Osmophobic Effect

In document Betaine analogues and related compounds for biomedical applications (Page 32-35)



2.2.3 Osmophobic Effect

Bolen and co-workers have tried to explore the mechanism of protein stabilization with their studies on osmolytes and related compounds. They studied the effects of osmolytes that are chemically similar to one another, choosing glycine based osmolytes and studying their stabilizing effect on ribonuclease A (RNase A) and hen egg white lysozyme (HEW lysozyme) against thermal unfolding (Santoro et al., 1992). They observed that Tm, the midpoint temperature of thermal unfolding transition for a protein, increased for both RNase A and HEW lysozyme in the presence of osmolytes. These osmolytes seemed to stabilize proteins even well beyond the physiological concentration and moreover, the degree of stabilization was extraordinary. They also showed that RNase A will refold in a completely reversible manner in the presence of up to 8 M sarcosine, which indicated that basic rules for RNase A refolding did not change as a function of the concentration of the osmolytes. Some of their results also suggested that there is a tendency for these osmolytes to destabilize the proteins at very high

concentration. They concluded that osmolytes should be able to stabilize any enzyme or protein to which it is exposed and it does not matter whether the protein evolved in the presence of osmolytes or not.

Baskakov and Bolen (1998) studied the effects of two osmolytes, urea (a denaturant) and trimethylamine-N-oxide (TMAO) (an effective stabilizer). They showed that urea is no better at solubilizing hydrophobic side chains than is TMAO, as the overall side chain interactions with urea and TMAO are favorable, which implies that both these solutes should denature proteins. They suggested that the magnitude of the favorable vs. unfavorable interactions of the protein backbone with the solute, primarily determines whether the solute will be a denaturing or a protecting osmolytes. Qu et al. (1998) raised the possibility that the backbone might play a role in protein folding in water and also whether solvophobicity of the backbone in water had been overlooked so far as a contributor to the folding of protein in aqueous solution.

Bolen and Baskakov (2001) wrote a comprehensive review of their studies with osmolytes and proteins, and their proposed mechanism. They classified the unfavorable interactions between a solvent component (an osmolytes) and a protein functional group (peptide backbone) as solvophobic and called the unfavorable interaction the ‘osmophobic effect’. They argued that experimental measurements made by Timasheff and co-workers could not distinguish a solvophobic mechanism from other mechanisms. By means of their measurements of transfer Gibbs energy of amino acid side-chains and peptide backbone from water to a variety of naturally occurring osmolytes, Bolen and co- workers presented the evidence that the mechanism of osmolytes action is solvophobic and proposed that osmophobic effect is a thermodynamic force of biological importance in protein folding.

They revealed the consequences of the preferential exclusion of osmolytes from native and denatured proteins species using a thermodynamic cycle (Figure 2.5). In the thermodynamic cycle, reactions 2 and 4 represent the transfer of denatured (D) and native protein (N) from water to a fixed concentration of osmolyte. They argued that, compared

to the native state, denatured protein exposes more protein fabric to solvent, thus resulting in denatured state of the protein being more solvophobic toward osmolyte than the native state, making ∆GD[Os] a significantly more positive quantity than ∆GN[Os].

Figure 2.5: Thermodynamic cycle of protein denaturation (Bolen and Baskakov, 2001). According to them, the thermodynamic cycle requires that ∆GD[Os] - ∆GN[Os] = ∆Gden[Os] -

∆Gden water, and this condition forces the conclusion that denaturation of protein (∆Gden [Os]) is less favorable (more positive quantity) in the presence of osmolyte than it is in water (∆Gden water). Because osmolytes raise the Gibbs energy of the denatured state far more than they do the native state, proteins are more stable in the presence of osmolytes than they are in water.

They obtained transfer Gibbs energy measurements of amino acid side-chains and backbone, from water to various osmolyte solutions, in order to determine the propensities of the side-chains and backbone to interact with several of naturally occurring osmolytes. They estimated the transfer Gibbs energy of native and denatured protein, ∆GN[Os] and ∆GD[Os], using transfer Gibbs energy data and models of denatured protein developed by Creamer et al. (1997).

The results of their measurements showed that the peptide backbone is responsible for the unfavorable interaction with osmolyte, and that the side chains collectively favour interaction with osmolyte (Liu and Bolen, 1995; Wang and Bolen, 1997; Qu et al., 1998). As suggested by Timasheff and co-workers, the origin of the hydrophobic effect in proteins lies in the unfavorable interaction between apolar side-chains and water. Similarly, Bolen and co-workers proposed that the origin of the hydrophobic effect in proteins lies in the unfavorable interaction between the peptide backbone and an osmolyte. The concept of both these forces is similar because an unfavorable interaction between a structural component of the protein and a component of solution is responsible for the force involved in protein folding.

Bolen and co-workers also suggested that the interactions of protecting osmolytes and urea (denaturant) with protein side-chains are of less importance during denaturation than their respective favorable and unfavorable interactions with the peptide backbone (Bolen and Baskakov, 2001). The principal difference between urea and protecting osmolytes is due to the fact that urea interaction with peptide backbone is favorable and dominant over its favorable interactions with side-chains, while the protecting osmolytes interact unfavorably with backbone but favorably with side-chains (Wang and Bolen, 1997). They concluded that osmolytes (through the osmophobic effect) focus on the peptide backbone, a part of protein separate from side-chains, for protein folding in dilute aqueous medium (Bolen and Baskakov, 2001).

2.2.4 Protein Stabilization at Low Temperatures (Freeze-thawing and

In document Betaine analogues and related compounds for biomedical applications (Page 32-35)