Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.
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PMID:Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system. 791 Dec 83

The chaperonin GroEL is able to mediate protein folding in its central cavity. GroEL-bound dihydrofolate reductase assumes its native conformation when the GroES cofactor caps one end of the GroEL cylinder, thereby discharging the unfolded polypeptide into an enclosed cage. Folded dihydrofolate reductase emerges upon ATP-dependent GroES release. Other proteins, such as rhodanese, may leave GroEL after having attained a conformation that is committed to fold. Incompletely folded polypeptide rebinds to GroEL, resulting in structural rearrangement for another folding trial in the chaperonin cavity.
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PMID:Protein folding in the central cavity of the GroEL-GroES chaperonin complex. 855 46

Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild-type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding.
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PMID:Hsp60-independent protein folding in the matrix of yeast mitochondria. 863 Dec 98

Ligand-induced conformational changes of GroEL alone and with bound rhodanese, citrate synthase, or dihydrofolate reductase were studied by limited proteolysis. Similar digestion patterns of GroEL, with or without bound substrate polypeptide, were obtained in the absence and presence of the chaperonin ligands, K+, Mg2+, or ATP. The rates of formation and degradation of the six produced proteolytic fragments were significantly different, however. Strikingly, only with Mg2+/ATP or K+/Mg2+/ATP an additional fragment of approximately 25 kDa was generated during digestion of GroEL alone or with bound rhodanese or dihydrofolate reductase, but not with bound citrate synthase. Most of the trypsin-sensitive sites in GroEL were localized in the flexible apical domain, which contains the putative polypeptide-binding region. Our data indicate that subtle structural changes in the trypsin-sensitive regions of GroEL occur as a result of the binding of the chaperonin ligands. However, these structural changes are influenced by the GroEL substrate polypeptides.
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PMID:Ligand-induced conformational changes of GroEL are dependent on the bound substrate polypeptide. 866 87

Most mitochondrial proteins are synthesized with an N-terminal signal sequence that targets these proteins to various compartments within the mitochondria. Signal sequences have been shown to be functional by fusing them to a nonmitochondrial passenger protein and observing import. In many cases, a signal sequence has been fused to passenger proteins, such as dihydrofolate reductase, and import occurred. There are, though, several unexplained instances in which a signal sequence was attached to a passenger protein and import was not observed. In this study, the N-terminal 23 residues of the matrix enzyme rhodanese could import several passenger proteins but were unable to import the mature form of mitochondrial aldehyde dehydrogenase (mALDH). However, if these same 23 residues were fused to the middle portion of mALDH, import was recovered, suggesting that the rhodanese signal sequence and N terminus of mALDH were incompatible for import. Circular dichroism data indicated that a peptide corresponding to the region of fusion between rhodanese and mALDH had less structure than corresponding peptides from imported fusion proteins, suggesting that mALDH may alter the helix in the rhodanese signal sequence, thus preventing import.
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PMID:Influence of the mature portion of a precursor protein on the mitochondrial signal sequence. 870 95

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.
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PMID:A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria. 1515 70

Nuclear magnetic resonance (NMR) observation of the uniformly (2) H,(15) N-labeled stringent 33-kDa substrate protein rhodanese in a productive complex with the uniformly (14) N-labeled 400 kDa single-ring version of the E. coli chaperonin GroEL, SR1, was achieved with the use of transverse relaxation-optimized spectroscopy, cross-correlated relaxation-induced polarization transfer, and cross-correlated relaxation-enhanced polarization transfer. To characterize the NMR-observable parts of the bound rhodanese, coherence buildup rates by different magnetization transfer mechanisms were measured, and effects of covalent crosslinking of the rhodanese to the apical binding surface of SR1 were investigated. The results indicate that the NMR-observable parts of the SR1-bound rhodanese are involved in intracomplex rate processes, which are not related to binding and release of the substrate protein from the SR1 binding surface. Rather, they correspond to mobility of the stably bound substrate, which thus appears to include flexibly disordered polypeptide segments devoid of long-lived secondary structures or tertiary folds, as was previously observed also with the smaller substrate human dihydrofolate reductase.
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PMID:Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL. 2163 84