Jan M. Antosiewicz

Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pKa values are known. Mean pKa values for aspartates and glutamates are ≤ 3.4 (±1.0) and 4.1 (±0.8), respectively. For aspartates, mean pKa values are 3.9 (±1.0) and 3.1 (±0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pKa values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pKa values: pKa values for solvent‐exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pKa values and these correlations are not improved by including simple surface‐area‐based terms to account for the effects of desolvation. Mean aspartate pKa values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pKa values are > 5.5 and most are found in active sites or ligand‐binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N‐termini of helices have mean pKa values of 2.8 (±0.5) and 3.4 (±0.6), respectively, about 0.6 units less than the overall mean values. Proteins 2002;48:388–403.

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI = 3.5) and a basic variant (pI = 10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulombs Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulombs Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.

pK Values of Histidine Residues in Ribonuclease Sa: Effect of Salt and Net Charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.

ELECTROSTATICS OF HEMOGLOBINS FROM MEASUREMENTS OF THE ELECTRIC DICHROISM AND COMPUTER SIMULATIONS

Hemoglobins from normal human cells, from sickle cells, and from horse were investigated by electrooptical methods in their oxy and deoxy forms. The reduced linear dichroism measured as a function of the electric field strength demonstrates the existence of permanent dipole moments in the range of 250-400 Debye units. The reduced limiting dichroism is relatively small (< or = 0.1); it is negative for hemoglobin from sickle cells and positive for the hemoglobins from normal human cells and from horse. The dichroism decay time constants are in the range from about 55 to 90 ns. Calculations of the electrooptical data from available crystal structures are given according to models of various complexity, including Monte Carlo simulations of proton fluctuations with energies evaluated by a finite difference Poisson-Boltzmann procedure. The experimental dipole moments are shown to be consistent with the results of the calculations. In the case of human deoxyhemoglobin, the root mean square dipole is higher than the mean dipole by a factor of about 4.5, indicating a particularly large relative contribution due to proton fluctuations. The ratio of the root mean square dipole to the mean dipole is much smaller (approximately 1.1 to approximately 1.5) for the other hemoglobin molecules. The calculations demonstrate that the dichroism decay time constants are not simply determined by the size/shape of the proteins, but are strongly influenced by the orientation of the dipole vector with respect to the axis of maximal absorbance. The comparison of experimental and calculated electrooptical data provides a useful test for the accuracy of electrostatic calculations and/or for the equivalence of structures in crystals and in solutions.

Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

Abstract The methyl ester of m 7 G 5′ p was synthesized by a carbodiimide-catalyzed reaction of G 5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m 7 Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m 7 G 5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m 7 G 5′ p than to m 7 Gp · methyl ester. Conversion of the esterified analog to m 7 G 5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m 7 G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.

Prediction of Secondary Ionization of the Phosphate Group in Phosphotyrosine Peptides

A computational approach, based on a continuum molecular electrostatics model, for the calculation of the pK(a) values of secondary ionization of the phosphate group in phenyl phosphate derivatives is described. The method uses the ESP atomic charges of the mono-anionic and di-anionic forms of the ionizable phosphate group, computed with the use of the density functional method, and applies a new concept of the model group, being the reference state for the pK(a) calculations. Both conformational flexibility and tautomeric degrees of freedom are taken into account in the calculations. The method was parameterized using experimentally available pK(a) values of four derivatives of phenyl phosphates, and phosphotyrosine. Subsequently this parameterization was used to predict pK(a) of the phosphate group in a short peptide Gly-Gly-Tyr(P)-Ala, and in a longer peptide consisting of 12 residues, the latter in water, and in a complex with a protein-phospholipase. The agreement between the computed and the experimental pK(a) values is better than +/-0.3 pH units for the optimized solute dielectric constant of 11-13. This approach is promising and its extension to other phospho-amino acids is in progress.

UV–Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications

In Part 2 we discuss application of several different types of UV–Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

Poisson-Boltzmann continuum-solvation models: applications to pH-dependent properties of biomolecules.

All molecules can be viewed as either discrete or continuous assemblies of electric charges, and electrostatics plays a major role in intermolecular and intramolecular interactions. Moreover, charge distribution within molecules may fluctuate due to the presence of ionizable groups capable of exchanging protons with the environment, leading to pH-dependence of phenomena involving such molecules. Electrostatic aspects of complex shapes and environments of biological molecules, in vitro and in vivo, are relatively well amenable to treatment by Poisson-Boltzmann models, which are attractive in that they possess a clear physical meaning, and can be readily solved by several mathematically sound methods. Here we describe applications of these models to obtain valuable insights into some biologically important pH-dependent properties of biomolecules, such as stability, binding of ligands (including potential drugs), enzymatic activity, conformational transitions, membrane transport and viral entry.

Hydration of alcohols by ultrasonic velocity measurements in ternary systems

Ultrasonic velocity maxima have been determined as a function of alcohol (methanol, ethanol, n-propanol or t-butanol) concentration in ternary aqueous alcoholic solutions of a number of mono- and polyhydroxyalcohols. The location of the maxima relative to those in the binary water-alcohol systems are related to the hydration of the solute molecule. It is established that correlations between the extent of hydration and some structural parameters of the solute molecules are of the same character in all binary solvents studied.

Poisson-Boltzmann model studies of molecular electrostatic properties of the cAMP-dependent protein kinase.

Abstract Protonation equilibria of residues important in the catalytic mechanism of a protein kinase were analyzed on the basis of the Poisson-Boltzmann electrostatic model along with a cluster-based treatment of the multiple titration state problem. Calculations were based upon crystallographic structures of the mammalian cAMP-dependent protein kinase, one representing the so called closed form of the enzyme and the other representing an open conformation. It was predicted that at pH 7 the preferred form of the phosphate group at the catalytically essential threonine 197 (P-Thr197) in the closed form is dianionic, whereas in the open form a monoanionic ionization state is preferred. This dianionic state of P-Thr197, in the closed form, is stabilized by interactions with ionizable residues His87, Arg165, and Lys189. Our calculations predict that the hydroxyl of the Ser residue in the peptide substrate is very difficult to ionize, both in the closed and open structures of the complex. Also, the supposed catalytic base, Asp166, does not seem to have a pKa appropriate to remove the hydroxyl group proton of the peptide substrate. However, when Ser of the peptide substrate is forced to remain ionized, the predicted pKa of Asp166 increases strongly, which suggests that the Asp residue is a likely candidate to attract the proton if the Ser residue becomes deprotonated, possibly during some structural change preceding formation of the transition state. Finally, in accord with suggestions made on the basis of the pH-dependence of kinase kinetics, our calculations predict that Glu230 and His87 are the residues responsible for the molecular pKa values of 6.2 and 8.5, observed in the experiment.