pH-dependent Dynamics Research Articles

Ultrafast spectroscopy of the hydrophilic carotenoid crocin at various pH.

This study delves into the pH-dependent effects on the excited-state behavior of crocin, a hydrophilic carotenoid with diverse functions in biological systems. Steady-state spectroscopy demonstrates notable changes in absorption and fluorescence spectra, characterized by a pH-dependent blue shift and altered resolution of vibrational bands. Transient absorption spectra further elucidate these effects, highlighting a significant blue shift in the S1-Sn peak with increasing pH. Detailed kinetic analysis shows the pH-dependent dynamics of crocin's excited states. At pH 11, a shortening of effective conjugation is observed, resulting in a prolonged S1/ICT lifetime. Conversely, at pH 9, our data suggest a more complex scenario, suggesting the presence of two distinct crocin species with different relaxation patterns. This implies structural alterations within the crocin molecule, potentially linked to the deprotonation of hydroxyl groups in crocin and/or saponification at high pH.

Thiol-5-methylene pyrrolones hydrogels with pH-tunable stress-relaxation and self-healing properties

Hydrogels containing dynamically crosslinked networks through covalent bonds have garnered substantial attention in both academia and technology sectors. This interest stems from their impressive mechanical stability and their unique spatiotemporal dynamic characteristics. Among the various type of dynamic covalent bonds used for preparation hydrogel, the 5-methylene pyrrolones (5MP) and thiol reaction stands out as one of the most prevalent. Given its reliability, efficiency and selectivity, thiol-5MP reaction has long been recognized as some of the most efficient Micheal additions. In this work, by utilizing thiol-5MP Michael addition with improved stability and specificity, a new type of dynamic hydrogel is easily prepared. Notably, the mechanical attributes of the resultant thiol-5MP hydrogels can be finely tuned by modulating the pH during their preparation process. Furthermore, hydrogels formulated under neutral (pH 7.5) or alkaline (pH 8.5) conditions display enhanced stress-relaxation response and superior self-healing capabilities compared to those generated under acidic conditions (pH 6.5). As revealed by single-molecule force spectroscopy assays, the pH-tunable mechanical properties are attributed to the pH-dependent dynamics of thiol-5MP bonds. This work showcases an innovative avenue for crafting dynamic hydrogels featuring pH-adjustable bulk characteristics, highlighting the versatility of thiol-5MP bonds as fundamental building blocks for the design of functional hydrogel materials.

Microscopic Insight into pH-Dependent Conformational Dynamics and Noncanonical Base Pairing in Telomeric i-Motif DNA.

Gene regulatory functions of noncanonical i-motif DNA are associated with dynamic i-motif formation in the cellular environment and pH variation. With atomistic simulations, we show the dramatic influence of solvent pH on the conformational dynamics of biologically relevant telomeric i-motif DNA coupled with protonation of cytosine bases in different conformations. We rationalized the pH-dependent dynamics and conformational variability of the i-motif in terms of base pairing and specific loop motions. The human telomeric i-motif is found to acquire various metastable folded conformations at pH values near the pKa of cytosine with the formation of a noncanonical C:C W:W trans base pair along with the hemiprotonated C:C+ pairs in the i-motif core. pH-dependent dynamics and the local solvent structure of i-motif DNA imply that the presence of a cosolvent or molecular crowding can promote i-motif formation in vivo by changing the conformational fluctuations and hydration state of the structure.

Effect of pH on the structure and function of cyclin-dependent kinase 6.

Cyclin-dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0-4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0-11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0-8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.

The removal of arsenic from solution through biochar-enhanced precipitation of calcium-arsenic derivatives

Arsenic (As) pollution remains a major threat to the quality of global soils and drinking water. The health effects of As pollution are often severe and have been largely reported across Asia and South America. This study investigated the possibility of using unmodified biochar derived from rice husk (RB) and aspen wood (WB) at 400 °C and 700 °C to enhance the precipitation of calcium/arsenic compounds for the removal of As(III) from solution. The approach was based on utilizing calcium to precipitate arsenic in solution and adding unmodified biochar to enhance the process. Using this approach, As(III) concentration in aqueous solution decreased by 58.1% when biochar was added, compared to 25.4% in the absence of biochar. Varying the pH from acidic to alkaline enabled an investigation into the pH dependent dynamics of the approach. Results indicated that significant precipitation was only possible at near neutral pH (i.e. pH = 6.5) where calcium arsenites (i.e. Ca(AsO2)2, and CaAsO2OH•½H2O) and arsenates (i.e. Ca5(AsO4)3OH) were precipitated and deposited as aggregates in the pores of biochars. Arsenite was only slightly precipitated under acidic conditions (pH = 4.5) while no arsenite was precipitated under alkaline conditions (pH = 9.5). Arsenite desorption from wood biochar was lowest at pH 6.5 indicating that wood biochar was able to retain a large quantity of the precipitates formed at pH 6.5 compared to pH 4.5 and pH 9.5. Given that the removal of As(III) from solution is often challenging and that biochar modification invites additional cost, the study demonstrated that low cost unmodified biochar can be effective in enhancing the removal of As(III) from the environment through Ca–As precipitation.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

Unveiling the pH-Dependent Dynamics of the Prepore-to-Pore Transition of a Tc Toxin

Tc toxins are 1.7 MDa protein complexes that are found in insect- and human-pathogenic bacteria. The complex consists of three subunits: the 1.4 MDa TcA pentamer, which mediates target cell association, membrane insertion and toxin translocation, and two smaller subunits, TcB and TcC, which form a 250 kDa cocoon that encapsulates the toxic enzyme. After endocytosis, Tc penetrates the membrane of the host's cells and translocates a deadly enzyme into the cytosol. However, the kinetics of the prepore-to-pore transition are unknown. Here we present continuous wave (cw) EPR and Double Electron-Electron Resonance (DEER) kinetics, on two crucial steps of pore formation: shell opening and linker contraction of TcA. Both steps are triggered by basic pH and proceed slowly with half-lives in the 10 hours range, with no indications of enrichment of an open, non-contracted intermediate. Moreover, we found that mutations in the TcA subunit, which prevent membrane insertion, caused an acceleration of pore formation by at least three orders of magnitudes and modified the pH-dependency of the reaction such that pores are also formed at acidic pH values in vitro. A new cryo-EM structure of the membrane-integration-deficient TcA variant shows a slight opening of the outer shell, which indicates that a structural rearrangement that might be caused by receptor binding in vivo, must precede the prepore-to-pore transition to effectively activate Tc.

PH-Dependent Morphology and Photoresponse of Azopyridine-Terminated Poly(N-isopropylacrylamide) Nanoparticles in Water.

A series of azopyridine-terminated poly(N-isopropylacrylamide)s (PNIPAM) (C12-PN-AzPy) (∼5000 < Mw < 20 000 g mol–1, polydispersity index 1.25 or less) were prepared by reversible addition–fragmentation chain-transfer polymerization of NIPAM in the presence of a chain-transfer agent that contains an AzPy group and an n-dodecyl chain. In cold water, the polymers form nanoparticles (5.9 nm < Rh < 10.9 nm) that were characterized by light scattering (LS), 1H NMR diffusion experiments, and high-resolution transmission electron microscopy. We monitored the pH-dependent photoisomerization of C12-PN-AzPy nanoparticles by steady-state and time-resolved UV–vis absorption spectroscopy. Azopyridine is known to undergo a very fast cis-to-trans thermal relaxation when the azopyridine nitrogen is quaternized or bound to a hydrogen bond donor. The cis-to-trans thermal relaxation of the AzPy chromophore in an acidic nanoparticle suspension is very fast with a half-life τ = 2.3 ms at pH 3.0. It slows down slightly for nanoparticles in neutral water (τ = 0.96 s, pH 7.0), and it is very slow for AzPy-PNIPAM particles in alkaline medium (τ > 3600 s, pH 10). The pH-dependent dynamics of the cis-to-trans dark relaxation, supported by Fourier transform infrared spectroscopy, 1H NMR spectroscopy, and LS analysis, suggest that in acidic medium, the nanoparticles consist of a core of assembled C12 chains surrounded by a shell of hydrated PNIPAM chains with the AzPy+ end groups preferentially located near the particle/water interface. In neutral medium, the shell surrounding the core contains AzPy groups H-bonded to the amide hydrogen of the PNIPAM chain repeat units. At pH 10.0, the amide hydrogen binds preferentially to the hydroxide anions. The AzPy groups reside preferentially in the vicinity of the C12 core of the nanoparticles. The morphology of the nanoparticles results from the competition between the segregation of the hydrophobic and hydrophilic components and weak attractive interactions, such as H-bonds between the AzPy groups and the amide hydrogen of the PNIPAM repeat units.

PH-dependent conformational dynamics of beta-secretase 1: A molecular dynamics study.

Beta-secretase 1 (BACE-1) is an aspartyl protease implicated in the overproduction of β-amyloid fibrils responsible for Alzheimer disease. The process of β-amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH-dependent dynamics of BACE-1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β-amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double-protonated, mono-protonated, and double-deprotonated states over the known pH range of higher activity. These differences include Tyr 71-aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE-1 activity.

Proton-Coupled Conformational Allostery Modulates the Inhibitor Selectivity for β-Secretase.

Many important pharmaceutical targets, such as aspartyl proteases and kinases, exhibit pH-dependent dynamics, functions and inhibition. Accurate prediction of their binding free energies is challenging because current computational techniques neglect the effects of pH. Here we combine free energy perturbation calculations with continuous constant pH molecular dynamics to explore the selectivity of a small-molecule inhibitor for β-secretase (BACE1), an important drug target for Alzheimer's disease. The calculations predicted identical affinity for BACE1 and the closely related cathepsin D at high pH; however, at pH 4.6 the inhibitor is selective for BACE1 by 1.3 kcal/mol, in excellent agreement with experiment. Surprisingly, the pH-dependent selectivity can be attributed to the protonation of His45, which allosterically modulates a loop-inhibitor interaction. Allosteric regulation induced by proton binding is likely common in biology; considering such allosteric sites could lead to exciting new opportunities in drug design.

Conformational Dynamics and Binding Free Energies of Inhibitors of BACE-1: From the Perspective of Protonation Equilibria.

BACE-1 is the β-secretase responsible for the initial amyloidogenesis in Alzheimer’s disease, catalyzing hydrolytic cleavage of substrate in a pH-sensitive manner. The catalytic mechanism of BACE-1 requires water-mediated proton transfer from aspartyl dyad to the substrate, as well as structural flexibility in the flap region. Thus, the coupling of protonation and conformational equilibria is essential to a full in silico characterization of BACE-1. In this work, we perform constant pH replica exchange molecular dynamics simulations on both apo BACE-1 and five BACE-1-inhibitor complexes to examine the effect of pH on dynamics and inhibitor binding properties of BACE-1. In our simulations, we find that solution pH controls the conformational flexibility of apo BACE-1, whereas bound inhibitors largely limit the motions of the holo enzyme at all levels of pH. The microscopic pKa values of titratable residues in BACE-1 including its aspartyl dyad are computed and compared between apo and inhibitor-bound states. Changes in protonation between the apo and holo forms suggest a thermodynamic linkage between binding of inhibitors and protons localized at the dyad. Utilizing our recently developed computational protocol applying the binding polynomial formalism to the constant pH molecular dynamics (CpHMD) framework, we are able to obtain the pH-dependent binding free energy profiles for various BACE-1-inhibitor complexes. Our results highlight the importance of correctly addressing the binding-induced protonation changes in protein-ligand systems where binding accompanies a net proton transfer. This work comprises the first application of our CpHMD-based free energy computational method to protein-ligand complexes and illustrates the value of CpHMD as an all-purpose tool for obtaining pH-dependent dynamics and binding free energies of biological systems.

Clarifying the Intersection Between Alpha-Crystallin B Function and Oligomerization by Altering Aggregation Conditions

Alpha-Crystallin-B (aXB) is a small heat shock protein found mainly in the lens of the eye. There it serves two purposes: acting as a chaperone to prevent the misfolding of the other protein and contributing to the high protein concentrations required to focus light. When aXB chaperone function fails cataract formation begins. Understanding the dependence of the mechanism of aXB chaperone function on its oligomerization state will aid in the delay or prevention of cataracts. Oligomerization of alpha-crystallinB is pH dependent. We present a mechanism for the chaperone function of aXB using insulin as a model system in which it is possible to induce aggregation, The relationship between oligomerization and chaperone function is tested by measuring the dependence of the onset of light scattering insulin aggregates on the oligomerization state of the aXB Different oligomerization states can be induced by pre-incubating aXB at a range of pHs. Attenuated chaperone function is observed for aXB pre-incubated at elevated pH even when the assay itself takes place at pH 7. These results are consistent with a model in which aXB is kinetically trapped into higher oligomer states at high pH and unable to return quickly to its functional equilibrium state upon dilution to neutral pH. We will also test the generality of this model by extending our studies to Gamma-Crystallin. Finally, we will measure the contribution of the C-terminal strand to chaperone function and oligomerization using fluorescence resonance energy transfer (FRET). Observation of pH dependent dynamics of the C-terminal strand will help to distinguish between stand-exchanged and dynamic states of the C terminus, allowing its contribution to oligomerization and chaperone function to be probed.

Toward Accurate Prediction of the Protonation Equilibrium of Nucleic Acids

The role of protonated nucleotides in modulating the pH-dependent properties of nucleic acids is one of the emerging frontiers in the field of nucleic acid biology. The recent development of a constant pH molecular dynamics simulation (CPHMDMSλD) framework for simulating nucleic acids has provided a tool for realistic simulations of pH-dependent dynamics. We enhanced the CPHMDMSλD framework with pH-based replica exchange (pH-REX), which significantly improves the sampling of both titration and spatial coordinates. The results from our pKa calculations for the GAAA tetraloop, which was predicted with lower accuracy previously due to sampling challenges, demonstrates that pH-REX reduces the average unsigned error (AUE) to 0.7 pKa units, and the error of the most poorly predicted residue A17 was drastically reduced from 2.9 to 1.2 pKa unit. Lastly, we show that pH-REX CPHMDMSλD simulations can be used to identify the dominant conformation of nucleic acid structures in alternate pH environments. This work suggests that pH-REX CPHMDMSλD simulations provide a practical tool for predicting nucleic acid protonation equilibrium from first-principles, and offering structural and mechanistic insight into the study of pH-dependent properties of nucleic acids.

PH-dependent dynamics of complex RNA macromolecules.

The role of pH-dependent protonation equilibrium in modulating RNA dynamics and function is one of the key unanswered questions in RNA biology. Molecular dynamics (MD) simulations can provide insight into the mechanistic roles of protonated nucleotides, but it is only capable of modeling fixed protonation states and requires prior knowledge of the key residue's protonation state. Recently, we developed a framework for constant pH molecular dynamics simulations (CPHMDMSλD) of nucleic acids, where the nucleotides' protonation states are modeled as dynamic variables that are coupled to the structural dynamics of the RNA. In the present study, we demonstrate the application of CPHMDMSλD to the lead-dependent ribozyme; establishing the validity of this approach for modeling complex RNA structures. We show that CPHMDMSλD accurately predicts the direction of the pKa shifts and reproduces experimentally-measured microscopic pKa values with an average unsigned error of 1.3 pKa units. The effects of coupled titration states in RNA structures are modeled, and the importance of conformation sampling is highlighted. The general accuracy of CPHMDMSλD simulations in reproducing pH-dependent observables reported in this work demonstrates that constant pH simulations provides a powerful tool to investigate pH-dependent processes in nucleic acids.

In Vitro Phosphorylation of the Focal Adhesion Targeting Domain of Focal Adhesion Kinase by Src Kinase

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

Relevance of extracellular and intracellular interactions of camptothecins as determinants of antitumor activity

Camptothecins are potent antitumor agents that stabilize the covalent binding of topoisomerase I to DNA forming a reversible ternary complex which, following collision with the replication forks, converts the single-strand breaks into lethal double-strand breaks. This cytotoxic mechanism has been originally ascribed to the closed lactone form, because opening of the lactone ring resulted in loss of antitumor activity. Since the lipophilic lactone favours passive diffusion into the cancer cells, the stability of the closed form is expected to be predictive for activity. Thus, the in vivo pharmacological behavior of camptothecins, which is dependent on the pH-dependent dynamics, is likely a critical determinant of their antitumor efficacy and therapeutic index. The physicochemical properties could influence a number of cellular and in vivo interactions, including stability of the ternary DNA–enzyme–drug complex, binding to serum proteins, recognition by transport systems. These interactions are also implicated in the processes responsible of toxic side effects and drug resistance which are major limitations of the efficacy of camptothecin-based therapy. A number of strategies have been developed to overcome the limitations associated with the peculiar in vivo reactivity and the reversibility of drug–target interaction. Modifications with hydrophilic or lipophilic substituents at specific positions may have a variable (and somewhat opposite) influence on interaction with the intracellular target and plasma proteins and on recognition by membrane transporters. Here, we highlight the interactions of camptothecins which could be exploited to optimize therapeutic efficacy.

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.