Changes In Secondary Structure Research Articles

RNA Secondary Structure Alteration Caused by Single Nucleotide Variants.

A point mutation in coding RNA can cause not only an amino acid substitution but also a dynamic change of RNA secondary structure, leading to a dysfunctional RNA. Although in silico structure prediction has been used to detect structure-disrupting point mutations known as riboSNitches, exhaustive simulation of long RNAs is needed to detect a significant enrichment or depletion of riboSNitches in functional RNAs. Here, we have developed a novel algorithm Radiam (RNA secondary structure Analysis with Deletion, Insertion, And substitution Mutations) for a comprehensive riboSNitch analysis of long RNAs. Radiam is based on the ParasoR framework, which efficiently computes local RNA secondary structures for long RNAs. ParasoR can compute a variety of structure scores over globally consistent structures with maximal span constraints for the base pair distance. Using the reusable structure database made by ParasoR, Radiam performs an efficient recomputation of the secondary structures for mutated sequences. An exhaustive simulation of Radiam is expected to find reliable riboSNitch candidates on long RNAs by evaluating their statistical significance in terms of the change of local structure stability.

Characterization of freeze-dried oxidized human red blood cells for pre-transfusion testing by synchrotron FTIR microspectroscopy live-cell analysis.

Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature. To better understand the effects of oxidation and freeze-drying/rehydration on RBC lipids and proteins, single-cell measurements were performed by synchrotron-based Fourier transform infrared (FTIR) microspectroscopy 'live-cell' (unfixed) analysis. Lipid and protein spectral data of tert-butyl hydroperoxide (TBHP)-oxidized RBCs (oxRBCs), FDoxRBCs and control (untreated) RBCs were compared using principal component analysis (PCA) and band integration ratios. The oxRBCs and FDoxRBCs samples had similar spectral profiles that were clearly different to control RBCs. Spectral changes in the CH stretching region of oxRBCs and FDoxRBCs indicated the presence of increased saturated and shorter-chain lipids, consistent with lipid peroxidation and stiffening of the RBC membrane compared to control RBCs. The PCA loadings plot for the fingerprint region of control RBCs corresponding to the α-helical structure of hemoglobin, shows that oxRBCs and FDoxRBCs have conformational changes in the protein secondary structure to β-pleated sheets and β-turns. Finally, the freeze-drying process did not appear to compound or induce additional changes. In this context, FDoxRBCs could become a stable source of reagent RBCs for pre-transfusion blood serology testing. The synchrotron FTIR microspectroscopic live-cell protocol provides a powerful analytical tool to characterize and contrast the effects of different treatments on RBC chemical composition at the single cell level.

Insight into the interaction between tannin acid and bovine serum albumin from a spectroscopic and molecular docking perspective.

In this study, the interaction mechanism of bovine serum albumin (BSA) with tannic acid (TA) was investigated by spectroscopic and computational approaches and further validated using circular dichroism (CD), differential scanning calorimetry (DSC) and molecular docking techniques. The fluorescence spectra showed that TA bound to BSA and underwent static quenching at a single binding site, which was consistent with the molecular docking results. And the fluorescence quenching of BSA by TA was dose-dependent. Thermodynamic analysis indicated that hydrophobic forces dominated the interaction of BSA with TA. The results of circular dichroism showed that the secondary structure of BSA was slightly changed after coupling with TA. Differential scanning calorimetry showed that the interaction between BSA and TA improved the stability of the BSA-TA complex, and the melting temperature increased to 86.67 °C and the enthalpy increased to 264.1 J g-1 when the ratio of TA to BSA was 1.2 : 1. Molecular docking techniques revealed specific amino acid binding sites for the BSA-TA complex with a docking energy of -12.9 kcal mol-1, which means the TA is non-covalently bound to the BSA active site.

Protective effect of pangasius myosin on thermal stability of lycopene and their interaction mechanism

In this study, the influence of extracted pangasius myosin on the thermal stability of lycopene and the mechanism of their interaction were examined. The addition of pangasius myosin positively affected the thermal stability of lycopene. After adding myosin, under heating conditions, the L* value of lycopene increased by 6.2–10.04%, a* value increased by 20–27%, and ΔE decreased by 42–51%. Lycopene inhibited the fluorescence of ovalbumin by static burst. Binding and thermodynamic parameters demonstrated that lycopene bound spontaneously to myosin through hydrophobic interaction with a complex stoichiometry ratio of 1:1. Circular dichroism and Fourier infrared transform spectroscopy were used to examine the secondary structure changes that occurred in myosin after it bound to lycopene. Particle size and electron microscopy analysis showed that the addition of low concentrations of lycopene resulted in smaller particle size and improve dispersion stability; however, high concentrations of lycopene promoted myosin aggregation. This study provides a theoretical foundation for improving the thermal stability of lycopene in minced fish products.

Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A.

Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the β-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded β-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 μs of all-atom thermal unfolding MD simulations of wild-type ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the β-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the β-tongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the β-tongue as well as N-terminal mutants that lower the tendency to unfold and disorder the β-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the β-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the β-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.

Investigation of novel cold atmospheric plasma sources and their impact on the structural and functional characteristics of pea protein

The impact of three cold atmospheric plasma (CAP) sources, atmospheric pressure plasma jet (APPJ), two- dimension dielectric barrier discharge (2D-DBD), and nanosecond pulsed discharge (ns-pulsed) on the structure, functionality, and amino acid composition of pea protein was evaluated. Different plasma sources and associated reactive species resulted in protein denaturation, increased surface hydrophobicity, formation of soluble aggregates mostly by disulfide linkages, and changes in secondary structures. Enhancement in surface properties, presence of soluble aggregates, and increase in β-sheet contributed to significant improvement in gelation and emulsification. Enhanced emulsion stability was attributed to relatively small droplet sizes and high surface-charge. Differences among CAP-treated samples were attributed to differences in fluence and composition of plasma-produced reactive species. While all three plasma treatments could be appreciable functionalization approaches, 2D-DBD (Ar + O2) treatment for 30 min had insignificant effect on the amino acid composition.

Probing the interaction of zinc oxide nanorods with human serum albumin: A spectroscopic approach

Bio-functionalized metal oxide nanoparticles (NPs) have been taken great importance in biomedical fields. The use of nanoparticles as delivery agents of therapeutic molecules led the researchers to emphasize the potential impact of these NPs on bio-macromolecules as protein–nanoparticle complexes, which also extended their importance as vehicles in targeted drug delivery systems due to increased ease of administration, firmness, reduced toxic side effects, and half-life of drugs. Since human serum albumin is the blood protein responsible for transporting materials in the blood system, the interaction of these particles with HSA is essential to be understood before considering the nanoparticles for any individual biomedical application. In the present study, we synthesized zinc-oxide nanorods (ZONRs) using a microwave-assisted synthesis technique, and characterized them by XRD, FTIR, Raman, SEM-EDX, UV-Vis spectroscopy, and photoluminescence (PL) spectroscopy methods. The interaction studies were carried out using fluorescence spectroscopy, and the change in secondary structure was analyzed using CD spectroscopy. The results of MTT cell viability assay demonstrated that the ZONRs has potential cytotoxic properties.

Fibrillization kinetics and rheological properties of panda bean (Vigna umbellata (Thunb.) Ohwi et Ohashi) protein isolate at pH 2.0

Recently, research interests are growing regarding the formation and mechanisms of amyloid fibrils from plant proteins. This study investigated the fibrillization kinetics and rheological behaviors of panda bean protein isolate (PBPI) at pH 2.0 and 90 °C for various heating times (0–24 h). Results showed that PBPI formed two distinct classes of fibrils after heating for 10 h, including flexible fibril with a contour length of ∼751 nm, and rigid fibril with periodicity of ∼40 nm. The secondary structural changes during fibril formation were monitored by circular dichroism spectroscopy and indicated that β-sheet content increased first (0−12 h) and then decreased (>12 h), which coincided with similar changes in thioflavin T fluorescence. The gel electrophoresis revealed that the polypeptides of PBPI were progressively hydrolyzed upon heating, and the resulting short fragments were involved in fibril formation rather than PBPI monomer. PBPI-derived fibrils showed extremely high viscosity and storage modulus. A plausible molecular mechanism for PBPI fibrillation process was hypothesized, including protein unfolding, hydrolysis, assembly into matured fibrils, and dissociation of the fibrils. The findings provide useful information to manipulate the formation of legume proteins-based fibrils and will benefit future research to explore their potential applications.

Ligand-receptor interaction in the specific targeting of biomimetic peptide nanoparticles to lysophosphatidylcholine

As nanotechnology is applied clinical medicine, nanoparticle-based therapy is emerging as a novel approach for the treatment of atherosclerosis. Ligand–receptor interaction affects the effectiveness of nanoparticle targeting therapy. In this study, the biomimetic peptide (BP-KFFVLK-WYKDGD) ligand specifically targeting the lysophosphatidylcholine (LPC) receptor in atherosclerotic plaques was constructed. The corresponding ligand–receptor interaction under different pH values was investigated by molecular dynamics simulation and experimental measurements. Results show that the interaction force between the peptide and LPC is greater than that of the peptide and human umbilical vein endothelial cell, clearly demonstrating the specific targeting of the peptide ligand to the LPC receptor. The ligand–receptor binding of peptide and LPC dominantly depends on Coulomb and van der Waals interactions. The YKDG amino acids of the peptide are the main fragment that binds to LPC. Compared with neutral environment at pH 7.4, the interaction forces between the peptide and oxidized low-density lipoprotein (oxLDL) decreased by 18.22 % and 45.87 % under acidic environments at pH 6.5 and 5.5, respectively, because of the change in oxLDL secondary structure and the release of LPC from oxLDL. Nevertheless, the peptide still has a strong binding capacity with oxLDL for the treatment of atherosclerosis.

Three Different Interaction Patterns between MCM-41 and Proteins.

As one of the most studied mesoporous silica nanoparticles (MSNs) in drug delivery systems, Mobil Composition of Matter No. 41 (MCM-41) possesses unique properties including perfect channel architecture, excellent load capacity, and good biocompatibility. However, the applications of MCM-41 nanoparticles in drug delivery have not yet been industrialized, due to the interaction between MCM-41 and biomolecules (especially proteins) that affect their in vivo behaviors after dosing. To investigate the interactions between MCM-41 and proteins, this study selected bovine serum albumin (BSA), lysozyme (Lyso), and bovine hemoglobin (BHb) as model proteins and characterized the ultraviolet-visible, fluorescence, circular dichroism spectra and the protein adsorption of MCM-41-protein complex. The UV-Vis spectra exhibited the different absorption increment degrees of three proteins. The fluorescence spectra showed that the fluorescence intensity of proteins changed by different trends. The CD spectra indicated that the secondary structure changes were ranked as BSA > Lyso > BHb, which is consistent with the protein's adsorption capability on MCM-41. It was shown that there were three different patterns of MCM-41-proteins interactions. The hydrophilic and low-charged BSA followed the strong interaction pattern, the hydrophilic but heavily charged Lyso followed the moderate interaction pattern, and the hydrophobic BHb followed the weak interaction pattern. Different interaction patterns would lead to different effects on the structural properties of proteins, the surface chemistry of MCM-41, and the absorption capability of proteins on MCM-41. We believe our study will provide a better insight into the application of MCM-41 nanoparticles in drug delivery systems.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Exploring GPR109A Receptor Interaction with Hippuric Acid Using MD Simulations and CD Spectroscopy

Previous research has indicated that various metabolites belonging to phenolic acids (PAs), produced by gut microflora through the breakdown of polyphenols, help in promoting bone development and protecting bone from degeneration. Results have also suggested that G-protein-coupled receptor 109A (GPR109A) functions as a receptor for those specific PAs such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA). Indeed, HA has a molecular structural similarity with nicotinic acid (niacin) which has been shown previously to bind to GPR109A receptor and to mediate antilipolytic effects; however, the binding pocket and the structural nature of the interaction remain to be recognized. In the present study, we employed a computational strategy to elucidate the molecular structural determinants of HA binding to GPR109A and GPR109B homology models in understanding the regulation of osteoclastogenesis. Based on the docking and molecular dynamics simulation studies, HA binds to GPR109A similarly to niacin. Specifically, the transmembrane helices 3, 4 and 6 (TMH3, TMH4 and TMH6) and Extracellular loop 1 and 2 (ECL1 and ECL2) residues of GRP109A; R111 (TMH3), K166 (TMH4), ECL2 residues; S178 and S179, and R251 (TMH6), and residues of GPR109B; Y87, Y86, S91 (ECL1) and C177 (ECL2) contribute for HA binding. Simulations and Molecular Mechanics Poisson-Boltzmann solvent accessible area (MM-PBSA) calculations reveal that HA has higher affinity for GPR109A than for GPR109B. Additionally, in silico mutation analysis of key residues have disrupted the binding and HA exited out from the GPR109A protein. Furthermore, measurements of time-resolved circular dichroism spectra revealed that there are no major conformational changes in the protein secondary structure on HA binding. Taken together, our findings suggest a mechanism of interaction of HA with both GPR109A and GPR109B receptors.

The Effect of Irradiation on the Skin upon Breast Cancer Radiotherapy Studied by FTIR Spectroscopy

Breast cancer affects the female population worldwide. Radiotherapy (RT) is part of the therapeutic modality in the management of breast cancer, after radical mastectomy or conserving surgery. The FTIR spectroscopic “marker bands” will lead us to approach the mechanism of skin damage due to the interaction of ionizing radiation and skin, on a molecular level at the very early stages. FT-IR spectroscopy, breast digital pictures, and ImageJ software were used in the study. Healthy breast skin was irradiated <em>ex-vivo</em> with a 4 Gy dose of a γ-<sup>60</sup>Co course Gammachamber 4000A. The FT-IR spectra showed that the low-dose irradiation induces skin dehydration, collagen secondary structure changes and advanced glycation end products (AGEs) as a result of free radicals as mediated products. The infrared “marker bands” at about 1743, 1160, and 870 cm<sup>-1</sup> are characteristic, indicating the development of inflammation, glycations, and peroxidations respectively, due to ionizing radiation-induced oxidative stress. ImageJ analysis provided the sharp surface of the skin after RT irradiation in contrast to the smooth surface of the non-irradiated healthy skin. The most important damages, induced by radiotherapy, were connective tissue lesions, glycosylation, and phosphorylation processes in the skin. The reactive oxygen species (ROS) free radicals prefer to abstract H atoms from lipids, sugar rings of glycoproteins, and base ribose of DNA. The produced intermediate free radicals, as a result of ROS reactions, led to the formation of AGEs and peroxides.

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

Preparation and characterization of cationic hyperbranched maltodextrins as potential carrier for siRNA encapsulation

The present study sought to investigate the physicochemical properties of cationic branched maltodextrins with similar degrees of substitution but different degrees of branching and their siRNA delivery capacity. The results showed that the ratio of α-1,6 glycosidic bonds was significantly increased in the sample treated with dual enzymes. The structural characterization results showed that abundant short chains reassembled by 1,4-α-glucan branching enzyme (GBEs) hydrolysis formed hyperbranched short clustered structure. The absorption peaks that appeared in the FT-IR spectrum confirmed the occurrence of quaternization. The complexes formed by self-assembly of cationic maltodextrins and siRNA were verified by the gel retardation assay and atomic force microscopy, demonstrating a uniform spherical structure with a size close to 300–350 nm. Meanwhile, cationic branched maltodextrins could effectively reduce the change of secondary structure of siRNA. Overall, the results suggested that branched maltodextrins with a cationic surface had significant potential as siRNA carriers.

Immobilization of lipase on silica nanoparticles by adsorption followed by glutaraldehyde cross-linking.

In this study, Candida antarctica lipase B was immobilized on silica (SiO2) nanoparticles by physical adsorption, and then cross-linked with glutaraldehyde (GA) to prepare cross-linked immobilized lipase (CLIL). During the condition of 1.28mg/mL lipase concentration, 25℃ temperature, 2h adsorption time, 0.01% GA (V/V) 7.5mL and 2h cross-linking time, the highest recovery activity of CLIL reached 87.82 ± 0.07% (22.55 ± 0.025U/mg). Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) confirmed that lipase was immobilized on the surface of SiO2 nanoparticles. The changes in secondary structures of CLIL indicated that cross-linking changed the secondary structure of lipase protein, which made the structure of CLIL more stable. Compared with the free lipase, the thermal stability and storage stability of CLIL was significantly improved, and the t1/2 at 60°C was extended. Studies had shown that it was a feasible method to obtain CLIL by cross-linking after adsorbing lipase on SiO2 nanoparticles.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility

β-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus—epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.

Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice.

Unusually low temperatures caused by global climate change adversely affect rice production. Sensing cold to trigger signal network is a key base for improvement of chilling tolerance trait. Here, we report that Oryza sativa Calreticulin 3 (OsCRT3) localized at the endoplasmic reticulum (ER) exhibits conformational changes under cold stress, thereby enhancing its interaction with CBL-interacting protein kinase 7 (OsCIPK7) to sense cold. Phenotypic analyses of OsCRT3 knock-out mutants and transgenic overexpression lines demonstrate that OsCRT3 is a positive regulator in chilling tolerance. OsCRT3 localizes at the ER and mediates increases in cytosolic calcium levels under cold stress. Notably, cold stress triggers secondary structural changes of OsCRT3 and enhances its binding affinity with OsCIPK7, which finally boosts its kinase activity. Moreover, Calcineurin B-like protein 7 (OsCBL7) and OsCBL8 interact with OsCIPK7 specifically on the plasma membrane. Taken together, our results thus identify a cold-sensing mechanism that simultaneously conveys cold-induced protein conformational change, enhances kinase activity, and Ca2+ signal generation to facilitate chilling tolerance in rice.

Shedding light on the molecular interaction between the Hepatitis B virus inhibitor, clevudine, and human serum albumin: Thermodynamic, spectroscopic, microscopic, and in silico analyses

The molecular interaction between clevudine (CLV), a hepatitis B virus inhibitor, and human serum albumin (HSA), the major transporter in plasma, was investigated via fluorescence, UV–vis absorption, circular dichroism (CD), atomic force microscopy (AFM), and molecular modelling approaches. A steady reduction in the Stern-Volmer quenching constant (Ksv) with increasing temperature endorsed the static mode of the protein fluorescence quenching upon CLV addition, thus confirming CLV-HSA complex formation. This finding was supported by alteration in the UV–vis absorption spectrum of HSA upon CLV addition, higher value (>10 10 M−1 s−1) of the bimolecular quenching rate constant (kq), along with swelling of the HSA molecule in the presence of CLV, as revealed by AFM. Fluorescence quenching titration established a weak binding affinity from the values of the binding constant (Ka=8.65-11.20×103 M−1) at various temperatures (290, 300, 310 and 315 K) for the CLV-HSA system. According to the thermodynamic data (ΔS = +51.89 J mol−1 K−1 and ΔH = −7.69 KJmol−1) of the binding reaction, hydrophobic interactions, hydrogen bonds, and van der Waals forces stabilized the CLV-HSA complex. Lesser changes in both secondary and tertiary structures of HSA upon CLV binding were ratified from the far-UV and the near-UV CD spectral results. In addition, the microenvironment around HSA’s Trp and Tyr residues was also altered, as deduced from three-dimensional fluorescence spectra. The protein showed some resistance against thermal stress in the presence of CLV. The binding site of CLV in subdomain IIA (Site I) of HSA was identified by competitive displacement and molecular docking findings.