Polar or apolar--the role of polarity for urea-induced protein denaturation

Abstract

Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 micros of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea-protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.

Figure 1. CI2 in native conformation.

(A) C_α-RMSD. (B) SAS for the two simulations in water (blue) and the 3 simulations in aqueous urea with regular charges (green). The solid bold lines show traces smoothed by a running average over 500 ps; dim lines show raw data.

Figure 2. Interaction coefficient C_UW for all amino acids types in the CI2 protein, as well as the backbone average (“bb”).

The four panels show C_UW for the different urea partial charge scalings (A: 50%, B: 75%, C: 100%, D: 150%). The color characterizes the amino acids. Red: charged, yellow: polar, gray: aliphatic, blue: aromatic, green: apolar. For better comparability, all C_UW are sorted according to C_UW in urea_50%.

Figure 3. Solvent accessible surface area of the protein in all simulations.

Blue: water, orange: urea_50%, magenta: urea_75%, green: urea_100%, black: urea_150%. The lines show traces smoothed by a running average over 500 ps. The histogram in the right panel shows the frequency of the respective SAS.

Figure 4. Unfolding pathways of the CI2 for the simulations in urea_75%, displayed as native secondary structure content versus native contact content.

The numbers next to the protein structures denote the respective time of the snapshot in ns.

Figure 5. Per-residue C_α-RMSD in the initial unfolding phases.

Blue corresponds to low, red to high RMSD. The numbers on the left denote the start and end times of the respective displayed trajectory segment in ns. Top row: root-mean-square-fluctuations per residue in the native state. In the one-letter sequence code below, red marks the α-helix and blue β-strands.

Figure 6. Ramachandran plots for (A) CI2 in urea_100% (folded state), (B) CI2 in urea_75% (unfolded state).

The white circles show the areas which have been used for the calculation of populations densitites, which are shown in the lower panel for antiparallel β-sheet (“apβ”), PP2 or parallel β-sheet (“pp2/pβ”), and helical (α) configurations.