Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK a values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK a values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H þ transport. The pK a values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK a values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK a of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK a near 4.5; the other 23 titrated with elevated pK a values ranging from 5.2-9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.dielectric effect | electrostatics | hydration | pKa | bioenergetics T he transfer of an ion from water into a less polar and polarizable environment, such as the hydrophobic interior of a protein, is energetically unfavorable. Internal charges usually destabilize the folded states of proteins, which is primarily why charged groups are largely excluded from the hydrophobic interior and found instead at the protein-water interface, where they can interact with bulk water (1). Paradoxically, internal ionizable groups in proteins are essential for biological energy transduction. These type of ionizable groups are found in the active sites of enzymes (2), and are necessary for e − transfer and H þ transport in proteins such as ATPase (3) and cytochrome c oxidase (4), for ion homeostasis (5, 6), and for light-activated processes in proteins such as bacteriorhodopsin (7,8). The structural adaptations necessary for proteins to tolerate internal ionizable groups, and the factors that stabilize internal charges, are poorly understood. For this reason, our understanding of fundamental aspects of function and evolution of proteins is still limited, as is our ability to manipulate and design novel enzymes.To examine systematically the capacity of globular proteins to tolerate the presence of buried charges, we measured the pK a of 25 internal glutamic acid resid...

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pKa values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.electrostatics ͉ internal ionizable groups ͉ pKa values ͉ dehydration

Summary The pKa values of internal ionizable groups are usually very different than the normal pKa values of ionizable groups in water. To examine the molecular determinants of pKa values of internal groups, we compared the properties of Lys, Asp and Glu at internal position 38 in staphylococcal nuclease. Lys-38 titrates with a normal or elevated pKa whereas Asp-38 and Glu-38 titrate with elevated pKa values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys-38 side chain makes an ion pair with Glu-122; whereas, in the structure of the L38E variant, the buried carboxyl group of Glu-38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously we showed that the pKa of Lys-38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pKa of Asp-38 and Glu-38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions and structural reorganization as determinants of pKa values of internal groups in proteins. This study suggests that improvements to computational methods for pKa calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.

Summary In response to environmental stress cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. We show further that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical and cellular analyses reveal a new and unexpected function for G proteins as mediators of stress-response signaling.