Jamie L. Schlessman

Cavities determine the pressure unfolding of proteins

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

Molecular determinants of the pK(a) values of Asp and Glu residues in staphylococcal nuclease.

Prior computational studies of the acid‐unfolding behavior of staphylococcal nuclease (SNase) suggest that the pKa values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pKa values of carboxylic groups in SNase, the pKa values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups. Fourteen Asp and Glu residues titrate with relatively normal pKa values that are depressed by less than 1.1 units relative to the normal pKa of Asp and Glu in water. Only six residues have pKa values shifted by more than 1.5 units. Asp‐21 has an unusually high pKa of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pKa values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pKa values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pKa values; however, no major pH‐sensitive conformational reorganization of the backbone was detected using NMR spectroscopy. Proteins 2009.

Arginine residues at internal positions in a protein are always charged.

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pKa values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pKa values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pKa; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.

The pKa values of acidic and basic residues buried at the same internal location in a protein are governed by different factors

The pK(a) values of internal ionizable groups are usually very different from the normal pK(a) values of ionizable groups in water. To examine the molecular determinants of pK(a) values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK(a), whereas Asp38 and Glu38 titrate with elevated pK(a) values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK(a) of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK(a) values of Asp38 and Glu38 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 pK(a) values of internal groups in proteins. This study suggests that improvements to computational methods for pK(a) calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.

A buried lysine that titrates with a normal pKa: Role of conformational flexibility at the protein–water interface as a determinant of pKavalues

Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pKa values shifted by up to 5 pKa units in the direction that promotes the neutral state. In contrast, the internal Lys‐38 in SNase titrates with a normal pKa. The crystal structure of the L38K variant shows that the side chain of Lys‐38 is buried. The ionizable moiety is ∼7 Å from solvent and ion paired with Glu‐122. This suggests that the pKa value of Lys‐38 is normal because the energetic penalty for dehydration is offset by a favorable Coulomb interaction. However, the pKa of Lys‐38 was also normal when Glu‐122 was replaced with Gln or with Ala. Continuum electrostatics calculations were unable to reproduce the pKa of Lys‐38 unless the protein was treated with an artificially high dielectric constant, consistent with structural reorganization being responsible for the normal pKa value of Lys‐38. This reorganization must be local because circular dichroism and NMR spectroscopy indicate that the L38K protein is native‐like under all conditions studied. In molecular dynamics simulations, the ion pair between Lys‐38 and Glu‐122 is unstable. The simulations show that a minor rearrangement of a loop is sufficient to allow penetration of water to the amino moiety of Lys‐38. This illustrates both the important roles of local flexibility and water penetration as determinants of pKa values of ionizable groups buried near the protein–water interface, and the challenges faced by structure‐based pKa calculations in reproducing these effects.

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease.

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK(a) values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK(a) values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK(a) values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK(a) was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N(epsilon2) state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK(a) calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK(a) values are governed by short-range polar and Coulomb interactions.

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.

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.

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.

The 2/2 hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 with covalently attached heme: Comparison of X‐ray and NMR structures

The X‐ray structures of the hemoglobin from Synechococcus sp. PCC 7002 (GlbN) were solved in the ferric bis‐histidine (1.44 Å resolution) and cyanide‐bound (2.25 Å resolution) states with covalently attached heme. The two structures illustrate the conformational changes and cavity opening caused by exogenous ligand binding. They also reveal an unusually distorted heme, ruffled as in c cytochromes. Comparison to the solution structure demonstrates the influence of crystal packing on several structural elements, whereas comparison to GlbN from Synechocystis sp. PCC 6803 shows subtle differences in heme geometries and environment. The new structures will be instrumental in elucidating GlbN reactivity. Proteins 2014; 82:528–534.