Shifted pKa Values Research Articles

Atomistic Molecular Dynamics Simulations of Drosophila Orai in a Hydrated Lipid Bilayer

Orai channels are prominent (calcium) Ca2+ signal mediators in many cell types. In T lymphocytes, Orai1 and STIM1 form the Ca2+ release-activated Ca2+ (CRAC) channel. Following endoplasmic reticulum (ER) Ca2+ store depletion, ER membrane-resident Ca2+ sensor STIM1 physically interacts with plasma membrane-bound, pore-forming Orai1 subunits. The STIM1-Orai1 interaction results in Ca2+ influx and ultimately changes in gene expression that underlie the immune response. In 2012, the Drosophila Orai (dOrai) crystal structure was solved to 3.34 A resolution. To develop an atomistic view of Orai channel function, given the high sequence identity (73%) between dOrai and human Orai1, we constructed a complete dOrai structure by building in flexible loops missing in the crystal structure and adjusting the protonation state of several pore residues. The model was placed into a lipid bilayer with excess hydration and simulated for over 200 ns. We find shifted pKa values for several titratable residues in the pore interior, and that changing protonation states of these key residues is essential to maintain pore stability. To explore the mechanism of channel block, we also simulated the model with either Ca2+ or (gadolinium) Gd3+ in the selectivity filter. Taken together, our results help to address the stability of the closed structure in a lipid bilayer while also yielding novel atomistic insights into channel block and pH sensitivity.This work was supported by NIH grants F30CA171717 to M.L.W., P01GM86685 to D.J.T., and NS-14609 to M.D.C.1. Hou, X, et al. Science. (2012); 338; 1308-13.

Substituent Effects and pH Profiles for Stability Constants of Arylboronic Acid Diol Esters

Stability constants of boronic acid diol esters in aqueous solution have been determined potentiometrically for a series of meta-, para-substituted phenylboronic acids and diols of variable acidity. The constants β(11-1) for reactions between neutral forms of reactants producing the anionic ester plus proton follow the Hammett equation with ρ depending on pKa of diol and varying from 2.0 for glucose to 1.29 for 4-nitrocatechol. Observed stability constants (K(obs)) measured by UV-vis and fluorometric titrations at variable pH for esters of 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron) generally agree with those expected on the basis of β(11-1) values, but the direct fitting of K(obs) vs pH profiles gives shifted pKa values both for boronic acids and diol as a result of significant interdependence of fitting parameters. The subsituent effects on absorption and fluorescence spectra of Tiron arylboronate esters are characterized. The K(obs) for Tiron determined by (11)B NMR titrations are approximately 1 order of magnitude smaller than those determined by UV-vis titrations under identical conditions. A general equation, which makes possible an estimate of β(11-1) for any pair of boronic acid and diol from their pKa values, is proposed on the basis of established Brönsted-type correlation of Hammett parameters for β(11-1) with acidity of diols. The equation allows one to calculate stability constants expected only on basis of acid-base properties of the components, thus permitting more strict evaluation of contributions of additional factors such as steric or charge effects to the ester stability.

Backbone Conformational Fluctuations as Determinants of pKa Values of Surface Residues in Proteins

The pKa values of surface ionizable residues in proteins are usually similar to the pKa values of ionizable residues in water. This is still difficult to reproduce using structure-based electrostatics calculations with static structures, which tend to exaggerate the shifts in pKa values because the magnitude of Coulomb effects is exaggerated and the balance between Coulomb and dehydration effects is not reproduced correctly. These problems can be minimized by using artificially high protein dielectric constants, but sometimes the discrepancy persists even when the protein interior is treated with the dielectric constant of water. This suggests that pKa values are affected by the conformational dynamics of the protein, which are not reflected in the crystal structure. Molecular dynamics or Monte Carlo simulations can be used to attempt to reproduce these dynamic effects implicitly, but there are no useful data for testing this approach directly. To examine the role of fluctuations of the backbone in determining pKa values, NMR spectroscopy was used to measure the pKa values of all 20 Asp and Glu residues in variants of staphylococcal nuclease with Gly substituted at select locations. These substitutions were intended to enhance backbone fluctuations without affecting the overall protein structure. Some Gly substitutions detectably shifted the pKa values of some carboxylic groups. Crystal structures of these variants showed no significant changes relative to the structure of the reference protein. Calculations with continuum methods using these crystal structures do not reproduce the measured shifts in pKa values. NMR spectroscopy studies suggest Gly substitutions affect the dynamics of the protein. Efforts are underway to better characterize these changes. Our results suggest that the high apparent polarizability of proteins might be due to subtle structural fluctuations that are difficult to reproduce computationally.

Dynamics and Oligomerization Tune Seven-Transmembrane Protein Function: Studies Based on Proteorhodopsin

Seven-transmembrane (7TM) proteins have diverse and important functions, ranging from signaling receptors to ion pumps. They share a reversible switching property, epitomized by the solar-powered microbial proton pump Proteorhodopsin (PR), which uses light energy to facilitate transport by a conformational “switch”. Here, we use PR as a model to capture the elusive details of activation and oligomerization necessary for the function of physiologically important membrane proteins.We have preliminary spectroscopic evidence that suggests altered photocycle kinetics and shifted pKa values for hexameric and monomeric forms of detergent-solubilized PR. These findings prove that PR function is tuned by self-association, and we apply magnetic resonance together with functional assays of proton transport to further understand dynamics changes that occur upon oligomerization. Our unique magnetic resonance techniques of electron paramagnetic resonance (EPR) and dynamic nuclear polarization (DNP) provide insight into the protein segment mobility and local hydration water dynamics of an amino acid residue spin-labeled with nitroxide-based radicals.Using these methods, we have found that PR's third cytoplasmic (E-F) loop is a short α-helical segment that experiences conformational change upon photoactivation. This structure is a common motif to the non-homologous G-protein coupled receptor bovine rhodopsin (Rh), where it is a docking point for a signal G-protein. Towards understanding how function hinges on dynamics, we developed a PR-Rh chimera by replacing the E-F loop of PR with the corresponding loop of Rh. The chimera successfully expresses and maintains optical properties. We evaluate its capability to activate the G-protein transducin, and apply EPR and DNP to obtain unique information about the biophysics of receptor/G-protein interactions. By controlling the oligomeric form of the PR-Rh chimera, we measure any changes in G-protein activation caused by varying the amount of receptor-receptor interactions.

Structural Consequences of the Ionization of Internal Lys Residues in a Protein

Internal ionizable residues in proteins play important functional roles in a variety of biological processes. The molecular determinants of their pKa values are poorly understood. Previously we measured the pKa of Lys, Asp, Glu and Arg at 25 internal groups in staphylococcal nuclease. 98 of these 100 variants are fully folded and native-like at pH 7. The pKa values of the majority of these groups are perturbed, some by as much as 5 pKa units, all in the direction that favors the neutral state. NMR spectroscopy was used to examine the structural and dynamic consequences of ionization of the internal lysine residues. In 9 of 10 crystal structures of Lys-containing variants the Lys side chain is completely buried, some in entirely hydrophobic microenvironments. In some cases the buried amino group makes contact with polar residues. The NMR experiments showed that in two variants the ionization of an internal Lys causes global unfolding. In five variants the ionization of the internal Lys triggers local structural changes and increased dynamics. The presence of conformational exchange in response to ionization appears to be correlated with the global stability of the variant proteins. Surprisingly, in the majority of cases, the changes in structure coupled to the ionization of the internal Lys residues are modest. These data demonstrate that proteins can tolerate internal ionizable residues, even those that exhibit large shifts in pKa values, and even in their charged states. The internal charged groups somehow manage to become solvated without disrupting the overall fold of the protein.

Prediction and Rationalization of the pH Dependence of the Activity and Stability of Family 11 Xylanases

This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of 2-4.6. The influence of dynamical fluctuations of the protein structure is investigated for Bacillus agaradhaerens and Aspergillus kawachii xylanase using molecular dynamics (MD) simulations to provide snapshots from which average values can be computed. This decreases the respective predicted pH optima from 6.2-6.7 and 4.8 to 5.3 +/- 0.3 and 4.0 +/- 0.2, respectively, which are in better agreement with the observed values of 5.6 and 2, respectively. The change is primarily due to structural fluctuations of an Arg residue near the catalytic nucleophile, which lowers its pKa value compared to using the X-ray structure. The MD simulations and some X-ray structures indicate that this Arg residue can form a hydrogen bond to the catalytic base, and it is hypothesized that this hydrogen bond is stabilized by an additional hydrogen bond to another Glu residue present only in acidic xylanases. Formation of such a hydrogen bond is predicted to lower the pH optimum of A. kawachii xylanase to 2.9 +/- 0.3, which is in reasonable agreement with the observed value of 2. The predicted pH of optimal stability is in excellent agreement with the pH value at which the melting temperature (Tm) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and Tm, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pKa values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pKa values significantly lower than standard values.

Electrostatic Contributions to Residue-Specific Protonation Equilibria and Proton Binding Capacitance for a Small Protein

Charge-charge interactions in proteins are important in a host of biological processes. Here we use 13C NMR chemical shift data for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). Carbon chemical shifts are dominated by changes in the electron distribution within the side chain and therefore excellent reporters of the charge state of individual groups, and the data are of high precision. We demonstrate that it is possible to detect local charge interactions within this small protein domain that stretch and skew the chemical shift titration curves away from "ideal" behavior and introduce a framework for the analysis of such convoluted data to study local charge-charge interactions and electrostatic coupling. It is found that, due to changes in electrostatic potential, the proton binding affinity, Ka, of each carboxyl group changes throughout the titration process and results in a linearly pH dependent pKa value. This result could be readily explained by calculations of direct charge-charge interactions based on Coulomb's law. In addition, the slope of pKa versus pH was dependent on screening by salt, and this dependence allowed the selective study of charge-charge interactions. For PGB1-QDD, it was established that mainly differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pKa values within the protein environment.

Oxidation of Sb(III) to Sb(V) by O 2 and H 2O 2 in aqueous solutions

The rates of Sb(III) oxidation by O 2 and H 2O 2 were determined in homogeneous aqueous solutions. Above pH 10, the oxidation reaction of Sb(III) with O 2 was first order with respect to the Sb(III) concentration and inversely proportional to the H + concentrations at a constant O 2 content of 0.22 × 10 −3 M. Pseudo-first-order rate coefficients, k obs, ranged from 3.5 × 10 −8 s −1 to 2.5 × 10 −6 s −1 at pH values between 10.9 and 12.9. The relationship between k obs and pH was: log ⁡ k o b s = 1.0 ( ± 0.3 ) p H − 18.5 ( ± 3.4 ) R 2 = 0.89 No significant Sb(III) oxidation by O 2 was observed between pH 3.6 and 9.8 within 200 days. Oxidation of Sb(III) by H 2O 2 was found to be first order with respect to the total Sb(III), H 2O 2 and inversely proportional to the H + concentrations in a pH range of 8.1 to 11.7. Above pH 11.7 no pH dependence was observed. The rate law for the pH range 8.1 to 12.9 was determined to be: − d [ S b ( I I I ) ] tot d t = k × [ S b ( I I I ) ] tot × [ H 2 O 2 ] tot and k = k 3 × K 1 K a 1 + [ H + ] = k 4 × K 2 K a 2 + [ H + ] where K a1 and K a2 are the deprotonation constants of Sb(OH) 3 and H 2O 2, respectively, and k 3 (=365 M −1 s −1) and k 4 (=342 M −1 s −1) are the specific second-order rate coefficients. The results indicate that the rate-limiting step below pH 11.7 involves one deprotonated species, either Sb(OH) 4 − or HO 2 −. Since the pK a-values of Sb(OH) 3 and H 2O 2 are very close (11.8 and 11.6, respectively), it was not possible to determine which species is involved. Varying the ionic strength between 0.001 and 1.0 M at pH 10 and between 0.01 and 1.0 M at pH 12 resulted in a less than twofold increase in k obs. In both cases, the increase was attributed to a shift in pK a-values as a function of the ionic strength. It could be concluded that O 2 was unlikely to be a significant oxidant in homogenous solution, but that H 2O 2 might be responsible for the oxidation of Sb(III) in natural waters since half-lives could vary from 32 yr to 117 days at pH 8 with H 2O 2 concentrations between 10 −8 M and 10 −6 M, respectively.

The pH dependent adsorption of Coumarin 343 at the water/dichloroethane interface

The surface sensitive technique of optical second harmonic generation (SHG) and quasi-elastic laser scattering (QELS) have been applied to study the effect of pH on the adsorption of Coumarin 343 (C343) dye molecules at the water/1, 2-dichloroethane (DCE) interface. A pH dependent aggregation is observed for C343 molecules adsorbed at the interface. The surface spectrum shows that at pH 8 and lower, C343 adsorb at the interface in J-aggregated protonated form. At pH 9 and pH 10, C343 adsorb in both protonated and deprotonated forms at the interface, whereas, at pH 11, C343 is in H-aggregated-deprotonated form at the interface. The observed large shift in pKa value of C343 at the interface is attributed to the intramolecular hydrogen bonding along with the aggregation of dye molecules. From a light polarization analysis of the SHG signal, the average angle of orientation of C343 at pH 5 was determined to be 30°, and at pH 11, the angle was determined to be 42°. The QELS data show a weak adsorption of C343 at the interface for pH 11 and pH 3 and a strong adsorption at the intermediate pH values, reaching maximum for pH 10, and are consistent with SHG data. The calculated ratio of β values for protonated and deprotonated forms of C343, is in agreement with the experimentally measured ratio of β values. Both, experimental and calculated β values for the deprotonated form is lower than the β values for protonated one and is attributed to the electron-donating effect of the –COO− substituent.

Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA.

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.

PH titration studies of an SH2 domain-phosphopeptide complex: unusual histidine and phosphate pKa values.

Electrostatic interactions in a complex of the phospholipase C-gamma 1 C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the beta platelet-derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His beta D4) and the phosphotyrosine (pTyr) phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pKa of His beta D4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.

Nanometric design of extraordinary hydrophobic‐induced pKa shifts for aspartic acid: Relevance to protein mechanisms

Commonly a key element enabling proteins to function is an amino acid residue or residues with functional side chains having shifted pKa values. This article reports the results on a set of protein-based polymers (model proteins) that exhibit hydrophobic folding and assembly transitions, and that have been designed for the purpose of achieving large hydrophobic-induced pKa shifts by selectively replacing Val residues by Phe residues. The high molecular weight polypentapeptides, actually poly(tricosapeptides) with six varied pentamers in fixed sequence, were designed and synthesized to have the same amino acid compositions but different proximities between a single aspartic acid residue and 5 Phe residues per 30 residues. With the 5 Phe residues distal from the Asp residue, the observed pKa shift was 2.9 when compared to the Val-containing reference. With the 5 Phe residues within 1 nm of the Asp residue, the pKa shift was 6.2. This represents a free energy of interaction of 8 kcal/mole. To our knowledge, this is the largest pKa shift documented for an Asp residue in a polypeptide- or protein-water system. Data are reviewed that do not support the usual electrostatic arguments for pKa shifts of charge-charge repulsion and/or unfavorable ion self-energies arising from displacement of water by hydrophobic moieties, but rather the data are interpreted to indicate the presence of an apolar-polar repulsive free energy of hydration, which results from a potentially highly cooperative competition between apolar and polar species for hydration.