Michael S. Chimenti

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.

Direct Evidence for Deprotonation of a Lysine Side Chain Buried in the Hydrophobic Core of a Protein

We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear 1H-15N NMR data on the Lys-66 side chain amine (Nzeta) group in the delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nzeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hzeta and 15Nzeta chemical shifts at pH 8.0 and 6 degrees C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH3+) state. Further investigations using a model system suggested that the abnormal 1H and 15N chemical shifts represent the deprotonated (NH2) state of the Lys-66 Nzeta group. More straightforward evidence for the deprotonation was obtained with 2D F1-1H-coupled 1H-15N heteronuclear correlation experiments. Observed 15N multiplets clearly indicated that the spin system for the Lys-66 Nzeta group is AX2 (NH2) rather than AX3 (NH3+). Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 Nzeta group was found to be relatively rapid (93 s(-1) at -1 degrees C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on 15Nzeta multiplets due to the rapid hydrogen exchange is also discussed.

Structural origins of high apparent dielectric constants experienced by ionizable groups in the hydrophobic core of a protein.

The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pK(a) values of 5.7, 8.8, and 8.9, respectively. To reproduce these pK(a) values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pK(a) calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.

High-Pressure SAXS Study of Folded and Unfolded Ensembles of Proteins

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.

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 Plasticity of Staphylococcal Nuclease Probed by Perturbation with Pressure and pH

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X‐ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys‐66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys‐66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys‐66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys‐66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys‐66 is located. Above 2 kbar pressure at these pH values the protein with Lys‐66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys‐66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys‐66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure. Proteins 2011.

Strong Coulomb Interactions Between Internal and Surface Charges in Proteins

Internal ionizable groups in proteins are essential for many biological processes. The molecular determinants of their pKa values are poorly understood. To examine this problem, we measured previously the pKa values of Lys, Arg, Asp and Glu at 25 internal positions in staphylococcal nuclease. The pKa values are usually shifted substantially (as many as 6 pKa units) in the direction that favors the neutral state because dehydration experienced by the ionizable groups in their buried positions is not compensated by interactions with polar or charged groups. A subset of variants with internal Lys residues showed evidence of interaction of the internal Lys with surface carboxylic groups. Using NMR spectroscopy and crystallography we have shown that internal Lys residues can have significant Coulomb interactions with surface Asp and Glu. Upon ionization the internal Lys residue remains internal and the proteins remain fully folded. The interactions between the internal Lys and surface carboxylic groups are long range (6.3 A or more between charged atoms) and are mostly through protein. Long-range pairwise interactions as high as 2 kcal/mol have been measured. In some cases strong effects are governed by the sum of many weaker long-range interactions that cannot be decomposed experimentally into pairwise contributions. Overall, there is strong evidence that surface carboxylic groups stabilize the charged form of many internal Lys residues. The experimentally measured Coulomb interaction energies between internal and surface charges constitute invaluable constraints for benchmarking of structure-based electrostatics calculations. They also suggest a strategy for modifying the pKa value of active site residues by the engineering of surface charges.