Temperature-dependent Binding Research Articles

Transglutaminase–mucin binding dynamics in gastrointestinal mucus: Interfacial behaviour, thermodynamics and gelation mechanism

Transglutaminase, an enzyme present in epithelial cells and as a residual component in processed foods, is hypothesised to interact with gastrointestinal mucus, impacting its structure and function. To test the physical validity of this phenomenon, this study investigates the binding kinetics and thermodynamics between porcine gastric mucin (PGM) and microbial transglumatinse (TGM) in a model gastrointestinal mucus, followed by the rheological analysis of the resulting systems. At pH 7, TGM exhibited pronounced binding with the PGM interface, characterised by a high surface density (55.34 ± 1.86 µg/m−2) and a low dissociation constant (KD ∼ 4.03 ± 0.10 μM) as determined by surface plasma resonance (SPR). Conversely, at pH 3, TGM showed weak adhesion onto PGM, resulting in a less stable binding, reflected by a lower surface density (10.94 ± 0.67 µg/m−2) and a higher dissociation constant (KD ∼ 6.24 ± 0.22 μM). Regardless of the nature of interaction, the Hill coefficients (nH ≥ 1) indicated that the binding sites were markedly denser at pH 7 (1383.38 ± 46.47 pmol/m−2), than at pH 3 (273.68 ± 16.84 pmol/m−2). Fluorimetry analysis suggested temperature-dependent dynamic binding between PGM and TGM. The resulting Benesi – Hildebrand plots illustrated a linear correlation between PGM and increasing TGM concentration, suggesting a single-step interaction mechanism. The calculated thermodynamic parameters indicated spontaneous interaction between PGM and TGM (ΔG < 0) via endothermic (entropic) interactions (ΔH > 0). Notably, hydrophobic forces played a significant role in the network stabilisation of PGM−TGM complex (ΔS > 0). Rheometry analysis elucidates that the interaction maxima within TGM−PGM systems substantially elevate both shear viscosity (η), and the melting point (Tm), shifting from 6.75 ± 0.28 to 70.94 ± 2.21 (×10−3) Pa s (at 1 s−1), and 36.80 ± 0.80 to 48.20 ± 0.11 °C, respectively. Furthermore, the coexistence of these two macromolecular species results in a 3- to 4-fold dramatic increase in the viscoelastic moduli of the binary complex. These findings build a strong physicochemical basis for the interaction between TGM and PGM, with profound effects on the rhological behaviour of the latter; it also highlights the need to examine the biochemical/enzymatic aspects of said interactions, and their potential effects on mucosa and human physiology.

Recognition of Amino Acid Salts by Temperature-Dependent Allosteric Binding with Stereodynamic Urea Receptors.

Positive homotropic artificial allosteric systems are important for the regulation of cooperativity, selectivity and nonlinear amplification. Stereodynamic homotropic allosteric receptors can transmit and amplify induced chirality by the first ligand binding to axial chirality between two chromophores. We herein report stereodynamic allosteric urea receptors consisting of a rotational shaft as the axial chirality unit, terphenyl units as structural transmission sites and four urea units as binding sites. NMR titration experiments revealed that the receptor can bind two carboxylate guests in a positive homotropic allosteric manner attributed to the inactivation by intramolecular hydrogen-bonding between urea units within the receptor. In addition, the VT-CD spectra observed upon binding of the urea receptor with l- or D-amino acid salts in MeCN showed interesting temperature-dependent Cotton effects, based on the differences of the receptor shaft unit and the guest structure. The successful discrimination of hydrocarbon-based side chains of amino acid salts indicated that the input of chiral and steric information for the guest was amplified as outputs of the Cotton effect and the temperature-dependence of VT-CD spectra through cooperativity of positive allosteric binding.

Antiproliferative activity of platinum(II) and copper(II) complexes containing novel biquinoxaline ligands.

Nowadays, cancer represents one of the major causes of death in humans worldwide, which renders the quest for new and improved antineoplastic agents to become an urgent issue in the field of biomedicine and human health. The present research focuses on the synthesis of 2,3,2',3'-tetra(pyridin-2-yl)-6,6'-biquinoxaline) and (2,3,2',3'-tetra(thiophen-2-yl)-6,6'-biquinoxaline) containing copper(II) and platinum(II) compounds as prodrug candidates. The binding interaction of these compounds with calf thymus DNA (CT-DNA) and human serum albumin were assessed with UV titration, thermal decomposition, viscometric, and fluorometric methods. The thermodynamical parameters and the temperature-dependent binding constant (K'b) values point out to spontaneous interactions between the complexes and CT-DNA via the van der Waals interactions and/or hydrogen bonding, except Cu(ttbq)Cl2 for which electrostatic interaction was proposed. The antitumor activity of the complexes against several human glioblastomata, lung, breast, cervix, and prostate cell lines were investigated by examining cell viability, oxidative stress, apoptosis-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, in vitro migration and invasion, in vitro-comet DNA damage, and plasmid DNA interaction assays. The U87 and HeLa cells were investigated as the cancer cells most sensitive to our complexes. The exerted cytotoxic effect of complexes was attributed to the formation of the reactive oxygen species in vitro. It is clearly demonstrated that Cu(ttbq)Cl2, Pt(ttbq)Cl2, and Pt(tpbq)Cl2 have the highest DNA degradation potential and anticancer effect among the tested complexes by leading apoptosis. The wound healing and invasion analysis results also supported the higher anticancer activity of these two compounds.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Exploring novel Cd(II) complexes with 5-methyl-4-imidazolecarboxaldehyde: synthesis, structure, computational insights, and affinity to DNA through switchSense methodology.

A series of four Cd(II) complexes with 5-methyl-4-imidazolecarboxaldehyde (L) with different inorganic anions within or outside the coordination sphere of general formula: [CdCl2L2] (1), [CdBr2L2] (2), [CdI2L2] (3), and [CdL4](PF6)2·3H2O (4) was synthesized through one-step and two-step reactions, respectively. All complexes were obtained as colorless crystals without the need for recrystallization and exhibited solubility in aqueous solutions. Structural analysis revealed different coordination environments for each complex, with variations in bond lengths and angles. The crystal packing of the complexes was stabilized by hydrogen bonding and π-π stacking interactions. FT-IR analysis indicated coordination of the ligand to the metal ion, and UV-Vis studies confirmed the stability of the complexes in solution. Computational analysis has revealed the polar nature of the complexes and their favorable stability constants. Affinity studies with DNA using the switchSense technique demonstrated rapid association and dissociation processes for all complexes, with temperature-dependent binding constants. Thermodynamic analysis suggested spontaneous with positive entropy change and endothermic formation processes for the complexes. Overall, the study underscores the synthesis, examination, and interaction with DNA of Cd(II) complexes, demonstrating their promise within medicinal chemistry.

Alpha clustering in 41, 45, 49Ca* nuclei formed in neutron induced reactions

The light neutron-rich nuclei play a vital role in nucleosynthesis process and the extent of alpha (α) clustering significantly influence the astrophysical rates. Thus, it is significant to explore the α clustering in these nuclei and in the present work, we have studied the α clustering in 41,45,49Ca* nuclei formed in neutron induced reactions within the dynamical cluster decay model (DCM). The results present that with progression towards neutron-rich 45Ca* and 49Ca* nuclei, there is a significant decrease in the α-cluster preformation factor P0. The inclusion of relativistic mean field theory (RMFT) based microscopic temperature-dependent binding energies (T.B.E.) within DCM, give relatively enhanced α-cluster preformation factor for 41,45,49Ca* nuclei compared to the case of macroscopic T.B.E. based upon Davidson mass formula. The cross-section associated with α-cluster emission depicts strong isospin dependence and falls off significantly with increasing neutron number of Ca* nuclei. Further, for the first time, we inculcate the microscopic nuclear potential constructed via folding the standard Fermi form fitted RMFT cluster densities and M3Y nucleon-nucleon interaction within the DCM. The neutron skin thickness of the Ar cluster, complementary to α-cluster, is varied and its effect upon the nuclear interaction potential and α-cluster preformation factor is analysed. The results present that with growing neutron skin of Ar cluster, the α-cluster preformation factor decreases. It explores a strong correlation among the neutron skin thickness and α-cluster preformation factor in light mass 41,45,49Ca* nuclear systems.

Excitation Density Effects in the Luminescence Yield and Kinetics of MAPbBr3 Single Crystals

The luminescent Z-scan technique with time resolution is applied to the study of the luminescence properties of CH3NH3PbBr3 single crystals representative of the family of hybrid organic–inorganic lead perovskites successfully applied recently in photovoltaics and currently investigated as potential nanosecond scintillators. The third harmonic of Ti-sapphire laser (λ = 266 nm) with a pulse duration of 26 fs and 1 kHz frequency was applied for the luminescence excitation creating the charge carriers with the estimated density from 1017 to 1021 cm−3 in the temperature range from 13 to 300 K. Temperature and excitation density dependence of the luminescence yield and kinetics is interpreted with the consideration of the temperature-dependent binding of electrons and holes into excitons, a saturation of defects responsible for the non-radiative relaxation channel competing with exciton creation; absorption saturation resulting in the increased penetration depth of the excitation radiation and hence the increased contribution of the re-absorption.

Molecular docking and In-Ovo analysis of human amniotic fluid extracellular vesicles loaded with sulforaphane: A potential therapy for neurological disorders

Sulforaphane (SFN) is a bioactive component found in broccoli that has neuroprotective and anti-cancer effects. SFN has been encapsulated in various drug carriers to improve its stability and effectiveness. Specifically, this study presents an SFN delivery system based on human Amniotic Fluid-EVs (hAFEVs) for the treatment of neurological disorders. The binding affinity between hAFEVs (<200 nm) and SFN was investigated by spectroscopic analysis, revealing a temperature-dependent binding mechanism. The binding constants (ksv, kq, and kb) were also assessed, indicating a static quenching mechanism. Additionally, the docking studies of SFN and CD9, CD81, HSP70, and TSG101 were performed using AutoDock 4.2.6 software's Lamarckian genetics algorithm to investigate the type of interaction and their effects on the targetability of hAFEVs. The obtained results showed that SFN-loaded hAFEVs had a favorable effect on increasing the survival and proliferation of human PC12 cells (137%). The results of gene expression studies showed that this release system can upregulate the expression of Nrf2 and decrease the expression of IL-6, which shows the support of nerve cells against oxidative stress. In the In-Ovo study, treatment with SFN-hAFEVs resulted in improved grey and white matter cohesion and the improved orientation of bipolar neurons. The results indicate a significant increase in synaptophysin expression and the number of doublecortin-positive cells improved neuronal development and synapse formation. The findings of this study suggest that SFN-hAFEVs have great potential as a treatment for neurological disorders.

Effects of temperature and microwave on the stability of the blast effector complex APikL2A/sHMA25 as determined by molecular dynamics analyses.

Magnaporthe oryzae is the causal agent of rice blast, and understanding how abiotic stress affects the resistance of plants to this disease is useful for designing disease control strategies. In this paper, the effects of temperature and microwave irradiation on the effector complex comprising APikL2A from M. oryzae and sHMA25 from foxtail millet were investigated by molecular dynamics simulations using the GROMACS software package. While the structure of APikL2A/sHMA25 remained relatively stable in a temperature range of 290K (16.85°C) to 320K (46.85°C), the concave shape of the temperature-dependent binding free energy curve indicated that there was maximum binding affinity between APikL2A and sHMA25 at 300K-310K. This coincided with the optimum infectivity temperature, thus suggesting that coupling of the two polypeptides may play a role in the infection process. A strong oscillating electric field destroyed the structure of APikL2A/sHMA25, although it was stable and not susceptible to weak electric fields.

The temperature-dependence of host-guest binding thermodynamics: experimental and simulation studies.

The thermodynamic parameters of host-guest binding can be used to describe, understand, and predict molecular recognition events in aqueous systems. However, interpreting binding thermodynamics remains challenging, even for these relatively simple molecules, as they are determined by both direct and solvent-mediated host-guest interactions. In this contribution, we focus on the contributions of water to binding by studying binding thermodynamics, both experimentally and computationally, for a series of nearly rigid, electrically neutral host-guest systems and report the temperature-dependent thermodynamic binding contributions ΔGb(T), ΔHb(T), ΔSb(T), and ΔCp,b. Combining isothermal titration calorimetry (ITC) measurements with molecular dynamics (MD) simulations, we provide insight into the binding forces at play for the macrocyclic hosts cucurbit[n]uril (CBn, n = 7-8) and β-cyclodextrin (β-CD) with a range of guest molecules. We find consistently negative changes in heat capacity on binding (ΔCp,b) for all systems studied herein - as well as for literature host-guest systems - indicating increased enthalpic driving forces for binding at higher temperatures. We ascribe these trends to solvation effects, as the solvent properties of water deteriorate as temperature rises. Unlike the entropic and enthalpic contributions to binding, with their differing signs and magnitudes for the classical and non-classical hydrophobic effect, heat capacity changes appear to be a unifying and more general feature of host-guest complex formation in water. This work has implications for understanding protein-ligand interactions and other complex systems in aqueous environments.

Cu(I)-phospine complex exhibits temperature-dependent DNA intercalative binding: Insights from spectroscopic and molecular modeling studies

The synthesis and characterization of a Cu(I) phosphine complex, as well as spectroscopic and simulation studies of its interaction with the DNA helix, are described. Based on X-ray single-crystal diffraction data, the monoclinic phase [Cu(PPh3)(L)(I)], L = 2-((pyridin-2-ylmethylene)amino)isoindoline-1,3-dione, with a tetrahedral molecular geometry was synthesised. The experimental results suggested that the complex could target DNA with a high affinity via an intercalation binding mode. The Stern–Volmer analysis based on fluorimetry approved that raising the temperature from 283 to 293 K causes the Ke to increase, confirmed a dynamic mechanism in the interaction while a further increase of temperature to 303 K, causes the opposite consequence which confirmed a static nature for the interaction at the high range of temperatures. Furthermore, a concluded positive cooperativeness at ambient temperature would be particularly important to achieve a significant impact of the complex on DNA strands even at low complex concentrations. According to competitive fluorescence tests the complex interacts with DNA at intercalation binding sites on DNA. The resulting entropy and enthalpy shifts identify the van der Waals forces as the interaction's primary binding mechanism. Docking study confirmed the insertion of aromatic ring into the DNA double helix.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.

Graphene and NTCDA adsorbed on Ag(111): Temperature-dependent binding distance and phonon coupling to the interface state

Flat lying physisorbates on surfaces can be expected to be lifted by heating. The details of this lattice expansion depend on the competition between two mechanisms. On the one hand, the interaction potential of each atom with the substrate is anharmonic and constitutes the reason for lifting. On the other hand, the internal bending stiffness of the planar adsorbate reduces its tendency to lift with temperature. Here we show that this competition can be investigated in a simplified molecular-dynamics simulation which considers only the vertical motion of the atoms of the adsorbate in respective model potentials. For two prototypical systems, i.e., graphene and 1,4,5,8-naphthalene-tetracarboxylic acid dianhydride (NTCDA) on Ag(111), we observe lifting by 0.04 \AA{} (graphene) and 0.06 \AA{} (NTCDA) between zero and room temperature. For the latter system, the impact of the temperature-dependent geometrical and vibrational properties on the occurring interface state (IS) is discussed. By introducing a local electron-phonon interaction model, we combine the indirect dependence of the IS on the average binding distance with direct coupling to the vibrational modes and obtain good agreement with the experimental results.

Temperature-Responsive Nanoparticles Enable Specific Binding of Apolipoproteins from Human Plasma.

Apolipoproteins are an important class of proteins because they provide a so-called stealth effect to nanoparticles. The stealth effect on nanocarriers leads to a reduced unspecific uptake into immune cells and thereby to a prolonged blood circulation time. Herein, a novel strategy to bind apolipoproteins specifically on nanoparticles by adjusting the temperature during their incubation in human plasma is presented. This specific binding, in turn, allows a control of the stealth behavior of the nanoparticles. Nanoparticles with a well-defined poly(N-isopropylacrylamide) shell are prepared, displaying a reversible change of hydrophobicity at a temperature around 32°C. It is shown by label-free quantitative liquid chromatography-mass spectrometry that the nanoparticles are largely enriched with apolipoprotein J (clusterin) at 25°C while they are enriched with apolipoprotein A1 and apolipoprotein E at 37°C. The temperature-dependent protein binding is found to significantly influence the uptake of the nanoparticles by RAW264.7 and HeLa cells. The findings imply that the functionalization of nanoparticles with temperature-responsive materials is a suitable method for imparting stealth properties to nanocarriers for drug-delivery.

Effects of chain length, temperature, and ionic strength on association and dissociation thermodynamics of DNA

In this study, association of oligonucleotides of length 8-22 bases and their complementary oligonucleotides to form DNA was analyzed using isothermal titration calorimetry. Additionally, dissociation of DNA into its respective oligonucleotides was analyzed using differential scanning calorimetry. The effects of chain length, temperature, and ionic strength on the association and dissociation of DNA were evaluated using thermodynamic analyses. At 25 °C, the association of 10-base oligonucleotides largely depended on ionic strength, however, for oligonucleotides of 12-base and above, association was independent of ionic strength. In oligonucleotides of length 12-, 16-, and 22-bases, both binding enthalpy and entropy changes decreased with increase in chain length around 25 °C, providing similar binding affinity. The highest association constant was approximately 1 × 109 M–1. During association of DNA oligonucleotide of the same chain length, binding enthalpy change gradually decreased with increase in temperature, indicating a negative heat capacity change around 25 °C. As the temperature increased close to the melting temperature, the binding enthalpy change increased, indicating a positive heat capacity change. The temperature-dependent binding enthalpy change was apparently extrapolated to the calorimetric enthalpy change of DNA at the melting temperature. At the melting temperature, the effect of chain length and ionic strength could be observed clearly; the stability of DNA increased with increasing chain length and ionic strength.

Role of microscopic temperature-dependent binding energies in the decay of Si*32 formed in the O20+C12 reaction

The investigation of fusion reactions involving light neutron-rich exotic nuclei is of paramount significance to understand nucleosynthesis in astrophysical scenarios. It is also estimated as a possible heat source to ignite $^{12}\mathrm{C}+^{12}\mathrm{C}$ reaction and production of x-ray superbursts from accreting neutron star. Recently, the fusion of neutron-rich $^{20}\mathrm{O}$ with $^{12}\mathrm{C}$ target has been studied with measurement of fusion cross-section $({\ensuremath{\sigma}}_{\text{fus}})$. Bass model under predicts the ${\ensuremath{\sigma}}_{\text{fus}}$ and time-dependent Hartree-Fock model also fails to explain the experimental data. To explicate the same, the investigation of $^{20}\mathrm{O}+^{12}\mathrm{C}$ reaction at near barrier energies has been made within quantum mechanical fragmentation-based dynamical cluster-decay model (DCM). Within DCM, the fragmentation potential comprises temperature-dependent Coulomb, nuclear and centrifugal potentials, along with temperature-dependent binding energies (T.B.E.) calculated within the macroscopic approach of Davidson mass formula. Recently, we have explored the temperature-dependence of different nuclear properties and nuclear symmetry energy within microscopic relativistic mean-field (RMF) theory [M. Kaur et al., Nucl. Phys. A 1000, 121871 (2020)]. In the present work, we inculcate the microscopic T.B.E. from RMF theory within DCM and investigate the structure of fragmentation potential for $^{32}\mathrm{Si}^{*}$ formed in $^{20}\mathrm{O}+^{12}\mathrm{C}$ reaction, comparatively for macroscopic (mac) and microscopic (mic) T.B.E. obtained from Davidson mass formula and RMF theory, respectively. The structure and magnitude of fragmentation potential are found to change drastically/notably along with a change in energetically favored/minimized fragments for both choices of T.B.E. The $\ensuremath{\alpha}$ particles $(^{4}\mathrm{He}$, $^{5}\mathrm{He})$ are favored at lower angular momenta in fragmentation profile for mic T.B.E. case only, which is in the agreement with predictions of statistical model results. This change in the nuclear structure embodied via fragmentation potential energy carries its imprints in the preformation probability ${P}_{0}$ of different fragments and affects the contribution of individual light-charged particle (LCP) channel in the ${\ensuremath{\sigma}}_{\text{fus}}$. A comparison of the relative cross-section of different LCP channels toward ${\ensuremath{\sigma}}_{\text{fus}}$ is quite different for both cases of T.B.E. The cross-section of $^{2}\mathrm{H}$ and $^{4}\mathrm{He}$ LCP channels is relatively enhanced for mic T.B.E. compared to mac T.B.E. Among different LCP channels, the $^{5}\mathrm{He}$ channel is the major contributor in ${\ensuremath{\sigma}}_{\text{fus}}$, which is in line with the results of the statistical EVAPOR model. The DCM-calculated ${\ensuremath{\sigma}}_{\text{fus}}$ is in agreement with the experimental data.

Clumped isotope geochemistry of island carbonates in the South China Sea: Implications for early diagenesis and dolomitization

Studying the diagenesis of island carbonates can be hampered by the difficulty of accurately constraining the mineral formation temperature. The carbonate clumped isotope composition (measured as the Δ47 value) is based on the temperature-dependent binding of 13C to 18O, and offers a promising means of calculating the formation temperature. In this study, we report the Δ47 values of island carbonates from a ~1 km-long drill core from the Meiji Reef in the South China Sea, which are integrated with petrography, carbon (δ13C) and oxygen isotope (δ18O) compositions, and trace element contents to constrain the diagenetic environment. We find that most of the digenetic alteration of the initial carbonates likely occurred above a water depth of ~200 m below sea level at temperatures between ~15 and 28 °C, and that the fluids involved during the formation of the diagenetic calcites and dolomites have similar δ18O values to modern seawater. Considering the estimated secular variation in seawater δ18O, the dolomite likely formed from an evaporative fluid source via a brine reflux mechanism and with a high convection rate during the late Miocene. Although isotopic compositions of the reef carbonates were overprinted by early diagenetic alteration, the Δ47 values can reflect the temperature of their depositional environment if the diagenesis occurred at depths similar to the depositional depth. These results can help refine the existing dolomitization model of tropical islands and increase the utility of Δ47 values of diagenetic reef carbonates in shallow burial settings as valuable records of the shallow water and climate conditions.

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding.

Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.

Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.

Temperature dependence of the SARS-CoV-2 affinity to human ACE2 determines COVID-19 progression and clinical outcome

The SARS-CoV-2 virus and its homolog SARS-CoV penetrate human cells by binding of viral spike protein and human angiotensin converting enzyme II (ACE2). SARS-CoV causes high fever in almost all patients, while SARS-CoV-2 does not. Moreover, analysis of the clinical data revealed that the higher body temperature is a protective factor in COVID-19 patients, making us to hypothesize a temperature-dependent binding affinity of SARS-CoV-2 to human ACE2 receptor. In this study, our molecular dynamics simulation and protein surface plasmon resonance cohesively proved the SARS-CoV-2-ACE2 binding was less affinitive and stable under 40 °C (~18 nM) than the optimum temperature 37 °C (6.2 nM), while SARS-CoV-ACE2 binding was not (6.4 nM vs. 8.5 nM), which evidenced the temperature-dependent affinity and explained that higher temperature is related to better clinical outcome. The decreased infection at higher temperature was also validated by pseudovirus entry assay using Vero and Caco-2 cells. We also demonstrated the structural basis of the distinct temperature-dependence of the two coronaviruses. Furthermore, the meta-analysis revealed a milder inflammatory response happened in the early stage of COVID-19, which explained the low fever tendency of COVID-19 and indicated the co-evolution of the viral protein structure and the inflammatory response. The temperature dependence of the binding affinity also indicated that higher body temperature at early stages might be beneficial to the COVID-19 patients.