Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish.
TL;DR: 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.
About: This article is published in International Journal of Biological Macromolecules.The article was published on 2016-10-01. It has received 10 citations till now. The article focuses on the topics: Equilibrium unfolding & Denaturation (biochemistry).
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TL;DR: The zebrafish (Danio rerio) is a versatile model organism that has been used in biomedical research for several decades to study a wide range of biological phenomena and is one of the predominant models of human genetic diseases.
Abstract: The zebrafish (Danio rerio) is a versatile model organism that has been used in biomedical research for several decades to study a wide range of biological phenomena. There are many technical advantages of using zebrafish over other vertebrate models. They are readily available, hardy, easy, and inexpensive to maintain in the laboratory, have a short life cycle, and have excellent fecundity. Due to its optical clarity and reproducible capabilities, it has become one of the predominant models of human genetic diseases. Zebrafish research has made rapid strides in the United States and Europe, but in India the field is at an early stage and many researchers still remain unaware of the full research potential of this tiny fish. The zebrafish model system was introduced into India in the early 2000s. Up to now, more than 200 scientific referred articles have been published by Indian researchers. This review gives an overview of the current state of knowledge for zebrafish research in India, with the aim of promoting wider utilization of zebrafish for high level biological studies.
18 citations
TL;DR: This 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.
9 citations
Cites background from "Investigation of folding unfolding ..."
...expression at 37oC, zDHFR shows maximum expression at lower temperature which is about 25 oC.(6,9) hDHFR requires overnight incubation in incubator shaker at 37 oC and...
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...A number of studies have been carried out to study the effect pronounced by osmolytes on the biochemical and biophysical aspects of several different enzymes.(6,9,15) Subsequently, there are two classes of osmolytes: compatible or counteracting....
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...Besides having a role in stabilizing the proteins, osmolytes also actively participate in the induction of refolding of the proteins which are misfolded.(6,9,15) It has been elucidated that the osmolytes are able to grant stability to proteins by unfavorably interacting with the peptide backbone of the protein due to which preferred hydration of the protein domain takes place...
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TL;DR: The effect of chemical chaperones from four different classes on the stability and functionality of aggregation prone protein zebrafish dihydrofolate reductase (zDHFR) in vitro and in vivo conditions is observed.
5 citations
TL;DR: Minichaperone can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon and indicates that it enhances the thermal stability of the enzyme.
4 citations
TL;DR: 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.
Abstract: 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.
4 citations
Cites methods from "Investigation of folding unfolding ..."
...Ellman’s test for determination of free thiols in zDHFR using DTNB Ellman’s assay is a colorimetric reaction which involves protein’s free thiols and Ellman’s reagent [DTNB, 5, 5-dithiobis (2-nitrobenzoic acid)] [14]....
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...6 then there was addition of 1 mM IPTG for the induction of histidine tagged protein at 25 C [14]....
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References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
TL;DR: Anfinsen as discussed by the authors provided a sketch of the rich history of research that provided the foundation for his work on protein folding and the Thermodynamic Hypothesis, and outlined potential avenues of current and future scientific exploration.
Abstract: Stanford Moore, William Stein, and Anfinsen were awarded the Nobel Prize in Chemistry in 1972 for \"their contribution
to the understanding of the connection between chemical structure and catalytic activity of the active center of the ribonuclease
molecule.\" In his Nobel Lecture, Anfinsen provided a sketch of the rich history of research that provided the foundation
for his work on protein folding and the \"Thermodynamic Hypothesis,\" and outlined potential avenues of current and
future scientific exploration.
6,520 citations
2,210 citations
TL;DR: Evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions.
Abstract: The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are on average more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.
943 citations