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Showing papers on "Conformational change published in 1977"


Journal ArticleDOI
TL;DR: A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.
Abstract: The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.

436 citations


Journal ArticleDOI
01 Sep 1977-Nature
TL;DR: Protein phosphorylation is a reversible, energy-dependent membrane modification, but it differs from the other changes in that it takes the form of a specific chemical reaction involving certain identifiable chloroplast membrane polypeptides.
Abstract: ILLUMINATION of chloroplast thylakoids leads to the formation of the so-called high energy state of the membrane1–3. The establishment of this state is accompanied by several structural changes within the membrane, including a conformational change in the coupling factor4, increased accessibility of photosystem II to the chemical probe p-diazonium benzene sulphonate5, and a reduction in the thickness of the partition between stacked thylakoids6. I describe here a rather different type of structural change that has not previously been reported for chloroplast membranes—protein phosphorylation. Like the above changes, protein phosphorylation is a reversible, energy-dependent membrane modification, but it differs from the other changes in that it takes the form of a specific chemical reaction involving certain identifiable chloroplast membrane polypeptides. The most conspicuous of these polypeptides is the light-harvesting chlorophyll a/b binding protein, the most abundant thylakoid polypeptide7.

383 citations


Journal ArticleDOI
TL;DR: The results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation Bbeing related to the free enzyme E and conformation A being related to E∗, the enzyme species after a substrate-induced conformational change.

285 citations


Journal ArticleDOI
TL;DR: The energy-dependent release of bound [14C]nucleotides trom the chloroplast coupling factor CF1, has been used to monitor conformational changes in CF1 and these results may be explained tentatively by the following concept.

186 citations


Journal ArticleDOI
TL;DR: Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.

136 citations


Journal ArticleDOI
TL;DR: Solvent perturbation difference spectroscopy shows that the binding of heparin to AT-III results in exposure of two tyrosine residues and a partial burial of a tryptophan residue.

130 citations


Journal ArticleDOI
TL;DR: In this paper, low-energy conformations of methionine-enkephalin were generated by means of an empirical method of computation, and one conformation was found to have a potential energy about 5 kcal/mol (21 X 10(3) J/mol) below that of the large group of compact conformations.
Abstract: Low-energy conformations of methionine-enkephalin were generated by means of an empirical method of computation. Many compact conformations, including those containing various standard bends, were of comparable energy. However, one conformation was found to have a potential energy about 5 kcal/mol (21 X 10(3) J/mol) below that of the large group of compact conformations. In this conformation, the 3-glycyl and 4-phenylalanyl residues form a bend of type II'. The conformation is stabilized by a hydrogen bond between the OH group of the 1-tyrosine side chain and the C==O group of 3-glycine or 4-phenylalanine. The phenylalanine and methionine side chains are relatively unrestricted. The conformation is consistent with published nuclear magnetic resonance parameters--coupling constants, temperature dependence of the chemical shift, and spin-lattice relaxation times. It is likely that the molecule undergoes a conformational change when it is bound to the receptor. Leucine-enkephalin appears to have the same conformation as its methionine homolog.

118 citations


Journal ArticleDOI
TL;DR: A comparison with assembly in vivo demonstrated that the pathways of protein assembly in vitro and in vivo are very similar at the beginning, but diverge towards the end of the process.

100 citations


Journal ArticleDOI
TL;DR: Both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide.
Abstract: The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.

91 citations


Journal ArticleDOI
TL;DR: Spectroscopic evidence is presented regarding the dependence of β‐sheet formation on sulfoxide concentration, treatment duration, pH, and tissue hydration, and the relationship of this conformational change to the enhancement of skin permeability.
Abstract: Formation of the antiparallel-chain β-sheet protein conformation is induced in in vitro human stratum corneum by three homologous organic sulfoxides known to enhance skin permeability: dimethylsulfoxide (Me2SO), hexylmethylsulfoxide (HxMeSO), and decylmethylsulfoxide (DecMeSO). Me2SO and HxMeSO apparently function by displacing water molecules bound to polar protein side-chains, whereas DecMeSO probably interacts hydrophobically with the protein. The conformational transition does not result from lipid removal. The β-sheet protein, most likely formed in normally α-helical portions of the intracellular keratin filaments, is reconverted to α-helix upon rehydration of the tissue. Though neat Me2SO produces the most β-sheet of all treatments examined, the sequence of ability to promote β-sheet formation at the 1M level is HxMeSO > DecMeSO > Me2SO. Spectroscopic evidence is presented regarding the dependence of β-sheet formation on sulfoxide concentration, treatment duration, pH, and tissue hydration. The relationship of this conformational change to the enhancement of skin permeability is briefly discussed. The result of sulfoxide treatment is different from results of sodium dodecylsulfate and heat treatments of stratum corneum.

78 citations


Journal ArticleDOI
TL;DR: In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophile.
Abstract: A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 +/- 10A. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 A in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 A through the receptor molecule.


Journal ArticleDOI
TL;DR: Results indicate that the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase is associated with a reversible change in the conformation of the protein.

Journal ArticleDOI
TL;DR: Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergoes a change toward a more hydrophobic environment in the presence of L-histidine.
Abstract: The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates

Journal ArticleDOI
01 May 1977-Nature
TL;DR: In this paper, a hypothesis is advanced that the hormonal activity attributable to the heterogeneous group of plant growth compounds called auxins is a direct result of the ability of the bound auxin to undergo a simultaneous conformational change or reorientation with the receptor, while in the binding site.
Abstract: An hypothesis is advanced that the hormonal activity attributable to the heterogeneous group of plant growth compounds called auxins is a direct result of the ability of the bound auxin to undergo a simultaneous conformational change or reorientation with the receptor, while in the binding site.

Journal ArticleDOI
TL;DR: A scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme at neutral pH, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench.

Journal ArticleDOI
TL;DR: It can be concluded from results that the calcium binding centers are located in a more or less flexible zone of the molecule probably involving the C-terminal part of the Aalpha chain and that thecium divalent cation stabilizes a more compact structure of the fibrinogen molecule.

Journal ArticleDOI
TL;DR: The results suggest that EF-Ts and kirromycin induce a similar conformational change in EF-Tu, thereby "opening" the guanine nucleotide binding site.
Abstract: Alterations of the structure of EF-Tu have been investigated by using the rate of EF-Tu cleavage by trypsin as a conformational probe. The presence of EF-Ts bound to EF-Tu results in a 10-fold increase in the cleavage rate. The antibiotic kirromycin, which inhibits protein synthesis by virtue of its interaction with EF-Tu, mimics this effect of EF-Ts. Both kirromycin and EF-Ts also facilitate the exchange of free GDP with GDP bound to EF-Tu. The results suggest that EF-Ts and kirromycin induce a similar conformational change in EF-Tu, thereby "opening" the guanine nucleotide binding site. The trypsin-cleaved EF-Tu still can bind GDP and EF-Ts and can function in Qbeta replicase, but it no longer spontaneously renatures following denaturation in urea.


Journal ArticleDOI
Ko Bp, Yazgan A, Yeagle Pl, Lottich Sc, Henkens Rw 
TL;DR: Results from two experiments show that a tight binding site for I forms at an intermediate stage in the folding process and a subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding.
Abstract: In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.

Journal ArticleDOI
TL;DR: It is concluded that the terminal A of tRNA Phe (yeast) is responsible for the occurrence of the conformational change of the tRNA-synthetase complex.
Abstract: The function of the terminal A of tRNA Phe (yeast) with respect to complex formation with the cognate aminoacyl-tRNA synthetase has been studied using equilibrium and fast kinetic techniques. Removal of the terminal A influences the equilibrium parameters of the tRNA-synthetase interaction only slightly, the mechanism of complex formation, however, is changed significantly. The binding mechanism of unmodified tRNAPhe comprises a recombination step and a consecutive conformational change. In contrast, the reaction between tRNAPheCC and the cognate synthetase is characterized by a simple one step mechanism. It is concluded that the terminal A is responsible for the occurrence of the conformational change of the tRNA-synthetase complex. The conformational change is interpreted as a proper alignment of the terminal A of the tRNA to the active site of the synthetase.

Journal ArticleDOI
TL;DR: The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis and no conformational changes of protein C in the presence ofCa2+ could be detected by circular dichroism or nuclear magnetic resonance.
Abstract: The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.

Journal ArticleDOI
TL;DR: The fluorescence intensity of the single tyrosine residue in histone H1 increases from R TYR = 0.3 to RTYR = 1.3 as the protein undergoes a conformational change from the random coil state to a folded form.

Journal ArticleDOI
TL;DR: Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.
Abstract: A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.

Journal ArticleDOI
TL;DR: Results with mixed polypeptides show that these protein models assume different conformational forms upon complexation with heparin, the former shows a poly-L-lysine-like beta-structure circular dichroism spectrum and the latter an alpha-helical structure.

Journal ArticleDOI
TL;DR: In this paper, the authors used the difference spectra of tryosyl residues obtained on denaturation of tropomycosin with urea or guanidinium chloride to indicate that strong hydrophobic environments exist in the native coiled-coil state.

Journal ArticleDOI
TL;DR: The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.

Journal ArticleDOI
TL;DR: The influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations are discussed.
Abstract: It is first shown that the inactivation of Escherichia coli apotryptophanase by N-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer. The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein, in which the cysteine is reactive, and a second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomerization step involved in this change in conformation is measured by the stopped-flow technique (τ= 0.4 s). Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of ‘inactive’ conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.

Journal ArticleDOI
TL;DR: The results indicate that the specificity of the binding of sodium ions to monensin is reflectied in the relatively slow dissociation process, and the entropy changes indicates that the activatedmonensin-sodium complex undergoes a conformational change, but the existence of a conformular change in monens in anion prior to complexation is excluded.

Journal ArticleDOI
TL;DR: In this paper, the average hydrodynamic radius of synthetic phospholipid vesicles and the variance of the size distribution were measured by dynamic light scattering spectroscopy near the lipid phase transition.