Showing papers on "Conformational change published in 1980"
TL;DR: As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex, the Ca2- dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold ofCa2+ concentration for control of enzyme activity.
Abstract: Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.
327 citations
TL;DR: It is shown that loss of fluorescence of the labeled G-actin may be associated with inactivation of the actin and any conformational change induced by KCl is probably different from that induced by divalent metal ions.
113 citations
TL;DR: These findings confirm and extend previous reports regarding the ferric complex and characterize the ferrous complex for the first time and confirm that the iron binding sites are not equivalent and the binding of iron appears to be mostly intramolecular.
62 citations
TL;DR: The structure of a complex of 8-bromoadenosine monophosphate with hexokinase in crystal form BIII has been determined from a 3 A resolution difference electron density map, and model building suggests there would be additional contacts between the small enzyme lobe and the metal ion.
62 citations
TL;DR: It is concluded that a slow conformational change closing open synaptic current channels is likely to determine i.p.s.c. decay, and suggests that ACh removal, either by diffusion or hydrolysis, is not the rate‐limiting step.
Abstract: 1. In neurones BL and BR 3, 6, 8, 9, 10 and 11 of Aplysia buccal ganglia, cholinergic inhibitory post-synaptic potentials are produced by activity in either of two presynaptic cells. In order to analyse the synaptic conductance change, neurones were voltage-clamped inhibitory post-synaptic currents (i.p.s.c.) recorded. 2. The synaptic conductance change rises to an average peak value of 0.65 micromho and decays exponentially with single time constant tau of 19 msec. 3. We have attempted to identify the rate-limiting step responsible for i.p.s.c. decay from among the following possibilities: (1) acetylcholine (ACh) supply, (2) ACh removal by diffusion, (3) ACh removal by hydrolysis or (4) a slow unbinding or conformational change closing open synaptic current channels. 4. Cooling prolongs tau, with Q10 of 5.2. Cooling and eserine treatment together produce greatly prolonged, exponentially decaying i.p.s.c.s with tau > 150 msec. These results suggest that ACh removal, either by diffusion or hydrolysis, is not the rate-limiting step. 5. Prolonging synaptic action potential time course with intracellular injection of tetraethylammonium broadens the i.p.s.c. peak but does not affect the decay tail, suggesting that the rate-limiting step is not ACh release. 6. The spectrum of ACh-induced current fluctuations is fitted by a double Lorentzian with cut-off frequencies of 7.8 and 47 Hz. The frequency of the slower component corresponds to the macroscopic i.p.s.c. decay tau. 7. We conclude that a slow conformational change closing open synaptic current channels is likely to determine i.p.s.c. decay. We cannot, however, exclude either delayed diffusion or a late tail of slow ACh release as possibilities.
55 citations
TL;DR: It is established that sulfate is able to modify the environment of some critical amino acids located in the N-terminal half of the protein, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm.
Abstract: Anions and particularly sulfate are known to interact with 3-phosphoglycerate kinase and to induce an increase of its catalytic efficiency. The present work affords information on the location of the anionic site and on the conformational change produced by the sulfate binding. We have established that sulfate is able, first, to modify the environment of some critical amino acids (cysteine and arginines) located in the N-terminal half of the protein, second, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm. All these modifications are produced by sulfate concentrations required for the activation of the enzyme. The most striking result consists in a large change in the hydrodynamic properties of the protein upon sulfate interaction as determined by analytical ultracentrifugation studies. Thus, sulfate modifies the shape of the molecular, causing it to become more compact. Furthermore, a study of the binary and ternary complexes between yeast 3-phosphoglycerate kinase and its substrates suggests that such a change of the shape of the molecular only occurs in sulfate-enzyme with or without substrates and in ATP (with or without Mg2+)-3-phosphoglycerate-enzyme complexes.
53 citations
TL;DR: Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge, suggesting a dissociative mechanism for protein Kinase, although a mechanism with some associative character remains possible.
48 citations
TL;DR: Experimental evidence suggests that the anticodon region lies in a groove of the protein, and can be correlated to structural modifications of the enzyme as shown by the modification of circular dichroism and tryptophan fluorescence.
Abstract: The interaction between yeast tRNAPhe and phenylalanyl-tRNA synthetase was studied by monitoring different emission properties of the wybutine residue. The following results were established: (a) the isolated tRNAPhe exists in solution under at least two forms in a magnesium-dependent equilibrium; (b) in the complex with the cognate synthetase, the tRNA still has two different conformations, slightly different from the above conformers. Mg2+ ions appear to play a crucial role in the adaptation of both macromolecules one to another: for concentrations of the order of 1 mM, magnesium ions trigger a conformational change of the anticodon loop of tRNA, resulting in the expulsion of the wybutine from a stacked region. Furthermore, experimental evidence suggests that the anticodon region lies in a groove of the protein.
This local conformation change, occurring in the anticodon loop, can be correlated to structural modifications of the enzyme as shown by the modification of circular dichroism and tryptophan fluorescence.
During these different conformation changes, an energy transfer from tryptophan residues to wybutine is triggered in the critical magnesium concentration range.
46 citations
TL;DR: The carbohydrate portion of prothrombin fragment 1 has been removed by fluorolysis in anhydrous HF and the deglycosylated protein retains its calcium- and membrane-binding properties, suggesting that the calcium-dependent conformational change exposes a site on the protein which functions in secondary self-association.
44 citations
41 citations
TL;DR: H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.
Abstract: Calf thymus nucleosomes exhibit two different and independent hydrodynamic responses to diminishing salt concentration. One change is gradual over the range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-site cross-linking efficiency. The other change is abrupt, being centered between 1 and 2 mM Na+. We found only one abrupt change in sedimentation rate for particles ranging in DNA content fom 144 to 230 base pairs. This response to decreasing ionic strength is similar for particles of both 169 and 230 base pairs. Core particles (144 base pairs) exhibit a somewhat diminished response. The abrupt change is blocked by formaldehyde or dimethylsuberimidate cross-linking. The blockage by dimethylsuberimidate demonstrates that the abrupt conformational change requires the participation of the core histones. H1 completely blocks the abrupt but not the gradual conformational change. Thus H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.
TL;DR: This study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process and is postulate that a function of the post-polymerization epimerization of d -glucuronate to l -iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.
TL;DR: Results demonstrate that human and bacterial thymidylate synthetases undergo marked structural changes upon forming a ternary complex which is probably very similar to an activated complex formed with both substrates.
Abstract: The conformations of thymidylate synthetases from CCRF-CEM human leukemic cells and Lactobacillus casei were studied by hydrodynamic methods. Although the human enzyme has a molecular weight of 70 000--72 500, somewhat smaller than that of the L. casei enzyme, it has a larger Stokes radius and a lower sedimentation coefficient, indicating that the human enzyme is less spherical than the bacterial enzyme. Thymidylate synthetases from the human leukemic cells and the bacterial source both undergo substantial conformational changes upon the formation of a covalent ternary complex with the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. The Stokes radius of both proteins decreases by 3.5% when the ternary complex is formed in spite of the 1.8% increase in molecular weight, and the sedimentation coefficient increases by 3.5% after appropriate corrections for the bound ligands. Ternary complex formation results in a more compact structure for both enzymes, with approximately the same reduction in the frictional ratio. Experiments with the bacterial enzyme indicate that approximately 70% of the total conformational change occurs upon binding of 1 mol of ligands/mol of enzyme. These results demonstrate that human and bacterial thymidylate synthetases undergo marked structural changes upon forming a ternary complex which is probably very similar to an activated complex formed with both substrates. These investigations also provide evidence for fundamental similarities in the mechanism of ternary complex formation with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, despite the marked differences in amino acid composition and the dissimilar conformations of these two enzymes obtained from widely divergent sources.
TL;DR: Results indicate a local conformational change on the surface of the cytochrome c molecule in an area to the lower left of and below the heme cleft (standard front view of the molecule facing the exposed edge of the prosthetic group), which might be instrumental in the cy tochrome c-mediated electron shuttle between cyto chrome c1 and a in the final segment of the mitochondrial electron transport chain.
TL;DR: The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment and appears to be the result of a conformational change that is most likely an unfolding of the T and D loops.
Abstract: The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment. This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)-programed tight-couple 70S ribosomes of Escherichia coli. The tritium incorporation into specific sites of the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments. Ribosome binding leads to marked suppression in the exchange rate of a number of fragments. This delineates extensive regions of tRNA-ribosome contact. No change in exchange rates is seen for fragments from the corner of the molecule, indicating that this region of bound tRNA is readily accessible to the solvent. Ribosome binding results in an enhanced exchange rate at the T loop. This appears to be the result of a conformational change that is most likely an unfolding of the T and D loops. Additional tritium exchange reactions suggest this conformational change is induced by ribosomes and not by messenger.
TL;DR: It is postulate that thiamin pyrophosphate can increase the biological half-life of the in vivo, membrane-bound complex through conformational changes induced by the binding of this cofactor.
TL;DR: It is suggested that this malleability of the cAMP-dependent protein kinase might constitute the molecular basis for modulating the specificity of the enzyme and channeling its activity from one substrate to another in response to intracellular specifier signals.
TL;DR: It is found that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core.
Abstract: We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.
TL;DR: The data indicate the presence of multiple conformational states of the enzyme, Na+,K+-ATPase, which can be repeated by the readdition of ATP but not by ADP.
Abstract: Na+,K+-ATPase [EC 3.6.1.3] from pig kidneys was treated with the fluorescent reagent N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) in the presence of CKl. The resultant preparation showed 70% of the activity with only a small change in the apparent affinity for ligands of the enzyme. The addition of Na+ to the treated preparation induced a -2.1 +/- 0.1% change of the total fluorescence intensity observed in the absence of Na+. Further addition of both Mg2+ and ATP transiently increased the fluorescence to +0.5 +/- 0.1%. After the exhaustion of ATP, the fluorescence decreased to -3.1 +/- 0.1%. This cycle can be repeated by the readdition of ATP but not by ADP. Ouabain inhibits the fluorescence change. The ligands used reduced the fluorescence intensity as follows: Mg2+ + Na+ + ATP approximately K+, none, Mg2+ approximately ATP, Na+ + ATP, Na+ approximately Na+ + Mg2+, Na+ + Mg2+ + ADP approximately Na+ + ADP. The data indicate the presence of multiple conformational states of the enzyme.
TL;DR: In all cases, for the tRNA-Phe-enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry.
Abstract: The variations of several spectroscopic properties of yeast tRNA-Phe and phenylalanyl-tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions. In all cases, for the tRNA-Phe-enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.
TL;DR: A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles and effectively restores the specific binding activity of the receptor protein.
Abstract: A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles. This process is independent of the phase transition of the lipid and effectively restores the specific binding activity of the receptor protein. The insensitivity of the resulting lipid-protein complex to ionic strength provides evidence for a hydrophobic interaction. There is a perturbation of the lipid phase transition concomitant with addition of the protein. Circular dichroism studies indicate that the protein undergoes a conformational change on association with lipid. Binding of specific ligand produces further physical changes in the receptor as indicated by alterations in the tryptophan fluorescence quenching pattern.
TL;DR: In the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ion attack at the light meromyosin/heavy meromyOSin junction is favoured by phosphorylation.
Abstract: It has previously been shown that the binding of calcium and magnesium ions to the isolated metal-binding light chains, i.e. those dissociable by 5,5′-dithiobis(2-nitrobenzoate), of rabbit skeletal muscle myosin is moderated by phosphorylation and is accompanied by a sizeable conformational change. As judged by circular dichroism in the region of the aromatic Cotton effects, this conformational change occurs when calcium ions bind to the light chain in situ on the myosin head. Moreover the affinity for calcium is again changed by phosphorylation. The change in chymotryptic digestion patterns, in particular the protection of the head-rod junction in insoluble myosin, by divalent cations, has been used to obtain binding profiles. The results are consistent with the presence of a single class of independent sites, showing no cooperativity. The affinity of the site for both calcium and magnesium ions is enhanced by 1–2 orders of magnitude when the light chain is incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, being marked for calcium and small for magnesium. On phosphorylation the calcium binding constant falls from 8 × 106M−1 to 4 × 106M−1 at physiological ionic strength, compared with 2.5 × 105M−1 and 5 × 104M−1 for the isolated light chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. Thus in the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ions attack at the light meromyosin/heavy meromyosin junction is favoured by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions.
TL;DR: Two dynamic phenomena of polyelectrolyte systems, namely the rapid conformational changes of weak polyelectiolytes (polyacrylic acid and polyethylenimine, induced by pH-jump) and the complexation of HPAA with polyvinylpyrrolidone are investigated by means of the conductance stopped-flow (kappa-SF) technique.
TL;DR: It is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca1+-ATPase and that phospholipid bilayer is not essential for this conformational change.
TL;DR: It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed.
Abstract: The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH-dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH-dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile-E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His-G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III.
Although the change in chemical shift of the His-G2 C-2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range.
Considering the tertiary structure obtained from an X-ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His-G2 with the carboxyl group of the C-terminal methionine-H22.
TL;DR: Results indicate that the two E x glucose crystals are distinguished by a nucleotide-dependent conformational difference, which is stabilized by lattice forces, which appears to be necessary to produce the active ternary complex.
TL;DR: In this paper, second order rate constants were measured at several temperatures for the basic hydrolysis and for concurrent carbonyl-oxygen exchange with solvent for N-benzyl-N-methyl-α,α-dideuteropropionamide-18O.
Abstract: Second order rate constants were measured at several temperatures for the basic hydrolysis and for concurrent carbonyl-oxygen exchange with solvent for N-benzyl-N-methylformamide-18O, N-benzyl-N-methyltrideuteroacetamide-18O, and N-benzyl-N-methyl-α,α-dideuteropropionamide-18O. Carbonyl-oxygen exchange with solvent is rationalized by invoking a conformational change in the tetrahedral intermediate. Energy barriers for these conformational changes are estimated to be of the order of 5.6, 8.0, 8.2 kcal/mol respectively for the above amides.
TL;DR: Findings appear to rule out a function of the anion-sensitive Mg-ATPase in the transport of anions and protons across the plasma membrane in rabbit gastric mucosa and rat pancreas.
Abstract: Studies in our laboratory have shown that the anion-sensitive Mg-ATPase is located in mitochondria, but not in the plasma membrane of rabbit gastric mucosa, trout gill, rabbit kidney and rat pancreas; whereas in rabbit erythrocyte membrane, it is part of the Ca-Mg activated ATPase system. These findings appear to rule out a function of the anion-sensitive ATPase in the transport of anions and protons across the plasma membrane in these tissues. On the other hand, the K-activated ATPase in a gradient-purified vesicle fraction of pig gastric mucosa mediates proton uptake in exchange for K+ in the presence of ATP, in agreement with earlier findings of other investigators. The enzyme requires a phospholipid environment for its activity. Studies of arginine modification with butanedione in the presence or absence of ATP and its analogues, and of activating cations indicate that the enzyme contains an essential arginine group involved in ATP binding; and that K+ induces a conformational change, which leads to decreased ATP binding and probably coincides with enzyme dephosphorylation. Similar studies of sulfhydryl modification with DTNB indicate that the enzyme contains an essential sulfhydryl group, which does not appear to be directly involved in ATP binding, but rather that ATP binding may induce a conformational change which makes the sulfhydryl group less accessible.
TL;DR: An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes, finding that less than 15% of the tryosyls in the nucleOSome core particle can be labeled even at 200- to 300-fold M excess of label.