Aaron C. Robinson

High tolerance for ionizable residues in the hydrophobic interior of proteins

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.

Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Significance Charges buried in hydrophobic environments in proteins play essential roles in energy transduction. We engineered an artificial ion pair in the hydrophobic core of a protein to demonstrate that buried ion pairs can be charged and stabilized, in this instance, by a strong Coulomb interaction worth 5 kcal/mol. Despite this interaction, the buried charge pair destabilized the folded protein because the Coulomb interaction recovered the energetic penalty for dehydrating only one of the two buried charges. Our results suggest how artificial active sites can be engineered in stable proteins without the need to design or evolve specialized structural adaptations to stabilize the buried charges. Minor structural reorganization is sufficient to mitigate the deleterious consequences of charges buried in hydrophobic environments. An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pKa values near 7. When buried together their pKa values appear to be normal. The ionizable moieties of the buried Glu–Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu–Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu–Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins.

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H(+) binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK(a) values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK(a) values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK(a) values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.

Charges in Hydrophobic Environments: A Strategy for Identifying Alternative States in Proteins

In the V23E variant of staphylococcal nuclease, Glu-23 has a pKa of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands β1 and β2 separate from the β-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent pKa of an internal ionizable group can reflect the average of the very different pKa values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the pKa value of internal ionizable group reflects conformational reorganization. (3) The apparent pKa of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict pKa values of internal residues and to reproduce pH-driven conformational transitions of proteins.

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15N-Detect NMR Spectroscopy

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.