken together, these results from CD measurements reveal that osmolytes stabilize protein
global folds under heat by supporting retention of secondary structure elements and aid in
refolding of thermally unfolded proteins.
4. New insights into molecular dynamics of protein folding and
unfolding from Nuclear Magnetic Resonance (NMR) spectroscopy
Internal dynamics of proteins play an important role in their biological function. Proteins do
not only exist in well-defined natively folded or fully unfolded states, but also in partially
folded intermediate states. The conformational exchange between a folded state and partial‐
ly folded states is highly sensitive to changes in the environment such as temperature, pH,
solvent and co-solvent conditions. In the plant cell, proteins are predestinated to function in
environments crowded by macromolecules, metabolites and other co-solvents that facilitate
protein folding under non-stress and stress conditions [47]. By measuring protein dynamics,
it is therefore important to include (co-)solvent conditions (Figure 6). High-osmolyte accu‐
mulation upon stress conditions induces changes in the protein environment. Variable pro‐
tein folds may be affected slightly different according to their hydrophobic or hydrophilic
surface properties, compactness, flexibility, hydrogen bonding patterns, excluded volume
effects and the affinity of binding sites for co-solvents or the hydration water.
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