Secondary Structure Transition Research Articles

Interaction of Antimalarial Drug, Pyrimethamine and Sulphadoxine with Sickle Haemoglobin at pH 5.0 and pH 7.2: A Fourier Transform Infrared (FTIR) Study

Aggregation of sickle haemoglobin (HbS), upon deoxygenation, is responsible for an inherited genetic disorder in the human red blood cells (RBCs) known as Sickle cell disease (SCD). On the suggestion that this phenomenon could arise from some form of conformational changes in the structure of HbS arising from a pH-induced rearrangement of secondary structural elements of the haemoglobin, Fourier Transform Infrared (FTIR) spectroscopy (in the mid-infrared region) was used to measure the amino acid side chain absorbance and also, the secondary structural transitions in the protein. A comparative study between the interaction of HbS with Pyrimethamine and sulphadoxine at pH 5.0, (the acidic pH of malarial parasites’ digestive vacuole during the intra-erythrocytic development and proliferation), and the body’s physiological pH of 7.2 was carried out. The amide I region (1700 – 1600 cm-1) comprising the amino acids side-chains absorbance and the proteins secondary structural components (α-helix, β-sheets, random and turn structures) showed that at pH 5.0 for Pyrimethamine-sulphadoxine combination therapy, there were significant changes in absorbance for HbS while at pH 7.2, little or no significant absorbance changes were observed. The results suggest that HbS is easily destabilized at pH 5.0 than pH 7.2 on interaction with Pyrimethamine and sulphadoxine. These interactions may hinder the development of the Plasmodium falciparum at the intra-erythrocytic stage and may account for the novel strategies of monitoring HbS aggregation during malarial infection as well as improvements in administering effective antimalarial treatment.

Secondary Structure Transitions for a Family of Amyloidogenic, Antimicrobial Uperin 3 Peptides in Contact with Sodium Dodecyl Sulfate.

Secondary structure changes are an inherent part of antimicrobial (AMP) and amyloidogenic peptide activity, especially in close proximity to membranes, and impact the peptides' function and dysfunction roles. The formation, and stability of α-helical components are regarded as essential 'intermediates' for both these functions. To illuminate the conformational transitions leading to amyloid formation we use short cationic AMPs, from an Australian toadlet, Uperoleia mjobergii, (Uperin 3 family, U3) and assess the impact on secondary structural elements in the presence of a membrane mimetic surfactant, sodium dodecyl sulfate (SDS). Specifically, Uperin 3.x, where x=4, 5, 6 wild-type peptides and position seven variants for each, R7A or K7A, were investigated using a combination of experimental and simulation approaches. In water, U3 peptides remain largely unstructured as random coils, with the addition of salts initiating structural transitions leading to assembly towards amyloid. Solution NMR data show that an unstructured U3.5 wt peptide transitions in the presence of SDS to a well-defined α-helical structure that spans nearly the entire sequence. Circular dichroism (CD) and ThT fluorescence studies show that all six U3 peptides aggregate in solution, albeit with vastly varying rates, and a dynamic equilibrium between soluble aggregates rich in either α-helices or β-sheets may exist in solution. However, the addition of SDS leads to a rapid disaggregation for all peptides and stabilisation of predominantly α-helical content in all the U3 peptides. Molecular dynamics (MD) simulations show that the adsorption of U3.5 wt/R7A peptides onto the SDS micelle is driven by Coulombic attraction between peptide cationic residues and the negatively charged sulfate head-groups on SDS. Simulating the interactions of various kinds of β-sheet dimers (of both U3.5 wt and its variant U3.5 R7A) with SDS micelles confirmed β-sheet content decreases in the dimers after their attachment to the SDS micelle. Adsorbed peptides interact favourably with the hydrophobic core of the micelle, promoting intramolecular hydrogen bonds leading to stabilisation of the α-helical structure in peptides, and resulting in a corresponding decrease in intermolecular hydrogen bonds responsible for β-sheets.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum) together with the bathy-like phytochrome XccBphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

Effects of an Electric Field on the Conformational Transition of the Protein: Pulsed and Oscillating Electric Fields with Different Frequencies.

The effect of pulsed and oscillating electric fields with different frequencies on the conformational properties of all-α proteins was investigated by molecular dynamics simulations. The root mean square deviation, the root mean square fluctuation, the dipole moment distribution, and the secondary structure analysis were used to assess the protein samples’ structural characteristics. In the simulation, we found that the higher frequency of the electric field influences the rapid response to the secondary structural transitions. However, the conformational changes measured by RMSD are diminished by applying the electrical field with a higher frequency. During the dipole moment analysis, we found that the magnitude and frequency of the dipole moment was directly related to the strength and frequency of the external electric field. In terms of the type of electric fields, we found that the average values of RMSD and RMSF of whole molecular protein are larger when the protein is exposed in the pulsed electric field. Concerning the typical sample 1BBL, the secondary structure analysis showed that two alpha-helix segments both transit to turns or random coils almost simultaneously when it is exposed in a pulsed electric field. Meanwhile, two segments present the different characteristic times when the transition occurs in the condition of an oscillating electric field. This study also demonstrated that the protein with fewer charged residues or more residues in forming α-helical structures display the higher conformational stability. These conclusions, achieved using MD simulations, provide a theoretical understanding of the effect of the frequency and expression form of external electric fields on the conformational changes of the all-α proteins with charged residues and the guidance for anticipative applications.

Different Thermal Treatment Methods and TGase Addition Affect Gel Quality and Flavour Characteristics of Decapterus maruadsi Surimi Products.

Decapterus maruadsi surimi products were prepared using the thermal treatment methods of boiling (BOI), steaming (STE), back-pressure sterilization (BAC), roasting (ROA), microwaving (MIC), and frying (FRI), respectively. The effect of glutamine transaminase (TGase) addition was also investigated. The moisture distribution, water retention, microstructure, color, fracture constant, protein secondary structure, chemical forces, and flavor components of each sample were determined. The differences in gel and favor characteristics between D. maruadsi surimi products caused by thermal treatment methods were analyzed. The results showed that BOI, STE, and FRI had the largest protein secondary structure transitions and formed dense gel structures with high fracture constant. The kinds of flavour components in BOI and STE were completer and more balanced. The high temperature treatment available at BAC and FRI (110 °C and 150 °C) accelerated the chemical reaction involved in flavor formation, which highlighted the flavor profiles dominated by furans or esters. The open thermal treatment environments of ROA, MIC, and FRI gave them a low moisture content and water loss. This allowed the MIC to underheat during the heat treatment, which formed a loose gel structure with a low fracture coefficient. The addition of TGase enhances the gel quality, most noticeably in the ROA. The aldehyde content of the FRI was enhanced in the flavor characteristic. The effect of adding TGase to enhance the quality of the gel is most evident in ROA. It also substantially increased the content of aldehydes in FRI. In conclusion, different heat treatments could change the gel characteristics of surimi products and provide different flavor profiles. The gel quality of BOI and STE was consistently better in all aspects.

Effect of pH on the Conformational Transition of Silk Fibroin in Aqueous Solution Monitored by Thioflavin-T Fluorescence.

In this work, the potential application of the fluorescence dye Thioflavin-T (ThT), which can specifically bind to amyloid, as a powerful tool for monitoring secondary structural transitions of silk fibroin (SF) induced by pH in low solution concentrations was examined. Results showed that ThT emission intensities substantially increased when pH decreased from 6.8 to 4.8. This increase may be ascribed to conformational transitions from random coil to β-sheet. The morphology and secondary structure of SF were also investigated via TEM, AFM and circular dichroism spectroscopy. The information obtained herein can be utilized not only for the development of convenient and efficient noninvasive method for monitoring the assembly behavior of SF in aqueous solution but also for in vitro fluorescence imaging.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to β-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Investigation on the competition of duplex/ G-quadruplex/ i-motif in telomere sequences and c-MYC gene with a solid-state nanopore sensor

Apart from duplex helix, the coexisted noncanonical DNA may be involved in inhibiting DNA replication, and controlling gene expression. The competition of duplex/ G4/ i-motif depends on many factors, including sequence information, external conditions and binding effects. This work looked into the human telomere sequences of different length (hTelo-1, 2) and intramolecular G-rich and C-rich strands (hTelo-3, 4, 5). Nanopore translocation properties suggested that in the presence of their complementary sequences in the mixed K+/Cs+ buffer, G4 was the major species, and G4 from hTelo-2 that holds multiple G-quartets formed slower than shorter hTelo-1. The intramolecular G/C-rich strands hTelo-3, 4, 5 predominantly formed duplex rather than quadruplex under K+ or acidic conditions, indicating that intramolecular quadruplex is less stable than duplex due to the strand strain. Translocation behaviors of mixed proto-oncogene cMYC/c-cMYC illustrated that G4 was formed in both K+ and Cs+, and Cs+ inhibited the duplex formation in K+/Cs+ buffer. The increase of the ratio of Cs+ will decrease the duplexes species. Our work demonstrated the relative stability of duplex/quadruplex under various conditions with distinct strands, which is useful for the investigation of DNA secondary structural dynamics and transition mechanism in biological function regions, and paved the way for the potential of disease-targeted diagnosis.

Investigation on conformational variation and fibrillation of human serum albumin affected by molybdenum disulfide quantum dots

In this work, binding interaction between molybdenum disulfide quantum dots (MoS2 QDs) and human serum albumin (HSA) was researched deeply to dissect the conformational variation and fibrillation of HSA affected by MoS2 QDs. The results revealed that MoS2 QDs bound strongly with HSA with molar ratio of 1:1 under the joint actions of hydrogen bond and van der Waals force, leading to the static fluorescence quenching of HSA. MoS2 QDs caused the secondary structure transition of HSA from α-helix stepwise to β-turn, β-sheet, and random coil gradually. MoS2 QDs reduced both the molar enthalpy change and the melting temperature of HSA, reducing the thermal stability of HSA significantly. It is worth noting that MoS2 QDs inhibited the fibrillation process of HSA according to the reduced hydrophobic environment and the disturbance of disulfide bonds in HSA network structure. These results reveal the precise binding mechanism of MoS2 QDs with HSA at molecular level, providing indispensable information for the potential application of MoS2 QDs in biological fields.

Decoding the Structural Dynamics and Conformational Alternations of DNA Secondary Structures by Single-Molecule FRET Microspectroscopy.

In addition to the canonical double helix form, DNA is known to be extrapolated into several other secondary structural patterns involving themselves in inter- and intramolecular type hydrogen bonding. The secondary structures of nucleic acids go through several stages of multiple, complex, and interconvertible heterogeneous conformations. The journey of DNA through these conformers has significant importance and has been monitored thoroughly to establish qualitative and quantitative information about the transition between the unfolded, folded, misfolded, and partially folded states. During this structural interconversion, there always exist specific populations of intermediates, which are short-lived or sometimes even do not accumulate within a heterogeneous population and are challenging to characterize using conventional ensemble techniques. The single-molecule FRET(sm-FRET) microspectroscopic method has the advantages to overcome these limitations and monitors biological phenomena transpiring at a measurable high rate and balanced stochastically over time. Thus, tracing the time trajectory of a particular molecule enables direct measurement of the rate constant of each transition step, including the intermediates that are hidden in the ensemble level due to their low concentrations. This review is focused on the advantages of the employment of single-molecule Forster’s resonance energy transfer (sm-FRET), which is worthwhile to access the dynamic architecture and structural transition of various secondary structures that DNA adopts, without letting the donor of one molecule to cross-talk with the acceptor of any other. We have emphasized the studies performed to explore the states of folding and unfolding of several nucleic acid secondary structures, for example, the DNA hairpin, Holliday junction, G-quadruplex, and i-motif.

Purification and characterization of an amyloidogenic repeat domain from the functional amyloid Pmel17

The pre-melanosomal protein (Pmel17) is a human functional amyloid that supports melanin biosynthesis within melanocytes. This occurs in the melanosome, a membrane-bound organelle with an acidic intraluminal pH. The repeat region of Pmel17 (RPT, residues 315–444) has been previously shown to form amyloid aggregates under acidic melanosomal conditions, but not under neutral cytosolic conditions, when expressed and purified using a C-terminal hexa-histidine tag (RPT-His). Given the importance of protonation states in RPT-His aggregation, we questioned whether the histidine tag influenced the pH-dependent behavior. In this report, we generated a tagless RPT by inserting a tobacco etch virus (TEV) protease recognition sequence (ENLYGQ(G/S)) immediately upstream of a native glycine residue at position 312 in Pmel17. After purification of the fusion construct using a histidine tag, cleavage with TEV protease generated a fully native RPT (nRPT) spanning resides 312–444. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH ≤ 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/β-sheet at pH 4 to canonical β-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.

Water-Mediated Folding Behaviors and Chiroptical Inversion of Ferrocene-Conjugated Dipeptides.

The hydration effect on the folding behavior of oligopeptides is of vital importance both in the structure basis of biomolecules and in the rational design of peptide-based materials, which however has rarely been addressed. Here we present the hydration impact on the spontaneous folding of dipeptides conjugated by the ferrocene spacer. In organic phase, the ferrocene-glycine-phenylalanine dipeptide formed a parallel β-sheet structure and Herrick's conformation, which underwent conformational transformation encountering aqueous media, by significantly switching dipeptide arm angles around the ferrocene axis up to 72°. The conformational transformation behavior aroused inversion of the chiroptical activity. Solid X-ray structures, proton nuclear magnetic resonance, chiroptical spectroscopy, and the density functional theory calculation were employed to unveil the hydration effect in the secondary structure transition, in which the rearrangement of hydrogen bonds played the vital role. This work deepens the understanding of water functioning in the structure modulation of biomolecules and also provides an alternative protocol in designing novel chiroptical switches and adaptive peptide-based biomaterials.

Ultrasonics induced variations in molecular structure and tensile properties of silk fibers in a chemical free environment

AbstractUltrasonics can be used as an alternative energy supply for a green silk wet processing and for enhancement of some chemical/physical properties of bio‐polymers. In this work, ultrasonically treated silk fabric were compared with that of untreated and were investigated for the changes in fiber conformation structure and subsequent tensile property. Experiments were conducted under different time duration in a chemical free environment. Fiber surface energy distribution and transformation of silk secondary structure were analyzed using X‐ray photoelectron spectroscopy (XPS), energy dispersive spectroscopy (EDS), and Fourier transform infrared spectrum (FITR). Results showed that subject to treatment time, a surface oxidation effect and a light transformation of random coil to β‐sheet can be induced from ultrasonic irradiation. Tensile testing showed that under certain treatment time, ultrasonics can lead to silk fibers with a slightly increased strength and reduced extensibility, as a result of the transition of fiber secondary structure.

Stimuli-controlled peptide self-assembly with secondary structure transitions and its application in drug release

Short peptides can self-assemble into supramolecular materials and be used for promising biomedical applications.

Exploring the structural significance of molecular switch mechanism alias motif phosphorylation in Wnt/β-catenin and their crucial role in triple-negative breast cancer

β-Catenin, a key transcriptional factor involved in the canonical Wnt signaling pathway, is regulated by a cascade of phosphorylations and plays a major role in the progression of triple-negative breast cancer (TNBC). However, the phosphorylation induced conformational changes in a β-Catenin is still poorly understood. Hence, we adopted a conventional molecular dynamics approach to study phosphorylations present in a sequence motif Ser 552 675 and Tyr670 of the β-Catenin domain and analyzed in terms of structural transitions, bond formation, and folding-misfolding conformations. Our results unveil the β-Catenin linear motif 549–555 (RRTSMGG) of armadillo repeats domain prefers order to disorder state. In contrast, helix C associated with 670–678 (YKKRLSVEL) motif prefers disorder to order upon phosphorylation of Ser 552 675 and Tyr670. In addition, the crucial secondary structural transition from α-helix to coil induced by phospho Ser552 and phospho Tyr670 of β-Catenin ARM domain connecting helix C modifies conformational diversity and binding affinities of the complex interaction in functional regulation significantly. Moreover, the post phosphorylation disrupted the hydrogen bond interactions (Ser552-Arg549, Arg550-Asp546 and Ser675-Lys672) and abolished the residual alliance with hydrophobic interactions (Tyr670-Leu674) that easily interrupt in secondary structure packing as well as folding conformations connecting ARM and helix C (R10, 12 & R1C) compared to unphosphorylation. Our integrated computational analysis may help in shedding light on understanding the induced folding and unfolding pattern due to motif phosphorylations. Overall, our results provide an atomistic structural description of the way phosphorylation facilitates conformational and dynamic changes in β-Catenin, a fundamental molecular switch mechanism in triple-negative breast cancer pathogenesis.

How do Self-Assembling Antimicrobial Lipopeptides Kill Bacteria?

Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides was formed by attaching an acyl chain to the N-terminus of α-helix-forming peptides with the sequence Cx-G(IIKK)yI-NH2 (CxGy, x = 4-12 and y = 2). CxGy self-assemble into nanofibers above their critical aggregation concentrations (CACs). With increasing x, the CACs decrease and the hydrophobic interactions increase, promoting secondary structure transitions within the nanofibers. Antimicrobial activity, determined by the minimum inhibition concentration (MIC), also decreases with increasing x, but the MICs are significantly smaller than the CACs, suggesting effective bacterial membrane-disrupting power. Unlike conventional antibiotics, both C8G2 and C12G2 can kill Staphylococcus aureus and Escherichia coli after only minutes of exposure under the concentrations studied. C12G2 nanofibers have considerably faster killing dynamics and lower cytotoxicity than their nonaggregated monomers. Antimicrobial activity of peptide aggregates has, to date, been underexploited, and it is found to be a very promising mechanism for peptide design. Detailed evidence for the molecular mechanisms involved is provided, based on superresolution fluorescence microscopy, solid-state nuclear magnetic resonance, atomic force microscopy, neutron scattering/reflectivity, circular dichroism, and Brewster angle microscopy.

The contrasting effect of macromolecular crowding and confinement on fibril formation of globular protein: Underlying cause of proteopathies

Protein molecules need to maintain a proper native conformation to perform encoded role in sustaining life. Naturally, the cellular milieu is densely crowded by different macromolecules, which can alter the secondary structure of protein eventually leading to protein aggregation which have been reported liable to many proteopathies. Hemoglobin (Hb), being an important alpha helical dominant globular protein, can serve as an excellent model to carryout structural transition studies. The aggregation profiling of Hb was done using turbidometric, thioflavin T, and Congo red assays. The secondary structural transition was confirmed by circular dichroism. Microscopic analysis using scanning electron microscope (SEM) show morphology of ordered Hb aggregates of protofibrils and fibril types. Generation of ROS (reactive oxygen species), genotoxic and cytotoxic potentials of Hb aggregates affirmed by DCFH-DA (2′,7′-dichlorofluorescin diacetate), DAPI (4′,6-diamidino-2-phenylindole) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay respectively. Our findings suggested that macromolecular crowding promotes Hb aggregation and the morphology of aggregates goes with the type of crowding agent used (polyethylene glycol 4000 (P4), 6000 (P6) and dextran 70 (D70)). Moreover, Hb aggregates causes redox perturbation and cytotoxicity in isolated human peripheral blood cells (PBMCs) which concludes the toxic potential and the gained toxic properties of Hb aggregates correlating to in vivo clinical conditions involved in various proteopathies including neurodegenerative disorders.

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication.

Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

Microsecond scale sampling of Egr-1 conformational landscape to decipher the impact of its disorder regions on structure–function relationship

ABSTRACT Early growth response-1 (Egr-1) is one of the key zinc finger transcription factors involved in controlling cell growth, proliferation, differentiation, angiogenesis and apoptosis. It also plays a pivotal role in conditions like cancer, neurological disorders and ocular disorders. The structural features of Egr-1 DNA-binding domain (DBD) are well documented, however, the functional role of its intrinsically disordered regions (IDRs) is yet to be explored. Hence, in this study, 3D structure of Egr-1 was modelled in conjunction with its IDR and was probed for conformational changes at microsecond scale in order to explore the structural plasticity and its functional impact. The results revealed the distortion of Zinc binding was due to the secondary structure transitions and flexibility of the DBD residues. Moreover, the increased distance of the flap residues that contributes to the proper orientation of Zinc interactions were also inferred, which shall aid in proper binding of DNA. This study also portrays the conformations that can be considered as optimal near-natives for further protein–protein interaction studies to delineate its interacting partners involved in various pathological conditions. This study demonstrates the impact of IDRs in Egr-1 on structure–function studies and also provides insights on optimal druggable cavities..