Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase with E. coli ribosome.
16 Mar 1992-Biochemical and Biophysical Research Communications (Academic Press)-Vol. 183, Iss: 2, pp 774-780
TL;DR: Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.
Abstract: Fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase were denatured either by physical or by chemical means. In vitro reconstitution of these denatured enzymes was assisted by 70S E. coli ribosome, as shown by the recovery of their catalytic competence. Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.
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TL;DR: This review focuses on the current state of knowledge in protein folding in the cell with emphasis on the early stage of a protein's life, as the nascent polypeptide traverses and emerges from the ribosomal tunnel.
Abstract: Over five decades of research have yielded a large body of information on how purified proteins attain their native state when refolded in the test tube, starting from a chemically or thermally denatured state. Nevertheless, we still know little about how proteins fold and unfold in their natural biological habitat: the living cell. Indeed, a variety of cellular components, including molecular chaperones, the ribosome, and crowding of the intracellular medium, modulate folding mechanisms in physiologically relevant environments. This review focuses on the current state of knowledge in protein folding in the cell with emphasis on the early stage of a protein's life, as the nascent polypeptide traverses and emerges from the ribosomal tunnel. Given the vectorial nature of ribosome-assisted translation, the transient degree of chain elongation becomes a relevant variable expected to affect nascent protein foldability, aggregation propensity and extent of interaction with chaperones and the ribosome.
107 citations
TL;DR: Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese and this activity is related to the state or conformation of ribosome that is promoted by EF-G.
Abstract: Background Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes. Results Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds of refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by α -sarcin. Activation by either mechanism is strongly inhibited by the EF-G·GDP·fusidic acid complex. Conclusion Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G·GDP·fusidic acid complex.
93 citations
TL;DR: Overall, this review illustrates how prokaryotic organisms might provide the bedrock on which to understand the complexity of protein folding and aggregation in the cell.
Abstract: Proteins might experience many conformational changes and interactions during their lifetimes, from their synthesis at ribosomes to their controlled degradation. Because, in most cases, only folded proteins are functional, protein folding in bacteria is tightly controlled genetically, transcriptionally, and at the protein sequence level. In addition, important cellular machinery assists the folding of polypeptides to avoid misfolding and ensure the attainment of functional structures. When these redundant protective strategies are overcome, misfolded polypeptides are recruited into insoluble inclusion bodies. The protein embedded in these intracellular deposits might display different conformations including functional and β-sheet-rich structures. The latter assemblies are similar to the amyloid fibrils characteristic of several human neurodegenerative diseases. Interestingly, bacteria exploit the same structural principles for functional properties such as adhesion or cytotoxicity. Overall, this review illustrates how prokaryotic organisms might provide the bedrock on which to understand the complexity of protein folding and aggregation in the cell.
91 citations
Cites background from "Reactivation of denatured fungal gl..."
...the ribosome itself has the capability to assist protein folding [46, 47]....
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TL;DR: Observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydration from baker's yeast by ribosomes from E. coli, wheat germ and rat liver show loss of tertiary structure inhibited the protein-folding activity of 23S rRNA.
Abstract: Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydro-genase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein-folding activity of E. coli, ribosomes was found to be present in 50s particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.
82 citations
TL;DR: Domain V of 23S rRNA appears to play a crucial role in reactivation of denatured proteins in E. coli.
Abstract: Escherichia coli ribosome, its 50S subunit, or simply the 23S rRNA can reactivate denatured proteins in vitro. Here we show that protein synthesis inhibitors chloramphenicol and erythromycin, which bind to domain V of 23S rRNA of E. coli, can inhibit reactivation of denatured pig muscle lactate dehydrogenase and fungal glucose-6-phosphate dehydrogenase by 23S rRNA completely. Oligodeoxynucleotides complementary to two regions within domain V (which cover sites of chloramphenicol resistant mutations and the putative A site of the incoming aminoacyl tRNA), but not to a region outside of domain V, also can inhibit the activity. Domain V of 23S rRNA, therefore, appears to play a crucial role in reactivation of denatured proteins.
67 citations
References
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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
TL;DR: Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes and are implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants.
Abstract: An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.
1,277 citations
TL;DR: The significance of this pathway in animal tissues, its physiological control and the relative importance of the direct oxidative and glycolytic routes of carbohydrate metabolism are still, however, chiefly matters of conjecture.
Abstract: Renewed interest in the direct oxidative pathway of glucose 6-phosphate metabolism during the last few years has revealed that this pathway is by no means restricted to erythrocytes, yeast and microorganisms. The triphosphopyridine-nucleotide(TPN)-specific glucose 6-phosphate and 6-phosphogluconate dehydrogenases are also widely distributed in mammalian tissues (Dickens & Glock, 1950, 1951; Horecker & Smyrniotis, 1951), in a variety of lower plants and animals (Cohen, 1950) and also in higher plants (Conn & Vennesland, 1951; Gibbs, 1952). The significance of this pathway in animal tissues, its physiological control and the relative importance of the direct oxidative and glycolytic routes of carbohydrate metabolism are still, however, chiefly matters of conjecture. An essential preliminary step to such an investigation is to devise a satisfactory procedure for the assay of glucose 6-phosphate and 6-phosphogluconate dehydrogenases in animal tissues and it was with this object in view that the present work was undertaken.
1,186 citations
TL;DR: In vitro reconstitution of active ribulose bisphosphate carboxylase (Rubisco) from unfolded poly-peptides is facilitated by the molecular chaperones: chaperonin-60 from Escherichia coli, yeast mitochondria or chloroplasts, together with chaper onin-10 from E coli, and Mg-ATP.
Abstract: In vitro reconstitution of active ribulose bisphosphate carboxylase (Rubisco) from unfolded polypeptides is facilitated by the molecular chaperones: chaperonin-60 from Escherichia coli (groEL), yeast mitochondria (hsp60) or chloroplasts (Rubisco sub-unit-binding protein), together with chaperonin-10 from E. coli (groES), and Mg-ATP. Because chaperonins are ubiquitous, a conserved Mg-ATP-dependent mechanism exists that uses the chaperonins to facilitate the folding of some other proteins.
705 citations
TL;DR: The effect that the recognition of the essential roles played by these proteins in assembly processes may have on the principle of spontaneous self-assembly is discussed.
Abstract: Many polypeptides can self-assemble into functional structures while others assemble only in the presence of additional proteins (molecular chaperones) which are not components of the final structure. We discuss here the effect that the recognition of the essential roles played by these proteins in assembly processes may have on the principle of spontaneous self-assembly.
474 citations