Subcellular pH and predicted pH-dependent features of proteins

A characteristic of two-dimensional proteomics gels is a general bimodal distribution of isoelectric (pI) values. Discussion of this feature has focussed on the balance of acidic and basic ionisable residues, and potential relationships between pI distributions and organism classification or protein subcellular location. Electrostatics calculations on a set of protein structures with known subcellular location show that predicted folded state pI are similar to those calculated from sequence alone, but adjusted according to a general stabilising effect from interactions between ionisable groups. Bimodal distributions dominate both pI and the predicted pH of maximal stability. However, there are significant differences between these features. The average pH of maximal stability generally follows organelle pH. Average pI values are well removed from organelle pH in most subcellular environments, consistent with the view that proteins have evolved to carry (on average) net charge in a given subcellular location, and relevant to discussion of solubility in crowded environments. Correlation of the predicted pH of maximum stability with subcellular pH suggests an evolutionary pressure to adjust folded state interactions according to environment. Finally, our analysis of ionisable group contributions to stability suggests that Golgi proteins have the largest such term, although this dataset is small.     

pH-dependent processes in proteins

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.     

The hydrophobic cores of proteins predicted by wavelet analysis

MOTIVATION: In the process of protein construction, buried hydrophobic residues tend to assemble in a core of a protein. Methods used to predict these cores involve use or no use of sequential alignment. In the case of a close homology, prediction was more accurate if sequential alignment was used. If the homology was weak, predictions would be unreliable. A hydrophobicity plot involving the hydropathy index is useful for purposes of prediction, and smoothing is essential. However, the proposed methods are insufficient. We attempted to predict hydrophobic cores with a low frequency extracted from the hydrophobicity plot, using wavelet analysis. RESULTS: The cores were predicted at a rate of 68.7%, by cross-validation. Using wavelet analysis, the cores of non-homologous proteins can be predicted with close to 70% accuracy, without sequential alignment. AVAILABILITY: The program used in this study is available from Intergalactic Reality (http://www.intergalact.com). CONTACT: hirakawa@grt.kyushu-u.ac.jp, kuhara@grt.kyushu-u.ac.jp     

pH-dependent conformational changes in thrombocyte proteins

A study was made of the medium pH influence on structural states of platelets by optical methods. Within the pH range (6-8), two pH induced reversible changes of platelet state were observed. A conclusion is made that the structural rearrangements in platelets induced in the medium by changes in hydrogen ion concentration may involve some rearrangements in platelet proteins, and thus acting as a factor regulating platelet function.     

Calculating pH-dependent free energy of proteins by using Monte Carlo protonation probabilities of ionizable residues

Protein folding, stability, and function are usually influenced by pH. And free energy plays a fundamental role in analysis of such pH-dependent properties. Electrostatics-based theoretical framework using dielectric solvent continuum model and solving Poisson-Boltzmann equation numerically has been shown to be very successful in understanding the pH-dependent properties. However, in this approach the exact computation of pH-dependent free energy becomes impractical for proteins possessing more than several tens of ionizable sites (e.g.>30), because exact evaluation of the partition function requires a summation over a vast number of possible protonation microstates. Here we present a method which computes the free energy using the average energy and the protonation probabilities of ionizable sites obtained by the well-established Monte Carlo sampling procedure. The key feature is to calculate the entropy by using the protonation probabilities. We used this method to examine a well-studied protein (lysozyme) and produced results which agree very well with the exact calculations. Applications to the optimum pH of maximal stability of proteins and protein–DNA interactions have also resulted in good agreement with experimental data. These examples recommend our method for application to the elucidation of the pH-dependent properties of proteins.     

pH-dependent pore formation properties of pardaxin analogues

The interaction of pardaxin, a shark-repellent neurotoxin, and its charge-modified analogues with vesicles and human erythrocytes is described. The following six analogues and derivatives were synthesized by a solid phase method: [Glu8, Glu16]pardaxin, [N1-succinamido,Glu8,Glu16]pardaxin, [N1,Lys8,Lys16-triacetyl]pardaxin, des-[1----9]pardaxin (Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202-20209), and des-[1----9] [Glu16]pardaxin. The relative hydrophobic characteristics of the analogues were examined using reverse-phase high performance liquid chromatography. The pH-dependent spectroscopic and functional characteristics of the analogues were also investigated at either neutral or acidic pH. Spectroscopic characterization was achieved by measuring circular dichroism both before and after binding to vesicles, at either neutral or acidic pH. The ability of the peptides to dissipate a diffusion potential, to cause calcein release or the pH-dependent release of 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt/p-xylene-bis[pyridinium bromide] from sonicated unilamellar liposomes, as well as measurements of cytolytic activity on human erythrocytes, served to functionally characterize the peptides. We show a direct correlation between alpha-helical content, the analogues' hydrophobicity, and their pore-forming properties at the different pH values tested. We also demonstrate that the charge of the N terminus and of the peptide backbone, but not of the C terminus, affects the secondary structure as well as the activities of the analogues. Finally, we show that the cytolytic activity of pardaxin at neutral pH is not retained by any of the analogues.     

Detection of unrelated proteins in sequences multiple alignments by using predicted secondary structures

MOTIVATION: Multiple sequence alignments are essential tools for establishing the homology relations between proteins. Essential amino acids for the function and/or the structure are generally conserved, thus providing key arguments to help in protein characterization. However for distant proteins, it is more difficult to establish, in a reliable way, the homology relations that may exist between them. In this article, we show that secondary structure prediction is a valuable way to validate protein families at low identity rate. RESULTS: We show that the analysis of the secondary structures compatibility is a reliable way to discard non-related proteins in low identity multiple alignment. AVAILABILITY: This validation is possible through our NPS@ server (http://npsa-pbil.ibcp.fr)     

pH-dependent structures and properties of casein micelles

The association behavior of casein over a broad pH range has first been investigated by fluorescent technique together with DLS and turbidity measurements. Casein molecules can self-assemble into casein micelles in the pH ranges 2.0 to 3.0, and 5.5 to 12.0. The hydrophobic interaction, hydrogen bond and electrostatic action are the main interactions in the formation of casein micelles. The results show that the structure of casein micelles is more compact at low pH and looser at high pH. The casein micelle has the most compact structure at pH 5.5, when it has almost no electrostatic repulsion between casein molecules.     

Analysis of pH-dependent elements in proteins: geometry and properties of pairs of hydrogen-bonded carboxylic acid side-chains

A rather frequent but so far little discussed observation is that pairs of carboxylic acid side-chains in proteins can share a proton in a hydrogen bond. In the present article, quantum chemical calculations of simple model systems for carboxyl-carboxylate interactions are compared with structural observations from proteins. A detailed structural analysis of the proteins deposited in the PDB revealed that, in a subset of proteins sharing less than 90% sequence identity, 19% (314) contain at least one pair of carboxylic acids with their side-chain oxygen atoms within hydrogen-bonding distance. As the distance between those interacting oxygen atoms is frequently very short ( approximately 2.55 A), many of these carboxylic acids are suggested to share a proton in a strong hydrogen bond. When situated in an appropriate structural environment (low dielectric constant), some might even form a low barrier hydrogen bond. The quantum chemical studies show that the most frequent geometric features of carboxyl-carboxylate pairs found in proteins, and no or symmetric ligation, are also the most stable arrangements at low dielectric constants, and they also suggest at medium and low pH a higher stability than for isosteric amide-carboxylate pairs. The presence of these pairs in 119 different enzymes found in the BRENDA database is set in relation to their properties and functions. This analysis shows that pH optima of enzymes with carboxyl-carboxylate pairs are shifted to lower than average values, whereas temperature optima seem to be increased. The described structural principles can be used as guidelines for rational protein design (e.g., in order to improve pH or temperature stability).     

Graphical analysis of interactions between oxidation-reduction sites in two site oxidation-reduction proteins

Many enzymes that catalyze electron-transfer reaction contain multiple oxidation-reduction centers (sites). The oxidation-reduction potential of one site as well as the kinetics of electron transfer through this site may be altered by the state of reduction of a neighboring site. Oxidation-reduction site interactions may be mechanistically important and quantitation of site interactions would aid the interpretation of thermodynamic data and possibly kinetic data. A graphical means to detect and quantitate interactions between oxidation-reduction sites from oxidation-reduction equilibrium data (type A + B in equilibrium C + D) is described and has its roots in the Scatchard analysis of ligand binding equilibria (type A + B in equilibrium C). Oxidation-reduction sites often have distinct physical properties allowing the titration behavior of specific sites to be monitored. Equilibrium measurements on specific sites of a two site protein allow a further analysis of the data which can be combined with the oxidation-reduction Scatchard analysis to solve for all four specific site equilibrium constants. Ligand binding systems can usually measure only total site binding and simplifying assumptions of identical sites or noninteracting sites are required to solve for the site specific equilibrium constants. Thus, specific site equilibrium measurements offer a distinct advantage over total site measurements. The principles of the method are illustrated by applying the graphical analysis to the two site protein, thioredoxin reductase, which contains an oxidation-reduction active site disulfide in addition to FAD. The specific site oxidation-reduction midpoint potentials (Em) of the FAD and disulfide couples of thioredoxin reductase at pH 6.0, 12 degrees C, were found to be FAD/FADH2-enzyme-(S)2 = -0.183 V, FAD/FADH2-enzyme-(SH)2 = -0.199 V, (FAD)-enzyme-(S)2/(SH)2 = -0.202 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.218 V. Hence, at pH 6.0, the FAD and disulfide sites of thioredoxin reductase have Em values that differ by approximately 0.019 V and have a negative interaction of about 0.016 V.     

pH-dependent fusion of liposomes using titratable polycations

Polylysine promoted extensive membrane mixing of liposomes only if the buffer pH was below the pKa of the lysyl residues. This observation suggested that fusion could be regulated in a physiological pH range if the homopolymer of L-histidine was substituted as fusogen. Microgram quantities of polyhistidine were added to liposomes composed of soybean phospholipids, or to defined phospholipid-cholesterol mixtures which simulate the lipid composition of plasma membranes. A quantitative resonance energy transfer assay determined the extent of lipid phase mixing related to fusion. No fusion was detected at pH 7.4, but when the pH was lowered to 6.5 or below, fusion was rapid and substantial. The extent of membrane mixing increased with progressive acidification of the vesicle-fusogen suspension. The charge density of each polyhistidine molecule, not the total cationic charge per vesicle, influenced the extent of fusion. The kinetics of the fusion reaction were rapid, as membrane mixing was completed within 1 min. If the vesicle suspension was acidified before fusogen addition, the rate of membrane mixing slowed 4-fold. This, as well as a slight increase in light scattering noted whenever polyhistidine was added at pH 7.4, suggests an enhancement of fusion kinetics by preaggregation of vesicles at neutral pH. The lipid composition, regulation of membrane mixing by pH in a physiological range, and rapid kinetics suggest that this model of liposome fusion may be pertinent to understanding some biological fusion events.     

De novo isolation of antibodies with pH-dependent binding properties

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.     

Modelling the pH-dependent properties of Kv1 potassium channels

It is known that the pH dependence of conductance for the rat potassium channel Kv1.4 is susbstantially reduced upon mutation of either H508 or K532. These residues lie in the extracellular mouth of the channel pore. We have used continuum electrostatics to investigate their interactions with K(+) sites in the pore. The predicted scale of interactions between H508/K532 and potassium sites is sufficient to significantly alter potassium occupancy and thus channel function. We interpret the effect of K532 mutation as indicating that the pH-dependent effect requires not only an ionisable group with a suitable pK(a) value (i.e. histidine), but also that other charged groups set the potential profile at a threshold level. This hypothesis is examined in the context of pH dependence for other members of the Kv1 family, and may represent a general tool with which to study potassium channels.     

Enhancer binding proteins predicted by informational spectrum method

Enhancer sequences analysed using the informational spectrum method (ISM) show a characteristic frequency at .0488. It has been shown that the characteristic frequency for some DNA binding proteins overlaps the characteristic frequency of their target DNA sequences. We suggest here that two types of proteins, homeoproteins and the glucocorticoid receptors, might bind to enhancer sequences.     

Functional heterogeneity and pH-dependent dissociation properties of human transferrin

Human diferric transferrin was partially labeled with ⁵⁹Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte ⁵⁹ uptake experiments with chemically labeled preparations indicated that iron bound at near neutral ph was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe³⁺, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2–5.8) was required to effect dissociation of iron that had remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-binding properties of human transferrin and identifies that the near neutral iron-donating site initially surrenders its iron to these cells.     

De novo isolation of antibodies with pH-dependent binding properties

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.     

Graphical representation of hydrogen bonding patterns in proteins

A graphical representation of the intramolecular hydrogen bonding in a protein is described, which provides a direct and easily interpretable display of its secondary and tertiary structural elements. The representation is constructed by scanning the coordinate list for all potential proton donor (PD)--proton acceptor (PA) pairs, and any pair which satisfies certain preset distance and angle criteria is classified as being H-bonded. The resulting list of H-bonds is mapped onto an N x N matrix, where N is the number of residues in the protein, by assigning an element ij of the matrix to all the PA-PD pairs between atoms of residues i and j. Subsequently graphical objects are generated for all elements which are labeled as representing one or more H-bonds, and which can then be plotted or displayed in a way analogous to the graphical representation of the distance matrix (DM). In contrast to the DM, the hydrogen bonding matrix (HBM) is sparse, which allows the patterns representing secondary and tertiary structural motifs to be quickly and clearly recognized. In addition, changes in structure are easily identifiable from changes in the H-bonding patterns. The analysis and interpretation of the HBM is discussed using aspartate amino-transferase and calmodulin as examples.     

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.     

Theoretical study of the pH-dependent antioxidant properties of vitamin C

Molecules acting as antioxidants capable of scavenging reactive oxygen species (ROS) are of utmost importance in the living cell. Vitamin C is known to be one of these molecules. In this study we have analyzed the reactivity of vitamin C toward the $ \cdot OH $ and $ \cdot OOH $ ROS species, in all acidic, neutral and basic media. In order to do so, density functional theory (DFT) have been used. More concretely, the meta-GGA functional MPW1B95 have been used. Two reaction types have been studied in each case: addition to the ring atoms, and hydrogen/proton abstraction. Our results show that $ \cdot OH $ is the most reactive species, while $ \cdot OOH $ displays low reactivity. In all three media, vitamin C reactions with two hydroxyl radicals show a wide variety of possible products.