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Biology LibreTexts

Ion - Ion (Electrostatics) in Proteins

These could be investigated by altering pH or ionic strength. Why is that?

a. General Charge Interactions - Proteins denature at extremes of pH. At these extremes, proteins have a maximal positive or negative charge, as evident in graphs which show denaturation temperature vs pH for proteins.

Figure: Denaturation temperature vs pH for proteins

02tmvsph.gif

Electrostatic repulsions would cause the protein to denature. The folded, compact state has an increasing charge density at pH extremes, which could be alleviated by unfolding to a less dense state. But what about specific charge pair interactions? In contrast to the general charge interactions, these might actually stabilize a protein. Are they the predominant factor that determines stability?

b. Specific Charge Interactions (charge pairs) - If ion pairs are the source of protein stability, you would expect that high salt could disrupt them, and lead to denaturation. Although some salts do denature proteins, others stabilize them. Other evidence argues against this idea. Ion pairs are not conserved in evolution. In addition, the number of ion pairs in proteins is small (approx. 5/150 residues, with one of those buried). Also, the stability of a protein shows little dependence on pH or salt concentration (at low concentrations) near the isoelectric point, the pH at which proteins have a net zero charge.

The overall charge state affects not only the stability of a protein but also its solubility. Proteins are most insoluble at their isoelectric point since at that pH (where they have a net 0 charge) the proteins experience the least electrostatic repulsion and are most likely to aggregate and precipitate. Low salt concentration also promotes insolubility. Mutagenesis studies show that solubility can be increased by replacing nonpolar groups on the surface with polar ones. Pace (2009) cites studies on RNase Sa which has a maximally exposed Thr 76. If it was replaced with Asp, the solubility increased to 43 mg/ml but if it was replaced with Trp, it decreased to 3.6 mg/ml. His, Asn, Thr and Gln have a negative effect on solubility near the pI compared to Ala, a surprising result. Similar results were obtained compared to Ala when Arg and Lys were used. Smaller side chains, Asp and Ser, at position 76 increased the solubility over Ala.

The next two sections deal with H bonding and the hydrophobic effect. A theme of the course is that if you can understand the interactions among small molecules, you can apply that knowledge to the understanding of larger molecules like proteins. To understand if H bonds within proteins, often buried in the more hydrophobic interior of the protein, drive protein folding, we will examine the thermodynamics of H bond formation of a small molecule, N-methylacetamide, in water and in a nonpolar solvent. To understand if the hydrophobic effect, mediated by burying of nonpolar side chains within the more nonpolar center of the protein, drives protein folding, we will examine the thermodynamics of benzene solubility in water.