Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo.
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PMID:Novel 30 kDa protein possessing ATP-binding and chaperone activities. 929 Nov 33

Modification of the Escherichia coli chaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex. Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced. As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner. Folding of dihydrofolate reductase at 25 degrees C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely. At 37 degrees C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent. These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release.
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PMID:Role of the GroEL chaperonin intermediate domain in coupling ATP hydrolysis to polypeptide release. 951 31

Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) are shown to refold via several intermediate forms, each of which can bind to the chaperonin GroEL. When stable complexes with GroEL are formed, they consist of late-folding intermediates. In addition, we find that late-folding intermediates that are present in the native enzyme bind to GroEL. For the E. coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8 degreesC to 42 degreesC, at which temperature either protein is completely bound as the last (EcDHFR) or the last two (MuDHFR) folding intermediate(s). Thus for EcDHFR, the binding is transient at low temperature (<30 degreesC) and stable at high temperature (>35 degreesC). For MuDHFR, complex formation appears less temperature dependent. In general, the data demonstrate that the overall binding free energy for the interaction of GroEL with native DHFR is the sum of the free energy for the first step in DHFR unfolding, which is unfavorable, and the free energy of binding the non-native conformation, which is favorable. For EcDHFR, this results in an overall binding free energy that is unfavorable below 30 degreesC. Over the temperature range of 8 degreesC to 42 degreesC, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energy difference between two pre-existing non-native conformations of MuDHFR, resulting in binding to more than one folding intermediate.
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PMID:The chaperonin GroEL binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases. 991 11

Previous investigation has shown that at 22 degrees C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512-525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, approximately 80-90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0-50 microM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1). For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.
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PMID:Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL. 1054 63

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.
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PMID:Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding. 1193 2

The chaperonin GroEL binds to a large number of polypeptides, prevents their self-association, and mediates appropriate folding in a GroES and adenosine triphosphate-dependent manner. But how the GroEL molecule actually recognizes the polypeptide and what are the exact GroEL recognition sites in the substrates are still poorly understood. We have examined more than 50 in vivo substrates as well as well-characterized in vitro substrates, for their binding characteristics with GroEL. While addressing the issue, we have been driven by the basic concept that GroES, being the cochaperonin of GroEL, is the best-suited substrate for GroEL, as well as by the fact that polypeptide substrate and GroES occupy the same binding sites on the GroEL apical domain. GroES interacts with GroEL through selective hydrophobic residues present on its mobile loop region, and we have considered the group of residues on the GroES mobile loop as the key element in choosing a substrate for GroEL. Considering the hydrophobic region on the GroES mobile loop as the standard, we have attempted to identify the homologous region on the peptide sequences in the proteins of our interest. Polypeptides have been judged as potential GroEL substrates on the basis of the presence of the GroES mobile loop-like hydrophobic segments in their amino acid sequences. We have observed 1 or more GroES mobile loop-like hydrophobic patches in the peptide sequence of some of the proteins of our interest, and the hydropathy index of most of these patches also seems to be approximately close to that of the standard. It has been proposed that the presence of hydrophobic patches having substantial degree of hydropathy index as compared with the standard segment is a necessary condition for a peptide sequence to be recognized by GroEL molecules. We also observed that the overall hydrophobicity is also close to 30% in these substrates, although this is not the sufficient criterion for a polypeptide to be assigned as a substrate for GroEL. We found that the binding of aconitase, alpha-lactalbumin, and murine dihydrofolate reductase to GroEL falls in line with our present model and have also predicted the exact regions of their binding to GroEL. On the basis of our GroEL substrate prediction, we have presented a model for the binding of apo form of some proteins to GroEL and the eventual formation of the holo form. Our observation also reveals that in most of the cases, the GroES mobile loop-like hydrophobic patch is present in the unstructured region of the protein molecule, specifically in the loop or beta-sheeted region. The outcome of our study would be an essential feature in identifying a potential substrate for GroEL on the basis of the presence of 1 or more GroES mobile loop-like hydrophobic segments in the amino acid sequence of those polypeptides and their location in three-dimensional space.
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PMID:Factors governing the substrate recognition by GroEL chaperone: a sequence correlation approach. 1583 45

The reaction cycle and the major structural states of the molecular chaperone GroEL and its cochaperone, GroES, are well characterized. In contrast, very little is known about the nonnative states of the substrate polypeptide acted on by the chaperonin machinery. In this study, we investigated the substrate protein human dihydrofolate reductase (hDHFR) while bound to GroEL or to a single-ring analog, SR1, by NMR spectroscopy in solution under conditions where hDHFR was efficiently recovered as a folded, enzymatically active protein from the stable complexes upon addition of ATP and GroES. By using the NMR techniques of transverse relaxation-optimized spectroscopy (TROSY), cross-correlated relaxation-induced polarization transfer (CRIPT), and cross-correlated relaxation-enhanced polarization transfer (CRINEPT), bound hDHFR could be observed directly. Measurements of the buildup of hDHFR NMR signals by different magnetization transfer mechanisms were used to characterize the dynamic properties of the NMR-observable parts of the bound substrate. The NMR data suggest that the bound state includes random coil conformations devoid of stable native-like tertiary contacts and that the bound hDHFR might best be described as a dynamic ensemble of randomly structured conformers.
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PMID:Direct NMR observation of a substrate protein bound to the chaperonin GroEL. 1617 84

The chaperonin GroEL binds non-native polypeptides in an open ring via hydrophobic contacts and then, after ATP and GroES binding to the same ring as polypeptide, mediates productive folding in the now hydrophilic, encapsulated cis chamber. The nature of the folding reaction in the cis cavity remains poorly understood. In particular, it is unclear whether polypeptides take the same route to the native state in this cavity as they do when folding spontaneously free in solution. Here, we have addressed this question by using NMR measurements of the time course of acquisition of amide proton exchange protection of human dihydrofolate reductase (DHFR) during folding in the presence of methotrexate and ATP either free in solution or inside the stable cavity formed between a single ring variant of GroEL, SR1, and GroES. Recovery of DHFR refolded by the SR1/GroES-mediated reaction is 2-fold higher than in the spontaneous reaction. Nevertheless, DHFR folding was found to proceed by the same trajectories inside the cis folding chamber and free in solution. These observations are consistent with the description of the chaperonin chamber as an "Anfinsen cage" where polypeptide folding is determined solely by the amino acid sequence, as it is in solution. However, if misfolding occurs in the confinement of the chaperonin cavity, the polypeptide chain cannot undergo aggregation but rather finds its way back to a productive pathway in a manner that cannot be accomplished in solution, resulting in the observed high overall recovery.
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PMID:Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin cavity and free in solution. 1809 16

The original experiments reconstituting GroEL-GroES-mediated protein folding were carried out under "nonpermissive" conditions, where the chaperonin system was absolutely required and substrate proteins could not achieve the native state if diluted directly from denaturant into solution. Under "permissive" conditions, however, employing lower substrate concentration and lower temperature, some substrate proteins can be refolded both by the chaperonin system and while free in solution. For several of these, the protein refolds more rapidly inside the GroEL-GroES cis chamber than free in solution, suggesting that the chamber may have an active role in assisting protein folding. Here, we observe that the difference is caused by reversible multimolecular association while folding in solution, an avenue of kinetic partitioning that slows the overall rate of renaturation relative to the chaperonin chamber, where such associations cannot occur. For Rubisco, reversible aggregation during folding in solution was observed by gel filtration. For a mutant of maltose-binding protein (DM-MBP), the rate of folding in solution declined with increasing concentration, and the folding reaction produced light scattering. Under solution conditions where chloride was absent, however, light scattering no longer occurred, and DM-MBP folded at the same rate as in the cis cavity. In a further test, dihydrofolate reductase, thermally inactivated in the cis cavity or in solution, was substantially reactivated upon temperature downshift in the cis cavity but not in solution, where aggregation occurred. We conclude that the GroEL-GroES chamber behaves as a passive "Anfinsen cage" whose primary role is to prevent multimolecular association during folding.
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PMID:Chaperonin chamber accelerates protein folding through passive action of preventing aggregation. 1898 17

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


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