Derivation of molecular pK values from pH-dependences.
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ABSTRACT:
SUBMITTER: Dixon HB
PROVIDER: S-EPMC1186362 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:Ionisation equilibria in proteins are influenced by conformational flexibility, which can in principle be accounted for by molecular dynamics simulation. One problem in this method is the bias arising from the fixed protonation state during the simulation. Its effect is mostly exhibited when the ionisation behaviour of the titratable groups is extrapolated to pH regions where the predetermined protonation state of the protein may not be statistically relevant, leading to conformational sampling that is not representative of the true state. In this work we consider a simple approach which can essentially reduce this problem. Three molecular dynamics structure sets are generated, each with a different protonation state of the protein molecule expected to be relevant at three pH regions, and pK calculations from the three sets are combined to predict pK over the entire pH range of interest. This multiple pH molecular dynamics approach was tested on the GCN4 leucine zipper, a protein for which a full data set of experimental data is available. The pK values were predicted with a mean deviation from the experimental data of 0.29 pH units, and with a precision of 0.13 pH units, evaluated on the basis of equivalent sites in the dimeric GCN4 leucine zipper.
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Project description:Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent (13)C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK(a) values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.
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Project description:Five 8-(4-R-phenyl)-1-naphthol derivatives were prepared by PdCl(2)-catalysed electrophilic aromatic substitution. The pK(a)' values for these 1,8-disubstituted arene naphthols have been measured in acetonitrile/water (R = NO(2), 8.42; R = Cl, 8.52; R = H, 8.56; R = Me 8.68; and R = OMe, 8.71) and indicate a correlation with the electronic nature of the arene substituent, as determined through LFER analysis. Contributions to the relative pK(a)' values have been interpreted, using M06-2X DFT calculations, as consisting of two components: A small contribution from initial OH-? bonding in the starting materials and a larger contribution from anion-? interactions in the products. Such effects have implications for a range of other systems.
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Project description:Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pK(a) shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that (13)C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.
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Project description:An acid, HnA, with n ionizing groups is known to have the same titration curve as an equimolar mixture of n hypothetical monobasic acids, whose dissociation constants are known as the 'titration constants' of the real acid. We show that the pH-dependence of any property of HnA is also represented by the sum of one-site titration curves, characterized by these same titration constants. Since one such property is the degree of dissociation of one of the dissociating groups, a fraction of each group shows each of the various titration pK values, so that the group partitions among them. The n groups therefore share the same n titration pK values but differ in the fractions belonging to each. The one H+ ion per molecule that titrates with each pK is thus made up of the fractions, one from each group, that share this pK value. A group may possess a single pK value, in that it contributes virtually all of this pK and almost nothing to the others, only if either (1) in titrates in a different pH range from the other groups or (2) its affinity for H+ is unaffected by their ionization state.
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Project description:Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.
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Project description:Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles.
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Project description:Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production.
2024-01-08 | PXD046499 | Pride
Project description:Knowledge of the possible ionization states of a pharmaceutical substance, embodied in the pK(a) values (logarithm of the acid dissociation constant), is vital for understanding many properties essential to drug development. We compare nine commercially available or free programs for predicting ionization constants. Eight of these programs are based on empirical methods: ACD/pK(a) DB 12.0, ADME Boxes 4.9, ADMET Predictor 3.0, Epik 1.6, Marvin 5.1.4, Pallas pKalc Net 2.0, Pipeline Pilot 5.0, and SPARC 4.2; one program is based on a quantum chemical method: Jaguar 7.5. We compared their performances by applying them to 197 pharmaceutical substances with 261 carefully determined and highly reliable experimental pK(a) values from a literature source. The programs ADME Boxes 4.9, ACD/pK(a) DB 12.0, and SPARC 4.2 ranked as the top three with mean absolute deviations of 0.389, 0.478, and 0.651 and r(2) values of 0.944, 0.908, and 0.894, respectively. ACD/pK(a) DB 12.0 predicted all sites, whereas ADME Boxes 4.9 and SPARC 4.2 failed to predict 5 and 18 sites, respectively. The performance of the quantum chemical-based program Jaguar 7.5 was not as expected, with a mean absolute deviation of 1.283 and an r(2) value of 0.579, indicating the potential for further development of this type of approach to pK(a) prediction.
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