Ivan John Mercado Clement (U093544L)

LSM3243 Assignment 2
ivan.clement@nus.edu.sg

Q: Explain why proteins may be denatured by urea.

A:

Current literature on the subject urea-mediated denaturation of proteins proposed two mechanism
of action. The first one is by a direct mechanism, wherein urea binds to the protein and competes
with native non-covalent interactions with the protein. The other one proposes an indirect
mechanism, where urea perturbs the solvent environment and thus facilitates the exposure of the
protein's hydrophobic core. 1 While both of these mechanisms were initially elucidated using
molecular dynamics simulations, the latter one has not received much experimental validation. 2 In
the next paragraphs, we outline the possible interactions involved in both mechanisms proposed,
using these to explain how proteins can be denatured by urea.

In the direct mechanism, H bonding formed by the constituents of the protein backbone is disrupted
by urea. While in normal aqueous solution, water can form H bonds with the protein backbone as
well, it has been shown that the geometry of backbone-urea H bonds is very similar to backbone-
backbone H bonds.1 Hence, in the presence of urea, we may expect urea to form more favorable H
bonds with the protein backbone compared with water. This opens up the inner portions of the
protein to attack by more urea and water molecules. Once this happens, we may then imagine that
the hydrophilic residues of the protein would interact electrostatically with water and/or urea
molecules. Meanwhile, the hydrophobic residues would interact with urea, stabilized by dispersion
forces. This then facilitates the denaturation of the protein, with the aforementioned electrostatic
and dispersion interactions stabilizing the denatured state. 1

In the indirect mechanism, it has been observed in molecular dynamics simulations that introduction
of urea disrupts the water network wherein the protein is dissolved. 2 This then perturbs the
solvation shell of the protein because of the H bond formation between urea and water molecules.
This allows urea to be integrated into the water H-bonding network, thereby giving it access through
the solvation shell to attack the protein directly and disrupt the native H bonding in the protein. In
effect, what the literature is suggesting is that the indirect mechanism paves the way for the direct
mechanism to take place.1

In summary, addition of urea to a protein solutions gives the protein more interaction options with
its environment. This, undoubtedly, changes its thermodynamic stability, and thus the protein must
change shape in order to adapt to its altered environment. 1 Both the direct and indirect mechanisms
as mentioned above facilitates the protein's change of shape and effectively, its denaturation.

References:
1. Das, A. & Mukhopadhyay, C. Urea-Mediated Protein Denaturation: A Consensus View.
Journal of Physical Chemistry B 113, 12816-12824 (2009).
2. Rossky, P.J. Protein denaturation by urea: Slash and bond. Proceedings of the National
Academy of Sciences of the United States of America 105, 16825-16826 (2008).