A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.
Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.
The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2-4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pK(a) value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of alpha-helix. These data suggest that the protein dielectric constant needed to reproduce the pK(a) value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures.
The pKa values measured previously for the internal Lys-66, Asp-66 and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pKa units relative to normal pKa values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of the ionizable moieties in crystal structures. To examine the origins of these high apparent dielectric constants we showed that the pKa of these internal residues are sensitive to the global stability of the protein; the shifts tend to be smaller in less stable forms of nuclease. This implies that the apparent dielectric constants reported by these internal ionizable groups are high because they reflect conformational reorganization coupled to their ionization. To detect this directly, acid/base titrations monitored with Trp fluorescence, near-UV and far-UV CD spectroscopy were performed on variants with Lys-66, Glu-66 or Asp-66 in background proteins with different stability. Conformational reorganization coupled to the ionization of the internal groups was spectroscopically detectable, especially in the less stable background proteins. The data show that to improve the accuracy of structure-based pKa calculations of internal groups the calculations will have to treat explicitly all structural reorganization coupled to ionization. The data also suggest a novel approach to mapping the folding free energy landscape of proteins by using internal ionizable groups to stabilize partially unfolded states.
Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.
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