A versatile tool in controlling aggregation and Ag nanoparticles assisted in vitro folding of thermally denatured zDHFR.

BACKGROUND: Proteins have tendency to form inactive aggregates at higher temperatures due to thermal instability. Maintenance of thermal stability is essential to gain the protein in sufficient quantity and biologically active form during their commercial production. METHODS: BL21-DE3 Rosetta E. coli cells which contains plasmid pET43.1a vector was used for producing zDHFR protein commercially. The purification of N-terminal Histidine tagged zDHFR was performed by Immobilized Metal Ion chromatography (IMAC). Investigations were performed in existence and non existence of Silver nanoparticles (AgNPs). The inactivation kinetics of zDHFR in existence and non existence of AgNPs were monitored over a range of 40-80 °C as monitored by UV-Visible absorption spectroscopy. RESULTS: The protein completely lost its activity at 55 °C. Kinetics of inactivated zDHFR follows first order model in presence and absence of AgNPs. Decrease in rate constant (k) values at respective temperatures depicts that AgNPs contribute in the thermostability of the protein. AgNPs also assists in regaining the activity of zDHFR protein. CONCLUSIONS: AgNPs helps in maintaining thermostability and reducing the aggregation propensity of zDHFR protein. GENERAL SIGNIFICANCE: Result explains that AgNPs are recommended as a valuable system in enhancing the industrial production of biologically active zDHFR protein which is an important component in folate cycle and essential for survival of cells and prevents the protein from being aggregated.

DNA barcoding and Taxonomic account on some selected species of subfamily Plusiinae (Lepidoptera: Noctuidae) from India.

This study represents a detailed taxonomic account of 31 species of Plusiinae (Lepidoptera: Noctuidae) from India. The survey and collection of 39 localities from different regions of India between 2015 and 2018. The tribe Argyrogrammatini Eichlin Cunningham, 1978 with Ctenoplusia Dufey, 1970 was the most species rich genera with seven species, followed by Thysanoplusia Ichinose, 1973 and Chrysodeixis Hubner, 1821 with four and three species respectively. Among 31 species, 15 species are commonly found in Himalayan regions and while other species were distributed from subtropical to tropical region. Five species, T. orichalcea (Fabricius, 1775), Chrysodeixis eriosoma (Doubleday, 1843), C. acuta (Walker, 1858), C. chalcites (Esper, 1789), Trichoplusia ni (Hubner, 1803) are widespread throughout India and reported as serious crop pests. Present study also revealed range expansion of four species viz., Dactyloplusia impulsa (Walker, 1865), Ctenoplusia mutans (Walker, 1865), Ctenoplusia tarassota (Hampson, 1913) and Zonoplusia ochreata (Walker, 1865). Systematic accounts of all 31 species are discussed here with adult images, species diagnostic characters, collection localities, detailed distributions and reported larval host plants. In addition to morphological studies, for the first time, a preliminary barcode library for 25 species of Indian Plusiinae with average intra-specific distance (%), maximum intra-specific distance (%) and distance to nearest neighbor (%) for individual species is provided. Among 25 species, four species barcode data (Ctenoplusia mutans, C. kosemponesis, Plusiopalpa adrasta, Sclerogenia jessica) are novel to world and 18 species barcode sequences were novel to India.

Evolutionarily Related Dihydrofolate Reductases Perform Coequal Functions Yet Show Divergence in Their Trajectories.

The enzyme dihydrofolate reductase (DHFR) catalyzes NADPH dependent reduction of dihydrofolate to tetrahydrofolate. It plays a crucial role in the DNA synthesis. The investigation of evolution of DHFR generates immense curiosity. It aids in predicting how the enzyme has adapted to the surroundings of various cell types. In spite of great similarity in the structure of E. coli DHFR and human DHFR, their primary sequences are divergent to a great extent, which is evident in variations in the kinetics mechanism of their catalysis. In presence of physiological levels of ligands, they possess distinct kinetics and different rate limiting steps. We have reviewed the process of their unfolding and refolding, their behaviour in denaturing conditions and in presence of various chaperones. Although there is structural similarity between these two homologous enzymes yet they have established distinct mechanisms to accomplish the coequal functions.

Quantification of differential efficacy of chemical chaperones in ameliorating solubilization and folding of zebrafish dihydrofolate reductase.

Protein aggregation is a major hindrance in many in vivo and in vitro studies of proteins. It results in the formation of inclusion bodies and non-functional aggregates. Chemical chaperones also known as osmolytes which are accumulated during the stress conditions in the cells can influence the protein stability through various mechanisms. They act as osmoprotectants and contribute to the protein folding by enabling the protein to bury the backbone into the core of protein fold. In the current study, we observed the effect of chemical chaperones from four different classes on the stability and functionality of aggregation prone protein zebrafish dihydrofolate reductase (zDHFR). We also used UV-visible and circular dichroism (CD) spectroscopy to explore the protecting action of chemical chaperones on the structure and activity of zDHFR in vitro and in vivo conditions.

Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon.

Osmolyte induced enhancement of expression and solubility of human dihydrofolate reductase: An in vivo study.

The process of recombinant protein production in E. coli system is often hampered by the formation of insoluble aggregates. Human Dihydrofolate reductase (hDHFR), an enzyme involved in the synthesis of purine, thymidilate and several other amino acids like glycine, methionine and serine is highly aggregation prone. It catalyzes the reduction of dihydrofolate (H2F) in order to regenerate tetrahydrofolate (H4F) utilizing NADPH as a cofactor. We have attempted to ameliorate the production of soluble and functional protein by growing and inducing the cells under osmotic stress condition, in the presence of various osmolytes like glycerol, sorbitol, TMAO, proline and glycine at 37°C. The expression and yield of functional hDHFR protein were highly enhanced in the presence of these osmolytes. The specific activity of the purified recombinant hDHFR protein has also been increased to a cogent level in the presence of osmolytes. We also observed that protein expressed in presence of the osmolytes was stable in the denaturing conditions as compared to the protein expressed in absence of an osmolyte. We also observed using the intrinsic fluorescence spectroscopy that the osmolytes didn't interfere with the structure of the protein and in denaturing conditions the protein expressed in presence of osmolytes had more stability. Our study is consequential in increasing the production of functional and soluble protein in the cell extract and will also be appropriate to find a therapeutic agent against many neurodegenerative diseases.

Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish.

The folding and unfolding mechanisms of a small monomeric protein, Dihydrofolate reductase (1.5.1.3.) from a new variant, Zebrafish (zDHFR) has been studied through GdnHCl denaturation, followed by its refolding through dilution of the denaturant. Intrinsic and extrinsic fluorescence, far-UV CD and enzyme activity were employed to monitor structural and functional changes due to chemical denaturation. The unfolding transitions monitored by intrinsic fluorescence showed that GdnHCl based denaturation of zDHFR is reversible. At low concentration of the denaturant, zDHFR forms intermediate species as reflected by increased fluorescence intensity compared to the native and fully unfolded form. Equilibrium unfolding transition study of zDHFR induced by GdnHCl exhibited three- state process. The non- coincidence of fluorescence and far-UVCD based transitions curves support the establishment of three state model of zDHFR protein which involves native, intermediate and unfolded forms. Analysis of the equilibrium unfolding transition suggests the presence of non- native intermediate species. A comparative study of various species of DHFR shows that zDHFR has comparable thermodynamic stability with human counterpart and thus proved to be a good in vitro model system for structure- function relationship studies. Understanding various conformational states during the folding unfolding process of the zDHFR protein may provide important clues towards designing inhibitors against this important protein involved in cell cycle regulation.

Global survey of haplotype frequencies and linkage disequilibrium at the RET locus.

We have constructed haplotypes based on normal variation at six polymorphic sites-five single nucleotide polymorphisms (SNPs) and one short tandem repeat polymorphism (STRP)-at the RET locus for samples of normal individuals from 32 populations distributed across the major continental regions of the world. The haplotyped system spans 41.6 kilobases and encompasses most of the coding region of the gene. All of the markers are polymorphic in all regions of the world and in most individual populations. Expected heterozygosities for the six-site haplotypes range from 82 to 94% for all populations studied except for two Amerindian groups from the Amazon basin at 61 and 76%. Individual populations had from four to eight haplotypes with frequencies exceeding 5%. In general, African, southwest Asian and European groups have the highest numbers of total and of commonly occurring haplotypes; the lowest numbers are observed in Amerindian populations. Overall linkage disequilibrium (LD) for the five SNP sites was very significant (P