pH-induced Changes Research Articles

PH-Induced Structural Changes of Crystalline Curcumin Enhance Its Encapsulation in Emulsions

pH-Induced Structural Changes of Crystalline Curcumin Enhance Its Encapsulation in Emulsions

Insights into structural and proteomic alterations related to pH-induced changes and protein deamidation in hair.

The hair shaft is often exposed to shampoo and haircare products that have unknown or varying pH levels. These products contain a combination of surfactants and other active ingredients to treat the hair or the scalp. As amphoteric proteins, hair keratins have limited buffering capacity, so variations in pH can have multifaceted impacts on them. However, there is limited knowledge about how pH affects keratins and keratin-associated proteins (KAPs). Therefore, this study aims to examine the effects of varying pH levels (pH 3-pH 12) on hair structure and analyse consequent alterations in the hair proteome using mass spectrometry-based proteomics. A scanning electron microscope was used to examine changes in hair-shaft morphology due to exposure to various pH levels, while mass spectrometry was employed to analyse protein alterations. We found that exposing the hair shaft to varying pH levels led to specific effects on the cuticle, including cuticle lifting at certain pH levels, while proteomics analysis identified alterations in the hair proteome along with significant deamidation of keratins types I and II and KAPs. More pronounced effects were observed at extreme acidic conditions (pH 3) and alkaline conditions (above pH 8) on both hair morphology and hair proteins. pH levels between pH 5 and pH 7 had minimal impact on hair structure and proteins, suggesting that haircare products with pH in this range are ideal for hair-shaft health. In contrast, alkaline pH levels were found to negatively affect hair. The structure evaluation and proteomics data emphasize the critical role of pH in hair health. The extreme acidic or alkaline pH impacts the hair structure and hair proteins. The study highlights the optimal pH range for maintaining healthy hair.

PH-induced interface protein structure changes to adjust the stability of tilapia protein isolate emulsion prepared by high-pressure homogenization

The pH is a crucial external factor affecting the structure and emulsification characteristics of proteins. The current study aimed to reveal the correlation between the secondary structure changes and tilapia protein isolate (TPI) emulsion stability under different pH (3.0–10.0) prepared by high-pressure homogenization. The results showed that TPI with significantly increased solubility and emulsifying properties when the pH keep away from the isoelectric point (pH 5.0). Meanwhile, TPI emulsions presented significantly enhanced stability (with decreased particle size, increased zeta potential, creaming index close to 0, and uniform dispersion of droplets) at pH 3.0 and 10.0. Interface-adsorbed protein mainly consists of a myosin-heavy chain and actin, and the secondary structure was significantly influenced by pH and high-pressure homogenization. The α-helix will be transformed into β-sheet and β-turn when pH is closer to pH 5.0. However, the high-pressure homogenization induced α-helix conversion to β-sheet. The correlation analysis revealed that emulsion stability is positively correlated with α-helix and negatively correlated with β-sheet. This work provides a deep insight into the correlation between secondary structure changes and the stability of TPI emulsion as affected by pH to offer an alternative way to enhance TPI emulsion stability.

Simplified Electrochemical Approach for End-Point Yet Quantitative Detection of Nucleic Acids in Resource-Limited Settings.

Nucleic acid detection plays a crucial role in various aspects of health care, necessitating accessible and reliable quantification methods, especially in resource-limited settings. This work presents a simplified electrochemical approach for end-point yet quantitative nucleic acid detection. By elevating the concentration of redox species and choosing potential as the signals, we achieved enhanced signal robustness, even in the presence of interfering substances. Leveraging this robustness, we accurately measured pH-induced redox potential changes in methylene blue solution for end-point nucleic acid detection after loop-mediated isothermal amplification (LAMP). Our method demonstrated quantitative detection of the SARS-CoV-2 N gene and human ATCB gene and successful discrimination of the human BRAF V600E mutation, comparable in sensitivity to commercial kits. The developed user-friendly electrochemical method offers a simplified and reliable approach for end-point yet quantitative detection of nucleic acids, potentially expanding the benefits of nucleic acid testing in resource-limited settings.

Composite Materials Based on Biocompatible Metal-Organic Framework and Anthocyanins from Hibiscus sabdariffa for Active Food Packaging

The biocompatible metal-organic framework [Zn4(GA)4(H2O)4] · 4H2O (H2GA = glutamic acid) was used as a container for anthocyanins from Hibiscus sabdariffa in composite films based on kappa-carrageenan and hydroxypropyl methylcellulose. The obtained composite materials showed high antioxidant activity and ability to undergo pH-induced color change upon reactions with gaseous products of pathogen development and, hence, possess the potential for practical application as functional materials for food packaging.

Structural Characterization of Human Bufavirus 1: Receptor Binding and Endosomal pH-Induced Changes.

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo-lysosomal (pH 7.4-pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo-lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses.

Tailoring the geometry of visible and near-infrared light active CuSe nanostructure for enhanced photocatalytic activity

Semiconductor photocatalysts play a pivotal role in fostering a sustainable environment by harnessing solar energy directly into chemical energy to achieve various photocatalytic processes. Therefore, a visible-infrared active photocatalyst is the next level of demand for solving the alarming environmental issues. In this regard, the present article reports the investigations of crucial mechanisms and conditions leading to pure and defect-free hexagonal 2D CuSe nanosheets (NSs) and the influence of their structural architecture on visible-infrared active photocatalytic performance. Here, it was achieved by the conventional hydrothermal method by varying the pH of the reaction mixture. XRD analysis was used to examine the crystal structure and lattice parameters of CuSe NSs. The presence of H+ ions was suggested to significantly influence the observed anisotropic crystallite growth across different pH conditions. EDS and XPS analysis exhibited the purity and stoichiometry of 2D CuSe NSs, revealing that the obtained stoichiometry was consistent with CuSe composition. The morphological characteristics were investigated using FE-SEM as well as HR-TEM analyses, and the specific surface area was determined using Brunauer-Emmett-Teller analysis in which a significant change occurred in response to the variation in reaction conditions. The optical properties of optimized 2D CuSe NSs show excellent absorption in the visible and near-infrared region utilizing full solar with a notable decrease in emission intensity, suggesting a reduction in recombination rates due to pH-induced changes in the nanoarchitecture. Herein, methylene blue, a well-established model dye, was used as a probe to assess the photocatalytic activity of the synthesized CuSe NSs. Notably, the CuSe NSs synthesized under neutral conditions demonstrate a remarkable methylene blue degradation efficiency, reaching approximately 98 % within approximately 45 minutes, indicating a high degradation rate. Finally, an in-depth exploration into the intricacies of photocatalytic performance, degradation mechanisms, and the recyclability test was conducted, accompanied by a thorough investigation into the feasibility of CuSe NSs for their practical application in photocatalytic wastewater treatment.

PH-induced changes in IgE molecules measured by atomic force microscopy.

The environment surrounding proteins is tightly linked to its dynamics, which can significantly influence the conformation of proteins. This study focused on the effect of pH conditions on the ultrastructure of Immunoglobulin E (IgE) molecules. Herein, the morphology, height, and area of IgE molecules incubated at different pH were imaged by atomic force microscopy (AFM), and the law of IgE changes induced by pH value was explored. The experiment results indicated that the morphology, height and area of IgE molecules are pH dependent and highly sensitive. In particular, IgE molecules were more likely to present small-sized ellipsoids under acidic conditions, while IgE molecules tend to aggregate into large-sized flower-like structures under alkaline conditions. In addition, it was found that the height of IgE first decreased and then increased with the increase of pH, while the area of IgE increased with the increase of pH. This work provides valuable information for further study of IgE, and the methodological approach used in this study is expected to developed into AFM to investigate the changes of IgE molecules mediated by other physical and chemical factors. RESEARCH HIGHLIGHTS: The ultrastructure of IgE molecules is pH dependent and highly sensitive. IgE molecules were tend to present small-sized ellipsoids under acidic pH. Alkaline pH drives IgE self-assembly into flower-like aggregates.

Photo-modulated activation of organic bases enabling microencapsulation and on-demand reactivity

A method is developed for facile encapsulation of reactive organic bases with potential application for autonomous damage detection and self-healing polymers. Highly reactive chemicals such as bases and acids are challenging to encapsulate by traditional oil-water emulsion techniques due to unfavorable physical and chemical interactions. In this work, reactivity of the bases is temporarily masked with photo-removable protecting groups, and the resulting inactive payloads are encapsulated via an in situ emulsion-templated interfacial polymerization method. The encapsulated payloads are then activated to restore the organic bases via photo irradiation, either before or after being released from the core-shell carriers. The efficacy of the photo-activated capsules is demonstrated by a damage-triggered, pH-induced color change in polymeric coatings and by recovery of adhesive strength of a damaged interface. Given the wide range of potential photo-deprotection chemistries, this encapsulation scheme provides a simple but powerful method for storage and targeted delivery of a broad variety of reactive chemicals, promoting design of diverse autonomous functionalities in polymeric materials.

Quantifying pH-induced changes in plasma strong ion difference during experimental acidosis: clinical implications for base excess interpretation.

It is commonly assumed that changes in plasma strong ion difference (SID) result in equal changes in whole blood base excess (BE). However, at varying pH, albumin ionic-binding and transerythrocyte shifts alter the SID of plasma without affecting that of whole blood (SIDwb), i.e., the BE. We hypothesize that, during acidosis, 1) an expected plasma SID (SIDexp) reflecting electrolytes redistribution can be predicted from albumin and hemoglobin's charges, and 2) only deviations in SID from SIDexp reflect changes in SIDwb, and therefore, BE. We equilibrated whole blood of 18 healthy subjects (albumin = 4.8 ± 0.2 g/dL, hemoglobin = 14.2 ± 0.9 g/dL), 18 septic patients with hypoalbuminemia and anemia(albumin = 3.1 ± 0.5 g/dL, hemoglobin = 10.4 ± 0.8 g/dL), and 10 healthy subjects after in vitro-induced isolated anemia (albumin = 5.0 ± 0.2 g/dL, hemoglobin = 7.0 ± 0.9 g/dL) with varying CO2 concentrations (2-20%). Plasma SID increased by 12.7 ± 2.1, 9.3 ± 1.7, and 7.8 ± 1.6 mEq/L, respectively (P < 0.01) and its agreement (bias[limits of agreement]) with SIDexp was strong: 0.5[-1.9; 2.8], 0.9[-0.9; 2.6], and 0.3[-1.4; 2.1] mEq/L, respectively. Separately, we added 7.5 or 15 mEq/L of lactic or hydrochloric acid to whole blood of 10 healthy subjects obtaining BE of -6.6 ± 1.7, -13.4 ± 2.2, -6.8 ± 1.8, and -13.6 ± 2.1 mEq/L, respectively. The agreement between ΔBE and ΔSID was weak (2.6[-1.1; 6.3] mEq/L), worsening with varying CO2 (2-20%): 6.3[-2.7; 15.2] mEq/L. Conversely, ΔSIDwb (the deviation of SID from SIDexp) agreed strongly with ΔBE at both constant and varying CO2: -0.1[-2.0; 1.7], and -0.5[-2.4; 1.5] mEq/L, respectively. We conclude that BE reflects only changes in plasma SID that are not expected from electrolytes redistribution, the latter being predictable from albumin and hemoglobin's charges.NEW & NOTEWORTHY This paper challenges the assumed equivalence between changes in plasma strong ion difference (SID) and whole blood base excess (BE) during in vitro acidosis. We highlight that redistribution of strong ions, in the form of albumin ionic-binding and transerythrocyte shifts, alters SID without affecting BE. We demonstrate that these expected SID alterations are predictable from albumin and hemoglobin's charges, or from the noncarbonic whole blood buffer value, allowing a better interpretation of SID and BE during in vitro acidosis.

Structural Shifts of the Parvovirus B19 Capsid Receptor-binding Domain: A Peptide Study.

Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by the way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Versatile MXene Gels Assisted by Brief and Low-Strength Centrifugation

HighlightsA low-strength-centrifugation-assisted approach for rapid gelation of Ti3C2Tx MXene with a very low dispersion concentration into multifunctional 3D architectures is developed.On the basis of pH-induced surface termination changes, the MXene gels exhibit reconfigurable internal structures and tunable rheological, tribological, electrochemical, infrared-emissive and photothermal-conversion properties.By adopting a gel with optimized pH value, high lubrication, remarkable capacitance, long-term capacitance retention and high-precision screen- or extrusion-printing into high-resolution anticounterfeiting patterns can be achieved.

Conversion of raltegravir carrying a 1,3,4-oxadiazole ring to a hydrolysis product upon pH changes decreases its antiviral activity.

Raltegravir (RAL), a human immunodeficiency virus (HIV)-1 integrase inhibitor, has been administered as part of antiretroviral therapy. Studies in patients with HIV-1 have shown high variability in the pharmacokinetics of RAL, and in healthy volunteers, coadministration of proton-pump inhibitors has been shown to increase the plasma RAL concentrations. Here, we found that RAL containing a 1,3,4-oxadiazole ring is converted to a hydrolysis product (H-RAL) with a cleaved 1,3,4-oxadiazole ring at pH 1.0 and 13.0 conditions in vitro, thereby reducing the anti-HIV activity of the drug. The inclusion of cyclodextrins (beta-cyclodextrin [βCD], random methyl-βCD [RAM-βCD], and hydroxypropyl-βCD [HP-βCD]) can protect RAL from pH-induced changes. The conversion of RAL to H-RAL was detected by using various mass spectrometry analyses. The chromatogram of H-RAL increased in a time-dependent manner similar to another 1,3,4-oxadiazole-containing drug, zibotentan, using high-performance liquid chromatography. Oral bioavailability and target protein interactions of H-RAL were predicted to be lower than those of RAL. Moreover, H-RAL exhibited significantly reduced anti-HIV-1 activity, whereas combinations with βCD, RAM-βCD, and HP-βCD attenuated this effect in cell-based assays. These findings suggest that βCDs can potentially protect against the conversion of RAL to H-RAL under acidic conditions in the stomach, thereby preserving the anti-HIV-1 effect of RAL. Although clinical trials are needed for evaluation, we anticipate that protective devices such as βCDs may improve the pharmacokinetics of RAL, leading to better treatment outcomes, including reduced dosing, long-term anti-HIV-1 activity, and deeper HIV-1 suppression.

Novel Halochromic Disazo Stilbene Dye with Intramolecular Color Mixing Effect for Textile Sensor Material

A stilbene-based halochromic dye was synthesized, which possesses the effect of intramolecular color matching. By dyeing cotton fabric with the synthesized dye, a textile sensor material was prepared to visually monitor the acidity of the biologically significant pH range (4.5–8.0). The sensitivity of the sensor material to changes in the acidity of the environment, as well as the color stability to the action of saline solutions simulating biological fluids (sweat and blood plasma), were explored. A visually noticeable pH-induced color change of the material is observed in the pH range of 3.0–10.0. In the pH range of buffer solutions at pH 3, the color of the samples is green, from 6 to 8, orange, and from 9 and above, purple. The average color change time was 3 s. The results obtained show the promise of using the studied azo compound in technologies for designing multifunctional pH-sensitive textile sensor materials.

Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Orange Carotenoid Protein in Mesoporous Silica: A New System towards the Development of Colorimetric and Fluorescent Sensors for pH and Temperature.

Orange carotenoid protein (OCP) is a photochromic carotenoprotein involved in the photoprotection of cyanobacteria. It is activated by blue-green light to a red form OCPR capable of dissipating the excess of energy of the cyanobacterial photosynthetic light-harvesting systems. Activation to OCPR can also be achieved in the dark. In the present work, activation by pH changes of two different OCPs-containing echinenone or canthaxanthin as carotenoids-is investigated in different conditions. A particular emphasis is put on OCP encapsulated in SBA-15 mesoporous silica nanoparticles. It is known that in these hybrid systems, under appropriate conditions, OCP remains photoactive. Here, we show that when immobilised in SBA-15, the OCP visible spectrum is sensitive to pH changes, but such a colorimetric response is very different from the one observed for OCP in solution. In both cases (SBA-15 matrices and solutions), pH-induced colour changes are related either by orange-to-red OCP activation, or by carotenoid loss from the denatured protein. Of particular interest is the response of OCP in SBA-15 matrices, where a sudden change in the Vis absorption spectrum and in colour is observed for pH changing from 2 to 3 (in the case of canthaxanthin-binding OCP in SBA-15: λMAX shifts from 454 to 508 nm) and for pH changing from 3 to 4 (in the case of echinenone-binding OCP in SBA-15: λMAX shifts from 445 to 505 nm). The effect of temperature on OCP absorption spectrum and colour (in SBA-15 matrices) has also been investigated and found to be highly dependent on the properties of the used mesoporous silica matrix. Finally, we also show that simultaneous encapsulation in selected surface-functionalised SBA-15 nanoparticles of appropriate fluorophores makes it possible to develop OCP-based pH-sensitive fluorescent systems. This work therefore represents a proof of principle that OCP immobilised in mesoporous silica is a promising system in the development of colorimetric and fluorometric pH and temperature sensors.

PH-dependent endocytosis mechanisms for influenza A and SARS-coronavirus.

The ongoing SARS-CoV-2 pandemic and the influenza epidemics have revived the interest in understanding how these highly contagious enveloped viruses respond to alterations in the physicochemical properties of their microenvironment. By understanding the mechanisms and conditions by which viruses exploit the pH environment of the host cell during endocytosis, we can gain a better understanding of how they respond to pH-regulated anti-viral therapies but also pH-induced changes in extracellular environments. This review provides a detailed explanation of the pH-dependent viral structural changes preceding and initiating viral disassembly during endocytosis for influenza A (IAV) and SARS coronaviruses. Drawing upon extensive literature from the last few decades and latest research, I analyze and compare the circumstances in which IAV and SARS-coronavirus can undertake endocytotic pathways that are pH-dependent. While there are similarities in the pH-regulated patterns leading to fusion, the mechanisms and pH activation differ. In terms of fusion activity, the measured activation pH values for IAV, across all subtypes and species, vary between approximately 5.0 to 6.0, while SARS-coronavirus necessitates a lower pH of 6.0 or less. The main difference between the pH-dependent endocytic pathways is that the SARS-coronavirus, unlike IAV, require the presence of specific pH-sensitive enzymes (cathepsin L) during endosomal transport. Conversely, the conformational changes in the IAV virus under acidic conditions in endosomes occur due to the specific envelope glycoprotein residues and envelope protein ion channels (viroporins) getting protonated by H+ ions. Despite extensive research over several decades, comprehending the pH-triggered conformational alterations of viruses still poses a significant challenge. The precise mechanisms of protonation mechanisms of certain during endosomal transport for both viruses remain incompletely understood. In absence of evidence, further research is needed.

Exploring the pH Sensitivity of Ion-Pair Interactions on a Self-Assembled Monolayer by Scanning Electrochemical Microscopy

Insights into the chemical essence of weak interactions on the surface of biomacromolecules may help to regulate biological processes. In this work, the pH sensitivity of ion-pair interactions occurring on a cysteine self-assembled monolayer (Cys SAM) that simulates the local surface of a protein was probed by scanning electrochemical microscopy (SECM). Cys SAM and the ion-pair interactions subsequently formed with the introduced aspartic acid (Asp) were both pH-sensitive, as confirmed by the tip current changes in the feedback mode. After continuous pH measurements, the most significant negative feedback was observed at pH 5.50, indicating the most robust ion-pair interactions, which were simultaneously identified by voltammetry. In this case, the extra addition of the inorganic cation (i.e., Ca2+) did not disrupt the existing ion-pair interactions, and the binding constant (K) and Gibbs free energy (ΔGo) of the ion pair were finally determined to be 6.44 × 105 M-1 and -33.14 kJ mol-1, respectively. Overall, the pH sensitivity of ion-pair interactions was found to be mainly attributable to pH-induced changes in the deprotonated/protonated states of the α-amino acid moieties, which may provide insights into the artificial manipulation of complex binding events at the molecular level on the biological surface.

Exploring the mechanisms behind the E. coli pH-responsive riboswitch.

Riboswitches are 5’-untranslated regions of mRNA that change their conformation in response to ligand binding, dictating whether the downstream gene is transcribed and/or translated into protein. A “pH-responsive element” (PRE) riboswitch discovered in E. coli, however, does not require a ligand binding event in a traditional sense to rearrange its structure and instead senses the environmental H+ concentration. Specifically, at neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence (RBS), whereas at alkaline pH, the PRE adopts a translationally active structure with an exposed RBS. The precise mechanism that controls pH-responsive riboswitching has remained unclear, with two proposed models that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that alkaline pH enhances RNAP pause longevity during PRE synthesis to drive folding of structural motifs in the active conformer. I investigated this proposal by performing the first rigorous kinetic study of E. coli RNAP pausing at key sites for PRE folding under different pH conditions. I find that pH influences RNAP pausing, but not in the proposed manner; instead, my experiments uncovered that alkaline pH either decreases or has no effect on RNAP pause longevity. These results indicate that modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding and invite the possibility that the riboswitch RNA is actively involved in sensing the cytosolic pH (Stephen and Mishanina, J. Biol. Chem. 2022, 298:102302). I am currently conducting chemical probing and cryo-EM studies of PRE structure to explore the pH-induced RNA structural changes in rigorous detail, which will ultimately illuminate the mechanism for how pH-dependent riboswitching occurs.

EphH, a unique epoxide hydrolase encoded by Rv3338 is involved in the survival of Mycobacterium tuberculosis under in vitro stress and vacuolar pH-induced changes.

Mycobacterium tuberculosis (Mtb), one of the deadliest human pathogen, has evolved with different strategies of survival inside the host, leading to a chronic state of infection. Phagosomally residing Mtb encounters a variety of stresses, including increasing acidic pH. To better understand the host-pathogen interaction, it is imperative to identify the role of various genes involved in the survivability of Mtb during acidic pH environment. Bio-informatic and enzymatic analysis were used to identify Mtb gene, Rv3338, as epoxide hydrolase. Subsequently, CRISPRi knockdown strategy was used to decipher its role for Mtb survival during acidic stress, nutrient starvation and inside macrophages. Confocal microscopy was used to analyse its role in subverting phagosomal acidification within macrophage. The present work describes the characterization of Rv3338 which was previously known to be associated with the aprABC locus induced while encountering acidic stress within the macrophage. Bio-informatic analysis demonstrated its similarity to epoxide hydrolase, which was confirmed by enzymatic assays, thus, renamed EphH. Subsequently, we have deciphered its indispensable role for Mtb in protection from acidic stress by using the CRISPRi knockdown strategy. Our data demonstrated the pH dependent role of EphH for the survival of Mtb during nutrient starvation and in conferring resistance against elevated endogenous ROS levels during stress environment. To the best of our knowledge, this is the first report of an EH of Mtb as a crucial protein for bacterial fitness inside the host, a phenomenon central to its pathogenesis.