Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Effect of setting data collection parameters on the reliability of a circular dichroism spectrum.

Circular dichroism (CD) spectroscopy is a well-established biophysical technique used to investigate the structure of molecules. The analysis of a protein CD spectrum depends on the quality of the original CD data, which can be affected by the sample purity, background absorption of the additives/solvent/buffer, the choice of the parameters used for data collection, etc. In this paper, the CD spectrum of myoglobin was used as a model to exploit how variations on each data collection parameter could affect the final protein CD spectrum and, the subsequent effect of them on the quantitative analysis of protein secondary structure. Bioinformatics analysis carried out with SESCA package and PDBMD2CD server predicted a theoretical myoglobin CD spectrum, and a Monte Carlo-like model was implemented to estimate the uncertainty in secondary structure predictions performed with CDSSTR, Selcon 3 and ContinLL algorithms. An inappropriate choice of data collection parameters can lead to a misinterpretation of the CD data in terms of the protein structural content.

Investigation of binding mechanism and downregulation of elacestrant for wild and L536S mutant estrogen receptor-α through molecular dynamics simulation and binding free energy analysis.

The selective estrogen receptor downregulators (SERDs) are the new emerging class of drugs that are used for the treatment of endocrine resistance breast cancer. Elacestrant (ELA) is a new SERD, currently it is in phase II clinical trial. To understand the ELA-ERα interactions, the molecular docking analysis has been carried out. The ELA molecule binds with the helices H3, H5, H6, and H11 and forms important intermolecular interactions. In addition to this, the tetrahydronapthalene and phenyl rings of ELA are forming T-shaped π···π interactions with the Phe404 and Trp383 residues. Further to understand the stability and flexibility of ELA molecule in the active site of wild and mutated L536S ERα, 100ns molecular dynamics (MD) simulation was performed for both complexes. Interestingly, the MD analysis of wild complex revealed an interaction between ELA and the Asn532 of H11, which is an essential interaction for the downregulation/degradation of ERα, whereas this interaction is not observed in the mutated complex. The drug binding mechanism and H12 dynamics have been elucidated from the analysis of hydrogen bonding interactions and the secondary structure analysis. To explore the binding affinity of ELA molecule, the binding free energy and normal mode analyses were carried out. The per residue decomposition analysis also performed, which shows the contribution of individual amino acids. The principal component analysis and residue interaction network analysis were used to identify the modifications and the interaction between the residues. From the results of different analysis, the inhibition mechanism and downregulation of ERα-ELA complex has been investigated. © 2019 Wiley Periodicals, Inc.

FT-IR versus EC-QCL spectroscopy for biopharmaceutical quality assessment with focus on insulin-total protein assay and secondary structure analysis using attenuated total reflection.

For the quality control of biopharmaceutical products, which contain proteins as the most important active ingredients, shelf life may be limited due to inappropriate storage conditions or mechanical stress. For insulins as representatives of life-saving pharmaceuticals, analytical methods are needed, which are providing additional information than obtained by assays for total protein quantification. Despite sophisticated formulations, the chemical stability may be challenged by temperatures deviating from recommended conditions or shear rate exposure under storage, leading to misfolding, nucleation, and subsequent fibril formation, accompanied by a decrease in bioactivity. A reliable method for insulin quantification and determination of secondary structure changes has been developed by attenuated total reflection (ATR) Fourier-transform infrared spectroscopy of insulin formulations by a silver halide fiber-coupled diamond probe with subsequent dry-film preparation. A special emphasis has been placed on the protein amide I band evaluation, for which spectral band analysis provides unique information on secondary structure fractions for intact and misfolded insulins. Quantitative measurements are possible down to concentrations of less than 0.5 mg/ml, whereas the dry-film preparation delivers high signal-to-noise ratios due to the prior water evaporation, thus allowing a reliable determination of secondary structure information. Graphical abstract.

Solution and solid state conformational preferences of a family of cyclic disulphide bridged tetrapeptides.

A set of cyclic tetrapeptides of the general form cyclo (Boc-Cys-Pro-X-Cys-OMe) with X being L-/D-Ala, L-/D-Val, and L-/D-Trp was synthesized. These peptides serve as model systems for structure elucidation in solution and feature a variety of structural motifs - namely a ß-turn with intramolecular hydrogen bonding interactions, cis/trans isomerism, and a disulphide bond. In this work, we performed a comprehensive structural analysis focussing on their ß-turn conformational preferences using NMR, VCD, and Raman spectroscopy. Our results provide evidence for a strong influence of a single stereocenter on the structures of the peptides whereas solvent polarity does not significantly affect them. Additionally, the solid state conformational preferences were studied by crystal structure analysis. Overall, a general trend for the conformational preferences of this set of peptides can be concluded from the results of the complementary investigations.

Structural and functional characterization of peste des petits ruminants virus coded hemagglutinin protein using various in-silico approaches.

Peste des petits ruminants (PPR), a disease of socioeconomic importance has been a serious threat to small ruminants. The causative agent of this disease is PPR virus (PPRV) which belongs to the genus Morbillivirus. Hemagglutinin (H) is a PPRV coded transmembrane protein embedded in the viral envelope and plays a vital role in mediating the entry of virion particle into the cell. The infected host mounts an effective humoral response against H protein which is important for host to overcome the infection. In the present study, we have investigated structural, physiological and functional properties of hemagglutinin protein using various computational tools. The sequence analysis and structure prediction analysis show that hemagglutinin protein comprises of beta sheets as the predominant secondary structure, and may lack neuraminidase activity. PPRV-H consists of several important domains and motifs that form an essential scaffold which impart various critical roles to the protein. Comparative modeling predicted the protein to exist as a homo-tetramer that binds to its cognate cellular receptors. Certain amino acid substitutions identified by multiple sequence alignment were found to alter the predicted structure of the protein. PPRV-H through its predicted interaction with TLR-2 molecule may drive the expression of CD150 which could further propagate the virus into the host. Together, our study provides new insights into PPRV-H protein structure and its predicted functions.

Exploring the Origins of Association of Poly(acrylic acid) Polyelectrolyte with Lysozyme in Aqueous Environment through Molecular Simulations and Experiments.

This study provides a detailed picture of how a protein (lysozyme) complexes with a poly(acrylic acid) polyelectrolyte (PAA) in water at the atomic level using a combination of all-atom molecular dynamics simulations and experiments. The effect of PAA and temperature on the protein's structure is explored. The simulations reveal that a lysozyme's structure is relatively stable except from local conformational changes induced by the presence of PAA and temperature increase. The effect of a specific thermal treatment on the complexation process is investigated, revealing both structural and energetic changes. Certain types of secondary structures (i.e., α-helix) are found to undergo a partially irreversible shift upon thermal treatment, which aligns qualitatively with experimental observations. This uncovers the origins of thermally induced aggregation of lysozyme with PAA and points to new PAA/lysozyme bonds that are formed and potentially enhance the stability in the complexes. As the temperature changes, distinct amino acids are found to exhibit the closest proximity to PAA, resulting into different PAA/lysozyme interactions; consequently, a different complexation pathway is followed. Energy calculations reveal the dominant role of electrostatic interactions. This detailed information can be useful for designing new biopolymer/protein materials and understanding protein function under immobilization of polyelectrolytes and upon mild denaturation processes.

Rtools: A Web Server for Various Secondary Structural Analyses on Single RNA Sequences.

Predicting the secondary structures of RNA molecules is an essential step to characterize their functions, but the thermodynamic probability of any prediction is generally small. On the other hand, there are a few tools for calculating and visualizing various secondary structural information from RNA sequences. We implemented a web server that calculates in parallel various features of secondary structures: different types of secondary structure predictions, the marginal probabilities for local structural contexts, accessibilities of the subsequences, the energy changes by arbitrary base mutations, and the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp , which integrates software tools, CentroidFold, CentroidHomfold, IPknot, CapR, Raccess, Rchange, RintD, and RintW.

Infrared Spectroscopy for Structure Analysis of Protein Inclusion Bodies.

Infrared (IR) spectroscopy is a widely used technique for evaluation of protein secondary structure. In this chapter, we focus on the application of this analytical technique for analysis of inclusion bodies. After a general introduction to protein analysis by IR spectroscopy, different approaches for spectra acquisition, data processing, and secondary structure evaluation are presented.

The α-Synuclein Monomer May Have Different Misfolding Mechanisms in the Induction of α-Synuclein Fibrils with Different Polymorphs.

The aggregation of alpha-synuclein (α-Syn) is closely related to the occurrence of some neurodegenerative diseases such as Parkinson's disease. The misfolding of α-Syn monomer plays a key role in the formation of aggregates and extension of fibril. However, the misfolding mechanism of α-Syn remains elusive. Here, three different α-Syn fibrils (isolated from a diseased human brain, generated by in vitro cofactor-tau induction, and obtained by in vitro cofactor-free induction) were selected for the study. The misfolding mechanisms of α-Syn were uncovered by studying the dissociation of the boundary chains based on the conventional molecular dynamics (MD) and Steered MD simulations. The results showed that the dissociation paths of the boundary chains in the three systems were different. According to the reverse process of dissociation, we concluded that in the human brain system, the binding of the monomer and template starts from the C-terminal and gradually misfolds toward the N-terminal. In the cofactor-tau system, the monomer binding starts from residues 58-66 (contain ß3), followed by the C-terminal coil (residues 67-79). Then, the N-terminal coil (residues 36-41) and residues 50-57 (contain ß2) bind to the template, followed by residues 42-49 (contain ß1). In the cofactor-free system, two misfolding paths were found. One is that the monomer binds to the N/C-terminal (ß1/ß6) and then binds to the remaining residues. The other one is that the monomer binds sequentially from the C- to N-terminal, similar to the human brain system. Furthermore, in the human brain and cofactor-tau systems, electrostatic interactions (especially from residues 58-66) are the main driving force during the misfolding process, whereas in the cofactor-free system, the contributions of electrostatic and van der Waals interactions are comparable. These results may provide a deeper understanding for the misfolding and aggregation mechanism of α-Syn.

Digging deeper into the taxonomy of Cylindrospermum and description of Johanseniella tripurensis gen. et sp. nov. from India.

The wet soil-inhabiting cyanobacterium URH-6-PS was collected from the state of Tripura, India, and characterized using a polyphasic approach. Initial microscopic analysis indicated the strain to be a member of the genus Cylindrospermum, however, the 16S rRNA gene phylogenetic analysis showed some interesting results. The strain URH-6-PS clustered in the Clade I, which positioned itself outside the Cylindrospermum sensu stricto clade with strong probability/bootstrap support, indicating that the strain may not belong to the genus Cylindrospermum. Further, analysis of the 16S-23S ITS region using the folded secondary structures of the D1-D1', Box-B, and V3 helices and the 16S-23S ITS percentage dissimilarity values clearly indicated the distinctiveness of strain URH-6-PS from other members of the Clade I. The detailed investigations conducted in this study provided sufficient evidence that the taxonomic status of the members of Clade I need to be revised. Interestingly, all members of the clade I originated from tropical and subtropical habitats while members of the Cylindrospermum sensu stricto clade have been reported from temperate conditions. Previous studies had also indicated the possibility of the formation of tropical and subtropical Cylindrospermum-like genera, and thus this comes across as a much-awaited study to resolve the taxonomic complexities around the genus Cylindrospermum. Based on the results obtained in this study and the trends observed in the earlier studies, we describe a novel Cylindrospermum-like genus Johanseniella gen. nov., with the type species Johanseniellatripurensis sp. nov. in accordance with the International Code of Nomenclature for algae, fungi, and plants.

Quality Assurance of Commercial Insulin Formulations: Novel Assay Using Infrared Spectroscopy.

BACKGROUND: For insulins in commercial formulations, degradation can be observed within the certified shelf life when not stored at recommended conditions. Elevated temperatures and exposure to shear forces can cause changes in the secondary structure of the hormone, leading to a decrease in pharmaceutical potency. International pharmacopoeia recommendations for insulin quality monitoring assays mainly rely on liquid chromatography methods. These methods are unable to distinguish between active and inactive forms, both of which may exist in pharmaceutical insulins exposed to stress conditions. METHOD: Infrared attenuated total reflection spectroscopy has been used for the analysis of insulin dry film preparations using affordable instrumentation. This method can be applied to either formulated insulin specimens or pure insulins obtained by ultrafiltration. Such samples have been stored under different temperatures (0°C, 20°C, and 37°C), and degradation processes have been monitored up to a period of a few months. RESULTS: By analyzing specific shifts of absorption bands in the infrared spectra, which are sensitive to the protein secondary structure, even small structural changes in the hormone become evident. Another option is amide I band deconvolution into individual bands, which can be attributed to secondary structure subunits that are part of the insulin tertiary structure. CONCLUSION: A novel and innovative method based on infrared attenuated total reflection spectroscopy of insulin dry films is a promising analytical tool for quantifying the degree of insulin degradation, as it provides information on indicating a decrease in biological potency. The established methods for insulin potency assays require animal testing or clamp experiments on people with diabetes.

Systematic stability testing of insulins as representative biopharmaceuticals using ATR FTIR-spectroscopy with focus on quality assurance.

SIGNIFICANCE: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity. AIM: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation. APPROACH: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis. RESULTS: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity. CONCLUSIONS: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.

aeBlue Chromoprotein Color is Temperature Dependent.

BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.

Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Lipid Environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190 nm and producing information that is unobtainable by conventional CD instruments. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

Circular dichroism in functional quality evaluation of medicines.

Circular dichroism (CD) is a non-destructive and powerful technique for providing structure and ligand interaction information of small molecules as well as biotechnological medicines. While CD is a well-established technique in biomedical research, and different types and variants of CD do exist, the focus of this review is on the pharmaceutical quality control (QC) aspects of the classic electronic CD (ECD). The basic principles of the CD technique are initially described, followed by a systematic literature research on pharmaceutical aspects encompassing chiral small molecules, bio-polymers (i.e. proteins, peptides and nucleic acids), medicine-biotarget interaction (i.e. small molecule-albumin interaction, protein-receptor interaction and peptide-biotarget interaction) and medicine changes (i.e. chemical modification, biosimilar/bio-better with stability and aggregation). In addition, unstructured literature was also included covering the use of CD mainly in discovery and fundamental research, but which might shift towards the pharmaceutical QC field as well in the future.

High yield purification and first structural characterization of the full-length bacterial toxin CNF1.

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.

Functional Studies on Membrane Proteins by Means of H/D Exchange in Infrared: Structural Changes in Na+ NQR from V. cholerae in the Presence of Lipids.

H/D exchange kinetics at the level of the amide proton in the mid infrared (1700-1500 cm-1) make it possible to study the conformational flexibility of membrane proteins, independent of size or the presence of detergent or lipids. Slow, medium, and fast exchanging domains are distinguished, which reveal a different accessibility to the solvent. Whereas amide hydrogens undergo rapid exchange with solvent in an open structure, hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. Here, we describe the protocol that was used to study the effect of phospholipids on the overall structure of the Na+ NQR from V. cholerae, a sodium pumping membrane protein.

Secondary Structure Determination by Means of ATR-FTIR Spectroscopy.

Specialized infrared spectroscopic techniques have been developed that allow studying the secondary structure of membrane proteins and the influence of crucial parameters like lipid content and detergent. Here, we focus on an ATR-FTIR spectroscopic study of Af-Amt1 and the influence of LDAO/glycerol on its structural integrity. Our results clearly indicate that infrared spectroscopy can be used to identify the adapted sample conditions.

Anticancer property of sediment actinomycetes against MCF-7 and MDA-MB-231 cell lines.

OBJECTIVE: To investigate the anticancer property of marine sediment actinomycetes against two different breast cancer cell lines. METHODS: In vitro anticancer activity was carried out against breast (MCF-7 and MDA-MB-231) cancer cell lines. Partial sequences of the 16s rRNA gene, phylogenetic tree construction, multiple sequence analysis and secondary structure analysis were also carried out with the actinomycetes isolates. RESULTS: Of the selected five actinomycete isolates, ACT01 and ACT02 showed the IC50 value with (10.13±0.92) and (22.34±5.82) µg/mL concentrations, respectively for MCF-7 cell line at 48 h, but ACT01 showed the minimum (18.54±2.49 µg/mL) level of IC50 value with MDA-MB-231 cell line. Further, the 16s rRNA partial sequences of ACT01, ACT02, ACT03, ACT04 and ACT05 isolates were also deposited in NCBI data bank with the accession numbers of GQ478246, GQ478247, GQ478248, GQ478249 and GQ478250, respectively. The phylogenetic tree analysis showed that, the isolates of ACT02 and ACT03 were represented in group I and III, respectively, but ACT01 and ACT02 were represented in group II. The multiple sequence alignment of the actinomycete isolates showed that, the maximum identical conserved regions were identified with the nucleotide regions of 125 to 221st base pairs, 65 to 119th base pairs and 55, 48 and 31st base pairs. Secondary structure prediction of the 16s rRNA showed that, the maximum free energy was consumed with ACT03 isolate (-45.4 kkal/mol) and the minimum free energy was consumed with ACT04 isolate (-57.6 kkal/mol). CONCLUSIONS: The actinomycete isolates of ACT01 and ACT02 (GQ478246 and GQ478247) which are isolated from sediment sample can be further used as anticancer agents against breast cancer cell lines.