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Showing papers on "Conformational change published in 1978"


Journal ArticleDOI
TL;DR: It is found that the hydrophobic effect alone favors the active conformation of hexokinase in the presence and absence of sugar.
Abstract: The A isozyme of yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) crystallized as a complex with glucose has a conformation that is dramatically different from the conformation of the B isozyme crystallized in the absence of glucose. Comparison of the high-resolution structures shows that one lobe of the molecule is rotated by 12 degrees relative to the other lobe, resulting in movements of as much as 8 A in the polypeptide backbone and closing the cleft between the lobes into which glucose is bound. The conformational change is produced by the binding of glucose (R.C. McDonald, T.A. Steitz, and D.M. Engelman, unpublished data) and is essential for catalysis [Anderson, C.M., Stenkamp, R.E., McDonald, R.C. & Steitz, T.A. (1978) J. Mol. Biol. 123, 207-219] and thus provides an example of induced fit. The surface area of the hexokinase A-glucose complex exposed to solvent is smaller than that of native hexokinase B. By using the change in exposed surface area to estimate the hydrophobic contribution to the free energy changes upon glucose binding, we find that the hydrophobic effect alone favors the active conformation of hexokinase in the presence and absence of sugar. The observed stability of the inactive conformation of the enzyme in the absence of substrates may result from a deficiency of complementary interactions within the cavity that forms when the two lobes close together.

263 citations



Journal ArticleDOI
TL;DR: Circular dichroism experiments show that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin, as apparently only the fragment 1 portion of the molecule undergoes the transition.
Abstract: Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.

123 citations


Journal ArticleDOI
TL;DR: It is concluded that the large glucose-induced conformational change must be essential for subsequent catalytic steps and appears unlikely from this study that thiols play any direct role in catalysis or in substrate binding.

122 citations


Journal ArticleDOI
TL;DR: Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4, suggesting that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-Affinity sites are occupied.
Abstract: Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+-binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 × 107 and 4.5 × 10s M−1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (~30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. &Bothner-By, A. A. (1977) Biochemistry 16,4039–4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium o...

63 citations


Journal ArticleDOI
TL;DR: ATP induces a conformational change in the Escherichia coli K-12 restriction enzyme that allows it to discriminate between unmodified and modified DNA recognition sequences, and does not require ATP hydrolysis for DNA cleavage.
Abstract: ATP induces a conformational change in the Escherichia coli K-12 restriction enzyme that allows it to discriminate between unmodified and modified DNA recognition sequences. This conformational change does not require ATP hydrolysis. However, ATP hydrolysis is a requirement for DNA cleavage.

57 citations


Journal ArticleDOI
TL;DR: Evidence is reported for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2-Mn2+Con A and that the locked conformation of Con A is primarily responsible for saccharide-binding activity and the function of the bound metals is primarily to maintain the protein in the locked Con A conformation.
Abstract: The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

50 citations


Journal ArticleDOI
TL;DR: The Ca2+-dependent activator protein of myosin light-chain kinase, which was identified as the bovine brain modulator protein of cyclic nucleotide phosphodiesterase, was isolated from rabbit skeletal muscle.
Abstract: The Ca2+-dependent activator protein of myosin light-chain kinase, which was identified as the bovine brain modulator protein of cyclic nucleotide phosphodiesterase, was isolated from rabbit skeletal muscle. The purified protein binds about 2 Ca2+ per mol in a medium containing 5 mM MgCl2, 10 micron CaCl2, and 0.1 M KCl at pH 6.8. The Ca2+ binding caused a conformational change of the activator protein which was measured by difference ultraviolet absorption spectroscopy. In the same Ca2+ concentration range as that causing the conformational change, activation of myosin light-chain kinase was observed.

49 citations


Journal ArticleDOI
TL;DR: It is shown here that L16 indeed changes the conformation of a core particle lacking L16, and is related indirectly to many functions of this subunit.

48 citations


Journal ArticleDOI
TL;DR: It is concluded that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.
Abstract: GMP and native DNA were reacted with 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene. It was shown that the 7-halogeno derivatives react on C-8 of guanine. Furthermore the respective amount of arylamidation (covalent linkage on the C-8 of guanine) and arylation (covalent linkage on 2-NH2 groups of C3 of guanine) addition products was determined in both native and denatured DNA-[14C]AAIF. Two G containing deoxytrinucleotides modified by either AAFF or AAIF were studied comparatively by means of circular dichroism, and as a function of several parameters known to affect the conformation of the deoxytrinucleotides. The induced optical activity in fluorofluorene ring seemed to be very sensible to the conformational changes of the deoxytrinucleotides. On the other hand, the AAIF residue exhibit a lower induced optical activity which remained unchanged when the deoxytrinucleotides conformation was affected. The results presented in this paper led us to conclude that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.

44 citations


Journal ArticleDOI
TL;DR: The results with these modification reagents support earlier conclusions about a Photosystem II-linked conformational change based on work with diazonium benzenesulfonic acid and suggest that the conformational changes detected by tritium exchange does not involve the coupling factor complex.

Journal ArticleDOI
TL;DR: The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method and a model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop.
Abstract: The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method. In the range between -15 degrees C and +30 degrees C A NOVEL CONFORMATIONAL TRANSITION OF THE TRNA could be characterized. This conformational change was found in the absence of any artificial label; it is a characteristic property of tRNAPhe in its native structure. This transition accounts for 30% of the total fluorescence change. Its activation enthalpy is 16 kcal/mole (67 kJ/mole), and the transition enthalpy is between -2 kcal/mole and +2 kcal/mole (+/-8 kJ/mole). A model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop. The experimental findings are discussed with respect to several hypotheses about the molecular mechanism of protein biosynthesis which postulate conformational rearrangements of the anticodon loop.

Journal ArticleDOI
TL;DR: If the conformational change induced by carbamoylcholine observed in the snake toxin binding assay corresponds to desensitization of the receptor in vivo, facilitation of this conformationalchange by volatile anesthetics provides an attractive model for the pharmacological action of these compounds.
Abstract: Incubation of membrane fragments bearing acetylcholine receptors from Torpedo californica under an atmosphere of 3% halothane, 1% chloroform, or 6% diethyl ether greatly facilitates the carbamoylcholine-induced structural transition of the acetylcholine receptor reflected by alterations in the rate of binding of 125I-labeled α-bungarotoxin. The half-time of this ligand-induced conformational change is decreased to 10% of the original value after incubation of the membranes with these volatile anesthetics at or near their clinical concentrations. The synergistic effects observed with the general anesthetics and carbamoylcholine are abolished if the membranes are incubated under a stream of air after exposure to the inhalational agents. The antagonist d-tubocurarine exerts a smaller yet measurable time-dependent effect on the toxin-binding properties of the membrane fragments. Treatment of membranes with general anesthetics facilitates this antagonist-induced conversion of the receptor protein as well. The synergism between ligands and general anesthetics may be due to the disruption by these inhalational agents of interactions at the protein-lipid interface, which may play a significant role in determination of receptor conformation. In addition, if the conformational change induced by carbamoylcholine observed in the snake toxin binding assay corresponds to desensitization of the receptor in vivo, facilitation of this conformational change by volatile anesthetics provides an attractive model for the pharmacological action of these compounds.

Journal ArticleDOI
TL;DR: Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme.

Journal ArticleDOI
TL;DR: The spectral difference between normal and rapidly reacting deoxyhemoglobin is used to study the relationship between CO binding to hemoglobin and the conformational changes to the rapidly reacting form in a combined flow-laser flash experiment and good agreement between experimental results and simulations is obtained.

Journal ArticleDOI
TL;DR: Results indicate that hydrogen bonding between the protonated carboxylic group and carbonyl oxygen of the acetamido group is directly involved in the conformational change of the molecule.

Journal ArticleDOI
TL;DR: The hypothesis that Ca2+-binding to DNase A causes a conformational change that maintains a more active structure of the enzyme, especially when the pH-induced unfolding reduces its activity is supported.

Journal ArticleDOI
TL;DR: This simple tripeptide is able to distinguish between various base sequences in a single-stranded nucleic acid and is favored in AA sequence as compared with AC, CA, and CC sequence.
Abstract: The binding of the peptide Lys-Trp-Lys to various single-stranded copolymers of adenine and cytosine has been investigated using circular dichroism (CD) and fluorescence measurements. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the polynucleotide bases with the tryptophan residue whose fluorescence is quenched. The binding of the peptide leads to a conformational change of the copolymers as shown by the CD variations. The fluorescence and CD data have been analyzed according to the model involving the two types of complexes. The values of the binding constants have been studied as a function of the cytosine content of the copolymers. Analysis of the stacking process in term of nearest neighbor frequency demonstrates that this binding is sequence dependent and is favored in AA sequence as compared with AC, CA, and CC sequence. Thus this simple tripeptide is able to distinguish between various base sequences in a single-stranded nucleic acid.

Journal ArticleDOI
TL;DR: The results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA.

Book ChapterDOI
01 Jan 1978
TL;DR: The chapter discusses conformational forms of the estrogen receptor, an estradiol-dependent and temperature-dependent conformational change modifying the receptor's intrinsic activity and sedimentation characteristics.
Abstract: Publisher Summary The chapter discusses conformational forms of the estrogen receptor. The transformation or activation of the estrogen receptor is an estradiol-dependent and temperature-dependent conformational change modifying the receptor's intrinsic activity and sedimentation characteristics. The estrogen-induced transformation of the receptor may be attributed to a change in one or more of the properties of the 4S estrogen-binding protein (EBP): (1) shape, producing a form with hydrodynamic properties that permit faster sedimentation, (2) partial specific volume or density, for example, by phosphorylation or by dissociation of a lipid moiety, or (3) mass, by association with another macromolecule. A change in any of these properties could be provoked by the binding of estradiol to the 4S form of the receptor in association with or independent of the action of an enzyme. The 5S activated or transformed receptor is formed by the association of the 4S protein with another macromolecule, independently of the action of an enzyme.

Journal ArticleDOI
TL;DR: In order to determine from measured polymerization shifts the conformation of the polymer one needs to determine as precisely as possible the chemical shift variation which is due solely to the other nucleotides of the chain.

Journal ArticleDOI
TL;DR: N-6 spin-labeled derivatives of cAMP are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.
Abstract: Binding of adenosine 3':5'-monophosphate (cAMP) to protein kinase (type I) from rabbit skeletal muscle has been investigated using spin-labeled cAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the electron-spin resonance spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the cAMP site. Strong immobilization of the N-6 substituents up to a spacer length of seven atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilation. In addition the binding affinities of the spin-labeled cAMP derivatives have been determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the cAMP site. N-6 spin-labeled derivatives have also been investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.

Journal ArticleDOI
TL;DR: It is suggested that a conformational change in the abortive complex of enzyme, manganese, NADPH, malate, and malate is responsible for the malate inhibition and for the slow approach to the true steady state.

Journal ArticleDOI
TL;DR: ADP and AMP-PNP preferentially modify the A transition, which has been shown to involve the unfolding of a portion of spectrin, an erythrocyte membrane protein complex, which is characteristic of inhibitors of anion transport in the red cell.

Journal ArticleDOI
TL;DR: It is concluded that the presence of gamma-carboxyglutamate residues is a prerequisite for positive cooperative Ca2+ binding.

Journal ArticleDOI
TL;DR: The obtained results are consistent with the existence of two conformational forms of ferricytochrome c, cyt c and cyt c∗, and the conversion of cyt c to cyt c ∗ occurs by deprotonation (pK∼7), followed by a slow conformational change in the protein structure.


Journal ArticleDOI
TL;DR: Data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed, and it is proposed that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformationalchange in the absence of Ca2+.

Journal ArticleDOI
TL;DR: The experiments described here were designed to test the validity of the allosteric immunoglobulin model of Huber et al. (Nature 1976) according to which antigen could cause a stiffening of the flexible antibody molecule by the formation of longitudinal CH1–CH2 interdomain contacts.
Abstract: The experiments described here were designed to test the validity of the allosteric immunoglobulin model of Huber et al. (Nature 1976. 264: 415) according to which antigen could cause a stiffening of the flexible antibody molecule by the formation of longitudinal CH1–CH2 interdomain contacts. The largest structural change would involve the folding of the hinge peptide in-between the CH2 globules. Consequently, the accessibility of the hinge region to reduction and limited proteolysis should be considerably altered. Homogeneous rabbit antibodies to type II and III pneumococcal polysaccharide were reacted with corresponding oligosaccharide haptens and whole polysaccharide antigens to form monomeric and polymeric IgG antibody complexes, respectively. Reduction of the inter-heavy chain disulfide bond in the hinge region by a series of thiol reagents of increasing size and polarity was performed; the ratio of reduction rate in complexes with hapten or antigen compared to that of hapten-or antigen-free control was found to be close to unity, thus indicating that the susceptibility of the hinge disulfide bond was unaffected by the binding of hapten or antigen. Limited proteolysis in the hinge region of immune complexes by activator-free papain or pepsin in the absence of reducing agent was carried out, and the appearance of antibody fragments followed by sodium dodecyl sulfate gel electrophoresis. The rate of proteolysis in hapten-antibody complexes and in large soluble antigen-antibody aggregates (16–29 S) did not show any significant decrease when compared to that for unliganded antibody. The identity of the reduction and proteolysis rates in the presence and absence of antigen provides strong evidence for the lack of a major conformational change in the hinge region upon antigen binding.

Journal ArticleDOI
TL;DR: It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.
Abstract: The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.