Charges in Hydrophobic Environments in Proteins

Abstract

Ionizable groups buried in the hydrophobic interior of proteins play central roles in a wide variety of essential biochemical processes. Owing to the inherent differences in the dielectric properties of water and of protein, buried ionizable groups usually titrate with anomalous pKa values shifted relative to normal pKa values in water in the direction that favors the neutral state. The determinants of these unusual pKa values are being examined systematically with a family of variants of staphylococcal nuclease with Lys, Arg, His, Asp and Glu in 25 internal positions. Some of these ionizable groups titrate with normal pKa values, but the majority of them have anomalous pKa values, some shifted by as many as 6 units relative to the normal values. Dozens of crystal structures confirm that the ionizable groups are buried, at least when they are in the neutral state, often in complex with water molecules never before observed at internal locations in SNase. The structures show that the microenvironments of the ionizable groups range between the fully hydrophobic to the highly polar, but they do not fully explain the unusual properties of these groups. NMR spectroscopy has been used to examine the range of structural reorganization that can be coupled to the ionization of the internal ionizable. While the ionization of some residues is not easily detected in HSQC spectra, in most cases there is clear evidence of modest charge-induced changes in local structure or increased conformational fluctuations. Interactions between internal groups and polar groups, surface charges, and other internal groups have also been measured. These data constitute an extensive, quantitative description of origins of the apparent dielectric properties of proteins. They also show that proteins tolerate internal charges without the need for any special structural adaptations.