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Query: UNIPROT:Q86TM3 (cage)
 29,987 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluorescence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin antibodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin antibodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.
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PMID:Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro. 762 16

We have determined intersite distances from Cys374 of actin to Cys707 (SH1) and Cys697 (SH2) of myosin subfragment 1 (S1) in actosubfragment 1 (A.S1) by fluorescence resonance energy transfer for rigor complex A.S1 and complexes containing bound ADP and ADP plus orthovanadate (Vi), A.S1.ADP, and A.S1.ADP.Vi. A single energy acceptor (4-dimethylaminophenylazophenyl-4'-maleimide, DABMI) was attached to Cys374, and two different energy donors [(5-(iodoacetamideothyl)aminonaphthalene-1-sulfonic acid (IAEDANS) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)] were each attached to SH1 and SH2 for the distance determination. The two sites SH1 and SH2 of S1 were approximately equidistant (ca. 45 A) from actin Cys374 in rigor A.S1 when MIANS was the energy donor attached to the two thiols. The Cys374-SH1 distance decreased by 7-8 A in the presence of ADP plus Vi, but the distance Cys374-SH2 was essentially unaltered under identical conditions. Slightly different but similar distance results were obtained with AEDANS as energy donor. If the structure of actin monomer in A.S1 is assumed to be rigid [Miki, M. (1991) Biochemistry 30, 10878-10884], the present results indicate that MgADP plus Vi induced a movement of SH1 toward the actin site and that SH2 was insensitive to saturation of the active site pocket of S1 and relatively immobile. These results suggest that during the steady-state hydrolysis of ATP or in the weak-binding state of actomyosin, the short helical segment of S1 heavy chain containing SH1 moves closer to the COOH-terminal end of actin, while the adjacent helical segment containing SH2 remains stationary. The emission spectrum of MIANS attached to SH2 experienced a large red spectral shift (6-10 nm) in the presence of MgADP, MgADP + Vi, MgADP + beryllium fluoride, and ATP. A crude model of S1 based on the C alpha coordinates suggests that SH2 is located in a hydrophobic cage surrounded by three hydrophobic residues. Reorientation of one of these side chains could expose SH2 to the solvent. The observed red spectral shift of MIANS attached to SH2 could be explained by such a nucleotide-induced exposure, and this explanation would be consistent with the interpretation that SH2 is stationary.
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PMID:Internal movement in myosin subfragment 1 detected by fluorescence resonance energy transfer. 775 79

Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.
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PMID:Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. 791 27

An artificial membrane, blood-feeding technique was evaluated for Culicoides impunctatus Goetghebuer using wild-caught insects in Scotland. Chick skins and stretched Nescofilm were satisfactory as feeding membranes. The maximum mean feeding rate achieved was 61.7 +/- 4.0% through Nescofilm with < or = 50 female midges per cage. This rate decreased as a negative function of time and density. A 4 degrees C decrease in blood temperature resulted in a 30% reduction in feeding. ATP applied to the outer membrane surface at 0.01 M and 0.1 M concentrations increased the feeding rate at 30 min after collection from 19.3 +/- 3.3% to 37.2 +/- 4.2% (0.01 M) and 34.5 +/- 4.9% (0.1 M). The Nescofilm membrane method will aid in colonization and may be useful in virus transmission experiments.
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PMID:Laboratory feeding of Culicoides impunctatus (Diptera: Ceratopogonidae) through natural and artificial membranes. 818 22

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

An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.
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PMID:Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin. 860 51

Free GroEL binds denatured proteins very tightly: it retards the folding of barnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight-binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES.nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES.nucleotide complex. Fast-folding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL.denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL.GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination of folding cage and an annealing machine.
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PMID:Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and -pulsed folding and annealing cage. 863 99

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.
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PMID:Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis. 894 33

The chaperonin system GroEL/GroES assists in the folding of proteins in the bacterial cytosol. Recent applications of biophysical techniques for the structural analysis of GroEL, GroES, and chaperonin-bound protein folding intermediates have provided the basis for understanding the molecular mechanism of GroEL/GroES action. GroEL, a double-ring complex, binds unfolded proteins at its inner ring surface. Protein folding proceeds in the central cavity of GroEL, after dissociation of the polypeptide has been triggered by ATP hydrolysis in GroEL. Premature release of unfolded protein into external solution is prevented by binding of the cofactor GroES on top of the GroEL cylinder, resulting in an enclosed cage. Upon ATP-dependent dissociation of GroES, substrate protein is eventually released from GroEL in a native or native-like conformation. While current in vitro results about the structure, function, and molecular mechanism of GroEL/GroES-assisted protein folding have led to a quite detailed picture of this complex process, the extent to which the GroEL/GroES system actually participates in the folding of newly-synthesized proteins in the cell is less defined and remains a subject for further studies. Ingenious biochemical and genetic approaches will be necessary to show whether our current view of chaperonin action indeed accurately reflects its modus operandi inside a living cell.
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PMID:Protein folding assisted by the GroEL/GroES chaperonin system. 955 20

Fragments encompassing the apical domain of GroEL, called minichaperones, facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. We have identified the smallest minichaperone that is active in vitro in chaperoning the refolding of rhodanese and cyclophilin A: GroEL(193-335). This finding raises the question of whether the minichaperones are active under more stringent conditions in vivo. The smallest minichaperones complement two temperature-sensitive Escherichia coli groEL alleles, EL44 and EL673, at 43 degreesC. Although they cannot replace GroEL in cells in which the chromosomal groEL gene has been deleted by P1 transduction, GroEL(193-335) enhances the colony-forming ability of such cells when limiting amounts of GroEL are expressed from a tightly regulated plasmid. Surprisingly, we found that overexpression of GroEL prevents plaque formation by bacteriophage lambda and inhibits replication of the lambda origin-dependent plasmid, Lorist6. The minichaperones also inhibit Lorist6 replication, but less markedly. The complex quaternary structure of GroEL, its central cavity, and the structural allosteric changes that take place on the binding of nucleotides and GroES are not essential for all of its functions in vivo.
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PMID:In vivo activities of GroEL minichaperones. 970 66


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