pH-induced Changes Research Articles

PH-Dependent Conformational Switch Impacts Stability of the PsbS Dimer.

The photosystem II PsbS protein triggers the photoprotective mechanism of plants by sensing the acidification of the thylakoid lumen. Despite the mechanism of action of PsbS would require a pH-dependent monomerization of the dimeric form, a clear connection between the pH-induced structural changes and the dimer stability is missing. Here, by applying constant pH coarse-grained and all-atom molecular dynamics simulations, we investigate the pH-dependent structural response of the PsbS dimer. We find that the pH variation leads to structural changes in the lumen-exposed helices, located at the dimeric interface, providing an effective switch between PsbS inactive and active form. Moreover, the monomerization free energies reveal that in the neutral pH conformation, where the network of H-bond interactions at the dimeric interface is destroyed, the protein-protein interaction is weaker. Our results show how the pH-dependent conformations of PsbS affect their dimerization propensity, which is at the basis of the photoprotective mechanism.

Critical Influences of Plasma pH on Human Protein Properties for Modeling Considerations: Size, Charge, Conformation, Hydrophobicity, and Denaturation

The fouling of biomaterials (e.g., membranes) by plasma proteins has always garnered attention because it renders biomedical devices ineffective and can jeopardize the patient’s well-being. Modeling the fouling process sheds light on its mechanisms and helps improve the biocompatibility of biomaterials. Assuming proteins to be hard spheres with uniform surface properties reduces the modeling complexity, but it seriously deviates from the accurate, real perspective. One reason for the inaccuracy is that proteins’ properties tend to change as environmental factors such as pH and ionic strength are varied. This study critically reviews the pH-induced changes in protein properties, namely size, charge, conformity, hydrophobicity, and denaturation. Though these properties may be interrelated, they are addressed individually to allow for a thorough discussion. The study illustrates the necessity of incorporating the protein property changes resulting from pH alteration to better explain and model the fouling process. The discussion is focused on human serum albumin and fibrinogen. Human serum albumin is the most abundant plasma protein, while fibrinogen plays a major role in blood clotting and triggering of the thrombogenic response.

Hidden symmetry of the flavivirus protein shell and pH-controlled reconstruction of the viral surface.

Using recent Zika virus structural data we reveal a hidden symmetry of protein order in immature and mature flavivirus shells, violating the Caspar-Klug paradigmatic model of capsid structures. We show that proteins of the outer immature shell layer exhibit trihexagonal tiling, while proteins from inner and outer layers conjointly form a double-shelled close-packed structure, based on a common triangular spherical lattice. Within the proposed structural model, we furthermore rationalize the structural organization of misassembled non-infectious subviral particles that have no inner capsid. We consider a pH-controlled structural reconstruction of the outer shell from the trimeric to the dimeric state, and demonstrate that this transition, occurring during the virus maturation, can be induced by changes in protein charges at lower pH, leading to a decrease in the electrostatic interaction free energy. This transition could also be assisted by electrostatic attraction of shell proteins to the interposed lipid membrane substrate separating the shells.

Photocatalytic activity and pH-induced morphological changes of ZnO/CuO nanocomposites prepared by chemical bath precipitation

Various morphologies of ZnO/CuO nanocomposites were obtained by altering the pH value and their influence on the optical and photocatalytic properties of this nanocomposite against organic dyes was examined.

Elucidation of pH-Induced Protein Structural Changes: A Combined 2D IR and Computational Approach.

The acid-base behavior of amino acids plays critical roles in several biochemical processes. Depending on the interactions with the protein environment, the pKa values of these amino acids shift from their respective solution values. As the side chains interact with the polypeptide backbone, a pH-induced change in the protonation state of aspartic and glutamic acids might significantly influence the structure and stability of a protein. In this work, we have combined two-dimensional infrared spectroscopy and molecular dynamics simulations to elucidate the pH-induced structural changes in an antimicrobial enzyme, lysozyme, over a wide range of pH. Simultaneous measurements of the carbonyl signals arising from the backbone and the acidic side chains provide detailed information about the pH dependence of the local and global structural features. An excellent agreement between the experimental and the computational results allowed us to obtain a residue-specific molecular understanding. Although lysozyme retains the helical structure for the entire pH range, one distinct loop region (residues 65-75) undergoes local structural deformation at low pH. Interestingly, combining our experiments and simulations, we have identified the aspartic acid residues in lysozyme, which are influenced the most/least by pH modulation.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Formulating Zwitterionic, Responsive Polymers for Designing Smart Soils.

The design of new remediation strategies and materials for treating saline-alkaline soils is of fundamental and practical importantance for many applications. Conventional soil remediation strategies mainly focus on the development of fertilizers or additives for water, nutrient, and heavy metal managements in soils, but they often overlook a soil sensing function for early detection of salinization/alkalization levels toward optimal and timely soil remediation. Here, new smart soils, structurally consisting of the upper signal soil and the bottom hygroscopic bed and chemically including zwitterionic, thermo-responsive poly(NIPAM-co-VPES) and poly(NIPAM-co-SBAA) aerogels in each soil layer are formulated. Upon salinization, the resultant smart soils exhibit multiple superior capacities for reducing the soil salinity and alkalinity through ion exchange, controlling the water cycling, modulating the degradation of pyridine-base ligands into water-soluble, nitrogenous salts-rich ingredients for soil fertility, and real-time monitoring salinized soils via pH-induced allochroic color changes. Further studies of plant growth in smart soils with or without salinization treatments confirm a synergy effect of soil remediation and soil sensing on facilitating the growth of plants and increasing the saline-alkaline tolerance of plants. The esign concept of smart soils can be further expanded for soil remediation and assessment.

Glycosylation of a key cubilin Asn residue results in reduced binding to albumin

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin’s albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin–albumin interaction.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

Effects of casein on the stability, antioxidant activity, and bioavailability of lotus anthocyanins.

Effects of casein on the stability, antioxidant activity, and bioavailability of lotus anthocyanins were investigated. Casein could inhibit the unsatisfactory pH-induced color change of lotus anthocyanins, and improved their photo, oxidation, and thermal stabilities. During the simulated digestion, the anthocyanin retention increased from 65.39 to 76.14 mg C3G/L with the protection of casein, while the DPPH and ABTS scavenging activities of lotus anthocyanins with casein increased to 62.33% and 46.58%, respectively. However, casein with lower concentration showed a better protective effect on lotus anthocyanins due to its self-aggregation tendency at high dose. The zebrafish model further verified that casein could enhance the bioavailability of lotus anthocyanins. Furthermore, molecular docking revealed that casein could interact with anthocyanin by hydrogen bond and hydrophobic interaction, which led to the stronger stability and bioavailability of lotus anthocyanins. The results conveyed that casein could be used as a wall material to protect anthocyanins. PRACTICAL APPLICATIONS: Anthocyanins are natural colorants with multiple biological activities, but the poor stability during processing and digestion limits their application in food industry. In the present research, casein exhibited conspicuous ability to enhance the stability of lotus anthocyanins toward detrimental conditions. Additionally, casein could preserve anthocyanins from degradation during digestion and thus improve the bioavailability. These findings indicated that casein could serve as a potential carrier for encapsulating and delivering anthocyanins. The better stability and bioavailability would promote the application of anthocyanins in food products and human health.

Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability.

pH-induced changes in the conformation, structural dynamics, and carbohydrate-binding activity of Coccinia indica agglutinin (CIA17), a PP2-type lectin, were investigated employing biophysical approaches. The secondary structure of CIA17 remains nearly unaltered over a wide pH range (2.0-8.5), while the tertiary structure of the protein exhibits considerable changes. A decrease in the fluorescence intensity and excited-state lifetime at low pH indicated perturbation in the local conformation (near Trp residues) of CIA17, which was further supported by enhancement in the Trp accessibility toward charged quenchers under acidic conditions. Fluorescence correlation spectroscopic studies indicated that at pH 2.0, CIA17 exists as a monomer over the concentration range of 10-200 nM and forms dimers at higher concentrations (KD ∼ 387 nM) but could not form higher oligomers even at ∼150-fold higher concentrations, unlike under native conditions at pH 7.4. Thermal unfolding of the low pH intermediate involves two distinct steps: dissociation of a dimer to a monomer, followed by the unfolding of the monomer. These results strongly suggest that the acid-induced unfolding pathway of CIA17 involves the formation of a monomeric molten globule-like intermediate, which retains appreciable carbohydrate-binding ability. These observations are of great physiological significance since the PP2 proteins are involved in plant defense responses.

PH-Induced Changes in the SERS Spectrum of Thiophenol at Gold Electrodes during Cyclic Voltammetry.

Thiophenol is a model compound used in the study of self-assembly of arylthiols on gold surfaces. In particular, changes in the surface-enhanced Raman scattering (SERS) spectra of these self-assembled monolayers (SAMs) with a change of conditions have been ascribed to, for example, differences in orientation with respect to the surface, protonation state, and electrode potential. Here, we show that potential-induced changes in the SERS spectra of SAMs of thiophenol on electrochemically roughened gold surfaces can be due to local pH changes at the electrode. The changes observed during the potential step and cyclic voltammetry experiments are identical to those induced by acid–base switching experiments in a protic solvent. The data indicate that the potential-dependent spectral changes, assigned earlier to changes in molecular orientation with respect to the surface, can be ascribed to changes in the pH locally at the electrode. The pH at the electrode can change as much as several pH units during electrochemical measurements that reach positive potentials where oxidation of adventitious water can occur. Furthermore, once perturbed by applying positive potentials, the pH at the electrode takes considerable time to recover to that of the bulk solution. It is noted that the changes in pH even during cyclic voltammetry in organic solvents can be equivalent to the addition of strong acids, such as CF3SO3H, and such effects should be considered in the study of the redox chemistry of pH-sensitive redox systems and potential-dependent SERS in particular.

Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B–C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.Graphical abstract

PH-induced changes in Raman, UV–vis absorbance, and fluorescence spectra of dipicolinic acid (DPA)

Dipicolinic acid (DPA) is an essential component for the protection of DNA in bacterial endospores and is often used as a biomarker for spore detection. Depending upon the pH of the solution, DPA exists in different ionic forms. Therefore, it is important to understand how these ionic forms influence spectroscopic response. In this work, we characterize Raman and absorption spectra of DPA in a pH range of 2.0–10.5. We show that the ring breathing mode Raman peak of DPA shifts from 1003 cm−1 to 1017 cm−1 and then to 1000 cm−1 as pH increases from 2 to 5. The relative peak intensities related to the different ionic forms of DPA are used to experimentally derive the pKa values (2.3 and 4.8). We observe using UV–vis spectroscopy that the changes in the absorption spectrum of DPA as a function of pH correlate with the changes observed in Raman spectroscopy, and the same pKa values are verified. Lastly, using fluorescence spectroscopy and exciting a DPA solution at between 210–330 nm, we observe a shift in fluorescence emission from 375 nm to 425 nm between pH 2 and pH 6 when exciting at 320 nm. Our work shows that the different spectral responses from the three ionic forms of DPA may have to be taken into account in, e.g., spectral analysis and for detection applications.

Spectroscopic and electrochemical studies of the pH-Induced transition in the CuA centre from Thermus thermophilus

A detailed study of the pH induced conformational changes at the Cu2S2 coordination site of the CuA centre of cytochrome oxidase from Thermus thermophilus has been carried out to unravel the role of the pH-induced changes in the coordination geometry on the redox potential and electron transfer kinetics of the metal centre. The pH dependence of the UV–visible absorption bands as well as of the visible circular dichroism spectra corresponding to the metal centre of the protein could be analyzed by the model involving two protonation/deprotonation steps associated with the pKa values of 3.5 and 9.6. The redox potential of the protein determined by direct electrochemistry also showed analogous pH dependence. The results suggested that the metal centre undergoes equilibrium conformational transition involving three forms, viz., ‘low pH’ form, ‘neutral pH’ form and ‘high-pH’ form characterized by different redox potentials of the metal centre. The rates of heterogeneous electron transfer were found to decrease drastically at low pH as well as at high pH. The electron transfer from cytochrome c to the CuA site of the cytochrome oxidase is proposed to be gated by conformation changes at the CuA site through a protonation-induced conformation change, which may act as a sensor for optimum proton transfer across the enzyme. The results also have been discussed in the light of understanding fast electron transfer from the CuA to the heme a in the intact enzyme.

Carbon dot with aggregation induced emission and pH triggered disintegration

Reportedly, Aggregation Induced Emission (AIE) from Carbon Dots (CDs) is mainly induced by hydrophobic interactions. Herein, we report AIE from pristine CDs that are predominantly hydrophilic. A multistage purification protocol was employed to separate the pristine CD sample from byproducts and CD mixture. At neutral pH, the aggregated CDs exhibit a large Stokes shift with absorption maxima at 405 nm and emission in the yellow region (λmax- 535 nm) which decays mono-exponentially with a time constant of 4.03 ns. The AIE from CD quenches on moving towards the acidic region. Significant changes in the UV–visible absorption and emission spectrum are seen on increasing the pH towards alkaline medium. While the pH-induced changes in the acidic medium are reversible in nature, the emission turns blue (λmax ~ 430 nm) in alkaline medium and the process is irreversible. Microscopic and Light Scattering studies suggest pH triggered disintegration of aggregates in alkaline medium.

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

Glycopeptide-Conjugated Aggregation-Induced Emission Luminogen: A pH-Responsive Fluorescence Probe with Tunable Self-Assembly Morphologies for Cell Imaging.

pH values play an important role in various cell biological processes. Abnormal pH values in living systems are frequently associated with the development of diseases such as cancers, infection, and other diseases. Real-time monitoring of the changes of pH values in vivo will give us the significant indication for these diseases' progression. Within those pH-sensitive imaging probes, aggregation-induced emission (AIE) molecules exhibit great potential in aqueous imaging environment due to their high fluorescence quantum yield and stability. However, the modulation of the AIE probe with pH sensitivity and light-up property face challenges. Here, we introduced a new glycopeptide-modified AIE probe (TGO) based on the optimized solid-phase peptide synthesis approach. The response to pH of the peptide: DDDD progression changed hydrophobicity and hydrophilicity, resulting in the change of the amphipathicity balance. When modulating the pH from 5.5 to 8.0, the adverse protonation of the peptide induced assembled nanostructure transformation from nanolamellae to nanomicelles. Meanwhile, the pH-induced charge change in peptides can greatly influence the microenvironment of the AIEgen, resulting in the increase of fluorescence intensity.

Smartphone-assisted colorimetric sensing of enzyme-substrate system using pH-responsive gold nanoparticle assembly

Gold nanoparticle (AuNP)-based colorimetric biosensors have been widely used for pH sensing and monitoring its changes. However, few AuNP-based pH sensors have been developed through the manipulation of the aggregation states of AuNPs via i-motif DNA. We herein report i-motif DNA-assisted pH-responsive gold nanoparticle assembly (termed RGA), which shows a reversible and highly sensitive response to pH variation between 6.20 and 7.40. The acidic pH triggers the disassembly of the RGA, thus converting the AuNPs from aggregation state to disperse state, which leads to a color transition from blue-purple to red. Therefore, the pH value can be estimated by naked-eye determination or UV–vis spectroscopy analysis. More significantly, the visually detectable color change is monitored using the built-in camera of a smartphone. The RGB (red, green, blue) values of the RGA solution are measured by a smartphone application (APP). Following data processing, the RGB values can be converted into pH value, providing a new strategy for the on-site and real-time pH sensing. Furthermore, the pH-induced conformation change of i-motif DNA allows the RGA to detect a slight pH fluctuation in the catalytic oxidation of glucose by glucose oxidase and the hydrolysis of urea by urease.

PH-induced changes in β-casomorphin 7 structure studied by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy

β-Casomorphins are opioid peptides that can be liberated from β-casein during digestion, with some suggested effects on human physiology linked to their properties. In this study, the structural behaviour of β-casomorphin 7 (BCM7; Tyr–Pro–Phe–Pro–Gly–Pro–Ile) was studied at pH 2.3 and 6.7 using 1H nuclear magnetic resonance (1H NMR) and Fourier-transform infrared (FTIR) spectroscopy. Cis–trans isomerism involving the Tyr1–Pro2, Phe3–Pro4 and Gly5–Pro6 bonds of BCM7 was observed. Cis and trans isomers of Tyr1–Pro2 and Phe3–Pro4 and trans isomers for Gly5–Pro6 were observed at both pH 6.7 and 2.3 but cis isomers of Gly5–Pro6 were only observed at pH 2.3 and not at pH 6.7. These pH-induced conformational changes at the Gly5–Pro6 bond of BCM7 may play important role in enzymatic release of this peptide from the parent protein β-casein under gastro-intestinal conditions, as well as further cleavage of BCM7 into smaller peptides.