Wesley E. Stites

High Apparent Dielectric Constants in the Interior of a Protein Reflect Water Penetration

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.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.

Experimental pKa 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.

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amines relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.

MUSTARD GAS CROSSLINKING OF PROTEINS THROUGH PREFERENTIAL ALKYLATION OF CYSTEINES

Mustard gas,bis(2-chloroethyl)sulfide, treatment of proteins is shown to generate significant amounts of covalently crosslinked protein dimers. This is due to the preferential alkylation of cysteine residues. Crosslinking does not occur in the model protein staphylococcal nuclease, which has no cysteine residues. Treatment of cysteine-containing mutants of staphylococcal nuclease with this chemical warfare agent did result in crosslinking. However, these dimers are slowly cleaved back to monomers by an unknown mechanism. The alkylation and crosslinking of cysteine-containing proteins by mustard gas may contribute to its toxicity.

Effects of excluded volume upon protein stability in covalently cross-linked proteins with variable linker lengths.

To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.

Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium.

Abstract Ross, C. C., MacLeod, S. L., Plaxco, J. R., Froude, J. W., Fink, L. M., Wang, J., Stites, W. E. and Hauer-Jensen, M. Inactivation of Thrombomodulin by Ionizing Radiation in a Cell-Free System: Possible Implications for the Radiation Responses in Vascular Endothelium. Radiat. Res. 169, 408–416 (2008). Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4–6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to γ radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.

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.

An electrophoretic mobility shift assay for methionine sulfoxide in proteins.

Study of the posttranslational modification of methionine to its sulfoxide has been receiving increasing attention because of its implication in regulation of protein activity, but techniques for the detection of this modification remain limited. In particular, there has been no method to detect the oxidation of methionine on polyacrylamide gels. Here we demonstrate that alkylation of methionine introduces a charge change that shifts the mobility of the protein on an acidic gel relative to the alkylation-resistant sulfoxide form.

The pH dependence of staphylococcal nuclease stability is incompatible with a three-state denaturation model

Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.2. Within experimental error the values of [Formula: see text] and mGuHCl for the various proteins measured over this wide range of pH maintain a constant offset from one another, tracing a series of approximately parallel curves. This data offers an independent means of determining the error of stabilities and slopes determined by guanidine hydrochloride denaturations and shows that previous error estimates are accurate. More importantly, this behavior cannot be reconciled with a three-state denaturation model for staphylococcal nuclease. The large variations in mGuHCl observed in these mutants must therefore arise from other causes.