Conformational consequences of ionization of Lys, Asp, and Glu buried at position 66 in staphylococcal nuclease.

The pK(a) 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 pK(a) units relative to normal pK(a) 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 pK(a) values 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 and near-UV and far-UV CD spectroscopy were performed on variants with Lys-66, Glu-66, or Asp-66 in background proteins with different stabilities. 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 pK(a) 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.

Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups.

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.

High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp.

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.

Human monoclonal antibody stability and activity at vaginal pH.

Antibodies can be delivered topically to the vagina to protect against pregnancy and sexually transmitted infections, but the acidity of vaginal secretions (pH 3.5-4.5) might inactivate them. To address this question, both experimental and computational methods were used to evaluate the effects of pH on human monoclonal antibody (MAb) stability and activity. To determine the acid-sensitivity of their antigen binding sites, human MAbs against human sperm (H6-3C4) and gp120 of HIV (1511) were tested by ELISA for binding to human sperm and recombinant gp120, respectively, at pH 3.0-7.0, after storing them for 1 or 20 h at the same pH. Binding was unaltered by acidic pH> or =4 even after 20 h, and at pH 3.5 both MAbs retained > or =40% antigen binding activity. A humanized MAb against HSV-2 glycoprotein B expressed both in Chinese hamster ovary (CHO) cells and in soybean cells was incubated for 1 or 24 h at pH 3.5-7.6, brought to neutral pH, and tested for ability to block HSV-2 infection of foreskin fibroblast cells. Loss in blocking activity occurred only when antibodies were incubated at pH 3.5 for 24 h and was independent of the expression cell type. Using empirical structure-based methods, net charge, Z, and electrostatic contributions to free energy, DeltaDeltaG(el), were calculated as a function of pH for 1 human and 8 murine F(ab)s. The calculations indicate that Z changes slowly between pH 5.0 and 9.0 and that DeltaDeltaG(el) is nearly constant between pH 4.0 and 10 for all the F(ab)s and, therefore, human antibodies should remain stable in this pH range. Taken together, our data and empirical calculations suggest that vaginally applied human MAbs are likely to remain stable and active throughout the duration they are likely to reside in the vagina.

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

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.