Binding Of Calcium Ions Research Articles

Binding of calcium ions to putative antibiotics.

Calcium-dependent antibiotics represent a family of antibiotics with so far only one clinically approved member, daptomycin. Daptomycin binds to anionic bacterial membrane in a calcium-dependent fashion and induces lipid clustering. The synthesis and design of related substances is a promising source of antibiotics at a time where antibiotic resistance looms larger than ever. By developing a series of substances that bind to anionic membranes in a manner that depends on the presence of calcium ions, we hope to determine the minimum requirements for antibiotic activity. Calcium dependent antibiotics must bind calcium to function, and therefore their affinity for calcium is essential to understanding their efficacy. We determined the calcium affinity of novel compounds by pH titration, in conjunction with UV-vis spectroscopy, in the presence and absence of calcium ions. The apparent shift in the measured pKa can be used to determine the binding constant of calcium. We then compared the calcium binding constants thus obtained with those measured using a standard Ca2+-selective electrode. The next step will be to measure Ca2+-binding constants of lipophilic analogues when associated with a lipid bilayer. Conclusions regarding the suitability of a selection of compounds are discussed.

Effect of Buffers with Multiple Binding Sites on Calcium Waves

The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and unbinding of calcium ions to a multitude of different buffers in the cell. These buffers can be mobile or immobile and, in general, have multiple binding sites that are not independent. Previous theoretical studies have focused on the case when each buffer molecule binds a single calcium ion. In this study, we analyze how calcium waves are affected by calcium buffers with two non-independent binding sites, and show that the interactions between the calcium binding sites can result in the emergence of new behaviors. In particular, for certain combinations of kinetic parameters, the profiles of buffer molecules with one calcium ion bound can be non-monotone.

Structure elucidation of Staphylococcus capitis lipase. Molecular dynamics simulations to investigate the effects of calcium and zinc ions on the structural stability

Cold-adapted and organic solvent tolerant lipases have significant potential in a wide range of synthetic reactions in industry. But there are no sufficient studies on how these enzymes interacts with their substrates. Herein, the predicted structure and function of the Staphylococcus capitis lipase (SCL) are studied. Given the high amino acid sequence homology with the Staphylococcus simulans lipase (SSL), 3D structure models of closed and open forms of the S. capitis lipase were built using the structure of SSL as template. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. The SCL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions. By following this model and utilizing molecular dynamics (MD) simulations, the stability of the S. capitis lipase at low temperatures could be explained in the presence and in the absence of calcium and zinc. Due to its thermolability, the SCL is extremely valuable for different biotechnological applications in a wide variety of industries from molecular biology to detergency to food and beverage preparation. Communicated by Ramaswamy H. Sarma

On the Binding Mechanisms of Calcium Ions to Polycarboxylates: Effects of Molecular Weight, Side Chain, and Backbone Chemistry.

We experimentally determined the characteristics and Langmuir parameters of the binding of calcium ions to different polycarboxylates. By using potentiometric titrations and isothermal titration calorimetry, the effects of side chain chemistry, pH value, and chain length were systematically investigated using the linear polymers poly(aspartic acid), poly(glutamic acid), and poly(acrylic acid). We demonstrate that for polymers with high polymerization degrees, the binding process is governed by higher-order effects, such as the change of apparent pKa of carboxyl groups, and contributions arising from the whole polymer chain while the chemistry of the monomer unit constituting the polymer plays a subordinate role. In addition, primary binding sites need to be present in the polymer, thus rendering the abundance and sequential arrangement of protonated and deprotonated groups important. The detection of higher-order effects contradicts the assumptions posed by the Langmuir model of noninteracting binding sites and puts a question mark on whether ion binding to polycarboxylates can be described using solely a Langmuir binding model. No single uniform mechanism fits all investigated systems, and the whole polymer chain, including terminal groups, needs to be considered for the interpretation of binding data. Therefore, one needs to be careful when explaining ion binding to polymers solely based on studies on monomers or oligomers.

Codelivery of minocycline hydrochloride and dextran sulfate via bionic liposomes for the treatment of spinal cord injury

After primary injury to the spinal cord, a series of microenvironmental changes can lead to secondary injury. The use of nano-targeted drug delivery systems to improve the postinjury microenvironment, inhibit inflammation and reduce neuronal apoptosis can be of great help during spinal cord injury (SCI) recovery. In this study, we prepared primary macrophage membranes bionic modified nanoliposomes (MH-DS@M−Lips) loaded with minocycline hydrochloride (MH) and dextran sulfate (DS) to target their delivery to the site of injury to bind calcium ions in situ and form metal ion complexes. Complex formation reduced calcium ion concentrations and calcium-associated neuronal apoptosis, while MH was slowly released to produce better anti-inflammatory effects. The successful preparation of MH-DS@M−Lips was verified using transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), western blotting and dynamic light scattering (DLS). The targeting capability of the MH-DS@M−Lips was demonstrated using a Transwell system and an in vivo imaging system. The therapeutic efficacy of MH-DS@M−Lips was examined in vitro and in vivo using flow cytometry, immunofluorescence, ELISA kits and western blotting. The results showed that SCI mice treated with MH-DS@M−Lips received high behavioral scores, which led to the conclusion that MH-DS@M−Lips have great potential for the treatment of SCI.

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 μM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection.We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Rational Design of a Calcium-Independent Trypsin Variant

Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential is based on its high catalytic activity, substrate specificity, and catalytic robustness in non-physiological reaction conditions. The latter is mainly due to its stable protein fold, to which six intramolecular disulfide bridges make a significant contribution. Although trypsin does not depend on cofactors, it essentially requires the binding of calcium ions to its calcium-binding site to obtain complete enzymatic activity and stability. This behavior is inevitably associated with a limitation of the enzyme’s applicability. To make trypsin intrinsically calcium-independent, we removed the native calcium-binding site and replaced it with another disulfide bridge. The resulting stabilized apo-trypsin (aTn) retains full catalytic activity as proven by enzyme kinetics. Studies using Ellmann’s reagent further prove that the two inserted cysteines at positions Glu70 and Glu80 are in their oxidized state, creating the desired functional disulfide bond. Furthermore, aTn is independent of calcium ions, possesses increased thermal and functional stability, and significantly reduced autolysis compared to wildtype trypsin. Finally, we confirmed our experimental data by solving the X-ray crystal structure of aTn.

Adsorption and diffusion properties of calcium ions at the van der Waals interface of NbSe2-graphene 2D heterostructure for multivalent battery applications: density functional theory calculations

Multivalent-ion batteries such as calcium-ion batteries show promise as a high-density alternative to lithium-ion batteries which currently dominate the portable electronics market. In this work, the adsorption and diffusion properties of calcium ion at the van der Waals (vdW) interface of the 2D heterostructure formed by vertically stacking NbSe2 monolayer and graphene were investigated via density functional theory (DFT) calculations. Results showed that calcium can be effectively adsorbed on the vdW interface of the 2D heterostructure, with the binding energy of most stable site at −2.77 eV, much higher than most metal ions’ binding on pristine graphene. Thus, the NbSe2-graphene 2D heterostructure reinforced the binding of calcium ions at the interface. It is revealed that due to the random stacking nature of NbSe2 and graphene, a multi-path minimum energy pathways were identified at the van de Waals region, with relatively low diffusion barriers of around 0.20–0.50 eV. These indicate the capabilities of the 2D vdW heterostructure for fast multivalent ionic mobility and charge-discharge rate, while maintaining strong binding at the vdW interface. The results reveal NbSe2-graphene 2D vdW heterostructure’s potential as a promising anode material for multivalent battery applications.

The migration and immobilization for heavy metal chromium ions in the hydration products of calcium sulfoaluminate cement and their leaching behavior

Calcium sulfoaluminate cement (CSA) produced from the solid wastes is an effective way to realize the resource utilization of solid waste. Aiming at the key issue that limits this technique, the negative effects of the heavy metals introduced from solid waste on the environment. In this research, Cr(VI) ions, as the heavy metal with great environmental toxicity, are introduced into the calcium sulfoaluminate cement, and their influence on the setting time, compressive strength are explored. The thermal stability, phase structure, micromorphology , and chemical composition of the Cr(VI)-containing hydration products of cement were studied by thermogravimetric analysis (TGA), X-ray diffraction (XRD), X-ray fluorescence spectroscopy (XRF), scanning electron microscopy and X-ray energy spectroscopy (SEM–EDS). After hydration for 7 days, chromium ion leaching experiments were performed in different solutions, including deionized water , sodium sulfate solution , sodium carbonate solution, acetic acid solution, NaCl solution, and 70 °C deionized water. As a result, the introduction of 3% sodium chromate and potassium dichromate increased the setting time of CSA by 47.2% and 117.9%, and the compressive strength was reduced by 15.3% and 8.1%, respectively. The leaching concentration of chromium ions gradually increases with the oscillation time and remains basically unchanged after 12 h. For the above solutions, in NaCl solution, the immobilization of Cr(VI) in CSA cement is the highest (20.3 mg/g) but the lowest that in NaCO 3 solution (14.2 mg/g). The research results confirmed that the chloride ions in the NaCl solution promote the crystal growth of ettringite , thus facilitating the immobilization of Cr(VI) ions in their lattice, while in NaCO 3 solution, Cr(VI) ions are easily released due to CO 3 2− ions binding calcium ions in ettringite crystals but still maintain a high curing amount. Therefore, this work provides a theoretical criterion for toxicity leaching and safety application in different environments for calcium sulfoaluminate cement from solid waste. The main hydration product of sulphoaluminate cement is rod-like ettringite. The columnar structure of ettringite can stably solidify heavy metal Cr ions through physical adsorption and chemical exchange. By studying Cr(VI) leaching in different environments, it can be found that carbonate and acidic conditions are easy to destroy the structure of ettringite and increase the leaching concentration of Cr(VI). NaCl does not destroy the structure of ettringite and can stabilize Cr(VI), with the lowest leaching rate. • Heavy metal Cr (VI) ions enter the lattice of ettringite crystal – one of the hydrated products, by ionic substitution. • Cr(VI) contained ettringite crystals can exist stably in NaCl solution. • The leach of Cr(VI) resulted from the ettringite structure destroyed in acid environment.

A nematode effector inhibits plant immunity by preventing cytosolic free Ca2+ rise.

The Meloidogyne enterolobii effector MeTCTP is a member of the translationally controlled tumour protein (TCTP) family, involved in M. enterolobii parasitism. In this study, we found that MeTCTP forms homodimers and, in this form, binds calcium ions (Ca2+ ). At the same time, Ca2+ could induce homodimerization of MeTCTP. We further identified that MeTCTP inhibits the increase of cytosolic free Ca2+ concentration ([Ca2+ ]cyt ) in plant cells and suppresses plant immune responses. This includes suppression of reactive oxygen species burst and cell necrosis, further promoting M. enterolobii parasitism. Our results have elucidated that the effector MeTCTP can directly target Ca2+ by its homodimeric form and prevent [Ca2+ ]cyt rise in plant roots, revealing a novel mechanism utilized by plant pathogens to suppress plant immunity.

Involvement of MdWRKY40 in the defense of mycorrhizal apple against fusarium solani

BackgroundApple (Malus domestica Borkh.) is an important economic crop. The pathological effects of Fusarium solani, a species complex of soilborne pathogens, on the root systems of apple plants was unknown. It was unclear how mycorrhizal apple seedlings resist infection by F. solani. The transcriptional profiles of mycorrhizal and non-mycorrhizal plants infected by F. solani were compared using RNA-Seq.ResultsInfection with F. solani significantly reduced the dry weight of apple roots, and the roots of mycorrhizal apple plants were less damaged when the plants were infected with F. solani. They also had enhanced activity of antioxidant enzymes and a reduction in the oxidation of membrane lipids. A total of 1839 differentially expressed genes (DEGs) were obtained after mycorrhizal and non-mycorrhizal apple plants were infected with F. solani. A gene ontogeny (GO) analysis showed that most of the DEGs were involved in the binding of ADP and calcium ions. In addition, based on a MapMan analysis, a large number of DEGs were found to be involved in the response of mycorrhizal plants to stress. Among them, the overexpressed transcription factor MdWRKY40 significantly improved the resistance of the apple ‘Orin’ callus to F. solani and the expression of the resistance gene MdGLU by binding the promoter of MdGLU.ConclusionThis paper outlines how the inoculation of apple seedlings roots by arbuscular mycorrhizal fungi responded to infection with F. solani at the transcriptional level. In addition, MdWRKY40 played an important role in the resistance of mycorrhizal apple seedlings to infection with F. solani.

Does It Bind? A Method to Determine the Affinity of Calcium and Magnesium Ions for Polymers Using 1H NMR Spectroscopy.

The binding of calcium and magnesium ions (M2+) by polymers and other macromolecules in aqueous solution is ubiquitous across chemistry and biology. At present, it is difficult to assess the binding affinity of macromolecules for M2+ without recourse to potentiometric titrations and/or isothermal titration calorimetry. Both of these techniques require specialized equipment, and the measurements can be difficult to perform and interpret. Here, we present a new method based on 1H NMR chemical shift imaging (CSI) that enables the binding affinity of polymers to be assessed in a single experiment on standard high-field NMR equipment. In our method, M2+ acetate salt is weighed into a standard 5 mm NMR tube and a solution of polymer layered on top. Dissolution and diffusion of the salt carry the M2+ and acetate ions up through the solution. The concentrations of acetate, [Ac], and free (unbound) M2+, [M2+]f, are measured at different positions along the sample by CSI. Binding of M2+ to the polymer reduces [M2+]f and hinders the upward diffusion of M2+. A discrepancy is thus observed between [Ac] and [M2+]f from which the binding affinity of the polymer can be assessed. For systems which form insoluble complexes with M2+, such as sodium polyacrylate or carboxylate-functionalized nanocellulose (CNC), we can determine the concentration of M2+ at which the polymer will precipitate. We can also predict [M2+]f when a solution of polymer is mixed homogeneously with M2+ salt. We assess the binding properties of sodium polyacrylate, alginate, polystyrene sulfonate, CNC, polyethyleneimine, ethylenediamenetetraacetic acid, and maleate.

Rationally designed bioactive milk-derived protein scaffolds enhanced new bone formation.

Recently, a number of studies reported that casein was composed of various multifunctional bioactive peptides such as casein phosphopeptide and β-casochemotide-1 that bind calcium ions and induce macrophage chemotaxis, which is crucial for bone homeostasis and bone fracture repair by cytokines secreted in the process. We hypothesized that the effects of the multifunctional biopeptides in casein would contribute to improving bone regeneration. Thus, we designed a tissue engineering platform that consisted of casein and polyvinyl alcohol, which was a physical-crosslinked scaffold (milk-derived protein; MDP), via simple freeze-thaw cycles and performed surface modification using 3,4-dihydroxy-l-phenylalanine (DOPA), a mussel adhesive protein, for immobilizing adhesive proteins and cytokines for recruiting cells in vivo (MDP-DOPA). Both the MDP and MDP-DOPA groups proved indirectly contribution of macrophages migration as RAW 264.7 cells were highly migrated toward materials by contained bioactive peptides. We implanted MDP and MDP-DOPA in a mouse calvarial defect orthotopic model and evaluated whether MDP-DOPA showed much faster mineral deposition and higher bone density than that of the no-treatment and MDP groups. The MDP-DOPA group showed the accumulation of host M2 macrophages and mesenchymal stem cells (MSCs) around the scaffold, whereas MDP presented mostly M1 macrophages in the early stage.

Calcium-chelated nanosystem reversing cancer chemoresistance via replenishing intracellular calcium ions

Chemoresistance is the major reason for the failure of chemotherapy, which is a major cause of death in patients with advanced cancer. However, the current approach to reversing cancer chemoresistance is limited. Calcium ions are indispensable for all physiological activities of the body. Because a calcium concentration was reduced in chemoresistant cancer cells, we fabricated calcium-chelated nanoparticles upon self-assembly to replenish intracellular calcium ions in the cells. Through employing an amphipathic polyethylene glycosylated (PEGylated) lipids covalent bonding chelator molecule to bind calcium ions, the chelate calcium ions are used for the first time to reverse the chemoresistance of cancer cells. This nanoparticle released calcium ions more rapidly in a weakly acidic environment than in neutral and weak base conditions, which ensures the effective release of calcium ions in the acidic microenvironment of tumors. As assessed by in vitro and in vivo experiments, this calcium-chelated nanoparticle indeed restored calcium homeostasis and reversed chemoresistance in a variety of chemoresistant cancer cells, whereas calcium chloride solution did not increase the concentration of calcium ions in chemoresistant cancer cells. Furthermore, the mechanism study showed that calcium-chelated nanoparticles affected the expression of chemoresistant genes and promoted cancer cell apoptosis by inhibiting the NF-κB signaling pathway. In conclusion, our data provide direct insight into an effective approach for the reversal of chemoresistance and calcium-chelated nanoparticles may have potential applications for treating chemoresistant patients.

Penggunaan Berbagai Jenis Antikoagulan Terhadap Mutu Hasil Pemeriksaan Activated Partial Thromboplastine Time (APTT)

Activated Partial Thromboplastine Time (APTT) is a type of hemostasis examination with citrate anticoagulant 3.2% ratio 1:9. Ethylene Diamine Tetraacetic Acid (EDTA) is one of the examination materials for hematology which is often used as an anticoagulant. Apart from EDTA, other types of anticoagulants are sodium citrate and potassium oxalate. The three anticoagulants work to bind calcium ions. The purpose of this examination is to determine the use of various anticoagulants on the quality of the results of the Activated Partial Thromboplastine Time (APCT) examination. The method used in this study is an experimental study with 3 sample groups, each consisting of 9 samples in one group. The SPSS results showed a probability value of 0.000 with the Anova test, the average results of the APTT examination were administration of sodium citrate anticoagulant 25.9 seconds, potassium oxalate 70.92 seconds, and EDTA 37.5 seconds. Based on the results of these studies, there are significant differences related to the use of various anticoagulants on the quality of the results of the Activated Partial Thromboplastine Time (APTT) examination.Keywords : Activated Partial Thromboplastine Time, Antikoagulans,

Calcium Ion Binding to the Mutants of Calmodulin: A Structure-Based Computational Predictive Model of Binding Affinity Using a Charge Scaling Approach in Molecular Dynamics Simulation.

The binding of calcium ions (Ca2+) to the calcium-binding proteins (CBPs) controls a plethora of regulatory processes. Among the roles played by CBPs in several diseases, the onset and progress of some cardiovascular diseases are caused by mutations in calmodulin (CaM), an important member of CBPs. Rationalization and prediction of the binding affinity of Ca2+ ions to the CaM can play important roles in understanding the origin of cardiovascular diseases. However, there is no robust structure-based computational method for predicting the binding affinity of Ca2+ ions to the different forms of CBPs in general and CaM in particular. In the current work, we have devised a fast yet accurate computational technique to accurately calculate the binding affinity of Ca2+ to the different forms of CaM. This method combines the well-known molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method and a charge scaling approach developed by previous authors that takes care of the polarization of CaM and Ca2+ ions. Our detailed analysis of the different components of binding free energy shows that subtle changes in electrostatics and van der Waals contribute to the difference in the binding affinity of mutants from that of the wild type (WT), and the charge scaling approach is superior in calculating these subtle changes in electrostatics as compared to the nonpolarizable force field used in this work. A statistically significant regression model made from our binding free energy calculations gives a correlation coefficient close to 0.8 to the experimental results. This structure-based predictive model can open up a new strategy to understand and predict the binding of Ca2+ to the mutants of CBPs, in general.

Identification of Saccharum CaM gene family and functioncharacterization of ScCaM1 during cold and oxidant exposure in Pichia pastoris.

Calmodulin (CaM) plays an essential role in binding calcium ions and mediating the interpretation of Ca2+signals in plants under various stresses. However, the evolutionary relationship of CaM family proteins inSaccharumhas not been elucidated. To deduce and explore the evolution and function ofSaccharumCaM family. A total of 104 typical CaMs were obtained fromSaccharum spontaneumand other 18 plant species. The molecular characteristics and evolution of those CaM proteins were analyzed. A typicalCaMgene,ScCaM1, was subsequently cloned from sugarcane (Saccharumspp. hybrid). Its expression patterns in different tissues and under various abiotic stresses were assessed by quantitative real-time PCR. Then the green fluorescent protein was used to determine the subcellular localization of ScCaM1. Finally, the function ofScCaM1was evaluated via heterologous yeast expression systems. Three typical CaM members (SsCaM1, SsCaM2, and SsCaM3) were identified from theS. spontaneumgenome database.CaMs were originated from the two last common ancestors before the origin of angiosperms. The number of CaM family members did not correlate to the genome size but correlated with allopolyploidization events. TheScCaM1was more highly expressed in buds and roots than inother tissues. The expression patterns ofScCaM1suggested that it was involved in responses to various abiotic stresses in sugarcane via different hormonal signaling pathways. Noteworthily, its expression levels appeared relatively stable during the cold exposure in the cold-tolerant variety but significantly suppressed in the cold-susceptible variety. Moreover,therecombinant yeast (Pichia pastoris) overexpressingScCaM1grew better than the wild-type yeast strain under cold and oxidative stresses. It was revealed that theScCaM1played a positive role in reactive oxygen species scavenging and conferred enhanced cold and oxidative stress tolerance to cells. This study provided comprehensive information on the CaM gene family inSaccharumand would facilitate further investigation of their functional characterization.

Biomineralization of Carbonates Induced by Mucilaginibacter gossypii HFF1: Significant Role of Biochemical Parameters

Although the precipitation of carbonate minerals induced by various bacteria is widely studied, the changes in the biochemical parameters, and their significant role in the biomineralization processes, still need further exploration. In this study, Mucilaginibacter gossypii HFF1 was isolated, identified, and used to induce carbonate minerals at various Mg/Ca ratios. The biochemical parameters were determined in order to explore the biomineralization mechanisms, including cell concentration, pH, ammonia, carbonic anhydrase activity, and alkaline phosphatase activity. The characteristics of extracellular minerals and intracellular inclusions were both analyzed. In addition, the amino acid composition of the extracellular polymeric substance was also tested. Results show that the biochemical parameters provide an alkaline environment for precipitation, due to the combined effect of ammonia, carbonic anhydrase, and alkaline phosphatase. Biotic minerals are characterized by preferred orientation, specific shape, and better crystalline and better thermal stability, indicating their biogenesis. Most of the amino acids in the extracellular polymeric substance are negatived charged, and facilitate the binding of magnesium and calcium ions. The particles with weak crystalline structure in the EPS prove that it acts as a nucleation site. Intracellular analyses prove the presence of the intracellular amorphous inclusions. Our results suggest that the changes in the biochemical parameters caused by bacteria are beneficial to biomineralization, and play a necessary role in its process. This offers new insight into understanding the biomineralization mechanism of the bacteria HFF1.

Probing terahertz dynamics of multidomain protein in cell-like confinement

The development of meaningful descriptions of multidomain proteins exhibiting complex inter-domain dynamics modes is a key challenge for understanding their roles in molecular recognition and signalling processes. Here we developed a generally applicable approach for probing the low frequency collective hydration dynamics of multidomain proteins that uses terahertz spectroscopy of a protein molecule confined in a phospholipid reverse micelles environment (named Droplet THz). With the combination of normal mode analysis, we demonstrated the binding of calcium ions modulates the local inter-domain motion of the human coagulant factor VIII protein in a concentration-dependent manner. These findings highlight the Droplet THz as a valuable tool for dissecting the ultrafast dynamics of domain motion in the multidomain proteins and suggest a modulating mechanism of calcium ions on the structural flexibility and function of human coagulant proteins.

Temperature effects on calcium binding to caseins

The kinetics of binding of calcium ions in molar excess to individual caseins and casein ingredients was studied in pH 6.4 aqueous solutions using stopped-flow absorption spectroscopy. An initial second-order reaction, faster for β-casein than for αs-casein due to lower energy of activation (ΔEa1,β = 8.2 kJ∙mol−1; ΔEa1,α = 18.1 kJ∙mol−1, respectively), is followed by a slower first-order reaction with similar energies of activation (ΔEa2,β = 25.3 kJ∙mol−1 and ΔEa2,α = 20.7 kJ∙mol−1) as determined from temperature dependence of rate between 25 °C and 50 °C. Sodium caseinate reacts faster with calcium than both αs-casein and β-casein in the first reaction of the two consecutive reactions, while the rate of the second falls between αs-casein and β-casein. Global spectral analysis showed the UV–visible spectra of the reaction intermediates of the caseins to be more similar to the final products than to the initial casein reactants. Dynamic and static light scattering indicated decreasing particle sizes and increasing particle surface upon calcium-binding most significantly at low temperatures. The calcium binding to casein was found endothermic by isothermal titration calorimetry. Calcium binding seems to be controlled by enthalpy/entropy compensation corresponding to an isoequilibrium temperature of 38 °C in agreement with binding of calcium to o-phosphoserine rather than to aspartate or glutamate side chains of the caseins. Binding capacity and affinity for calcium to αs-casein and sodium caseinate both increased with increasing temperature in agreement with the endothermic nature of the binding. Decreasing enthalpy of binding for each calcium indicating a decrease in heat capacity of the caseins upon calcium-binding. The small difference between binding enthalpy and energy of activation for association of calcium to αs-casein lead to the conclusion that calcium dissociation goes through an early transition state. The rate of calcium dissociation hardly depends on temperature also explaining why calcium binding to caseins is important for calcium bioaccessibility.