Hindered Refolding as Mechanism for Urea Induced Denaturation of the Cold Shock protein
Financial Support: Volkswagen Foundation.
Some proteins such as the Cold Shock protein Bc-CsP are not strongly affected by the presence of urea in molecular dynamics simulations within reachable simulation times. To tackle this problem and study urea-induced unfolding for the Cold Shock protein, we generate partially unfolded states by high-temperature unfolding and simulate these structures at physiological temperature in aqueous urea solution as well as in pure water. While the partially unfolded proteins remain stable or undergo further unfolding steps in aqueous urea solution, the simulations in water exhibit a step-by-step reduction of the proteins’ solvent accessible hydrophobic surface, indicating first minor refolding events in the form of hydrophobic collapses. These results suggest that urea interacts predominantly with the less charged residues of the (partially) unfolded state and thereby prevents hydrophobic collapse of these residues, and hence refolding. In this model, urea unfolds the Cold Shock protein not primarily by attacking the native state. Instead, the by thermal fluctuations partially unfolded proteins are stabilized and hindered from refolding. This leads to a shift of the equilibrium towards the unfolded state.
Urea is known to be a strong protein denaturant. Although it is widely used to unfold and study proteins in the lab, the molecular mechanism of urea-induced protein unfolding still remains unidentified. No significant effect of urea on the folded protein was observed in simulations of up to 400 ns at physiological temperatures. To study the effect of urea on partially unfolded proteins, high temperature was used to obtain partial unfolding of the Cold Shock protein.
The protein unfolded very quickly in the high temperature (700 K) simulation and the solvent accessible hydrophobic surface (SAS), as a measure for unfolding, reached a maximum level in the completely unfolded state. Four partially unfolded structure and one completely unfolded structure were extracted and used as starting structures in simulations at physiological temperatures with pure water on the one hand, where partial refolding might be expected, and with 8M urea solution on the other hand, where further unfolding might be expedted.
Figure 2 shows the SAS of two representative simulations of partially unfolded structure number 4. In the simulations with the partially unfolded structures, a profound effect on urea was evident. The protein collapsed to a more compact form in the simulations with water, as indicated by the decrease of SAS (blue line in Fig. 2). This reduction of SAS stems from local hydrophobic collapse events and is the first step of refolding into the native structure. In the simulations with urea, however, the SAS stays constant or even increases (green line in Fig. 2), indicating further unfolding.
To quantify interactions of urea and water with the protein on the molecular level, an interaction coefficient was defined by the number of atomics contacts with urea divided by the number of atomic contacts with water. This was calculated for every single amino acid in an 200 ns simulation of the unfolded state. The urea-like residues GLY and ALA (coded red and yellow, respectively, in Fig. 3) exhibited strong tendency to interact with urea. In contrast, the charged residues ASP and GLU (coded blue and green, respectively) showed strong tendency to interact with water and rather avoid contact with water. Furthermore, the protein backbone showed a significantly higher preference to interact with urea than the sidechains, on average.
Implications for the molecular mechanism of urea-induced protein denaturation: Urea prevents hydrophobic collapse events of the unfolded protein and thus its refolding. This shifts the equilibrium between folded and unfolded towards the unfolded state. On the molecular level, the unfolded state is stabilized by favorable interactions between urea and the (urea-like) peptide backbone or less polar sidechains.