Showing papers on "Conformational change published in 1983"

Transmission of conformational change in insulin.

Abstract: Crystal structures of insulin contain molecules that are similar but not identical in conformation. Packed helices move relative to each other, these shifts being accommodated by motions of side-chain atoms arising from small changes in torsion angles. Such low-energy conformational adjustments can accommodate shifts of no more than approximately 1.5 A. This limits the extent to which conformational changes can be dissipated locally, causing their transmission over long distances.

Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C.

Abstract: A detailed mechanism that fully accounts for the Mg2+-induced polymerization of actin in the presence or absence of Ca2+ at 20 degrees C and pH 8 is presented. In the absence of Ca2+, the mechanism of the Mg2+-induced polymerization is as follows: Mg2+ binds to a metal-binding site on G-actin and induces a conformational change, which is required for eventual polymerization. The overall dissociation constant for this binding is about 30 microM. This actin species then binds a second molecule of Mg2+ (Kd = 5 mM), which yields a species capable of polymerization. Dimer formation from this monomeric species is quite unfavorable, but trimer formation from dimer and monomer is much more favorable. The trimer may then elongate to give filaments. Ca2+, when present, binds at the same site as the tightly bound Mg2+ and must be displaced by Mg2+ before the conformational change can occur. The rate and dissociation constants for tight binding of Ca2+ and Mg2+ and for the conformational change are consistent with those observed previously by using a fluorescently labeled G-actin. With the mechanism proposed, it is possible to fit the full time course of polymerization over a wide range of actin concentrations, Mg2+ concentrations, and Ca2+ concentrations.

Interactions of Divalent Metal Ions with Bovine, Human, and Goat α-Lactalbumins

Abstract: Bovine, human, and goat alpha-lactalbumins prepared by the ordinary methods were found to contain 1.1-1.3 atoms of Ca per protein molecule. Removal of Ca2+ was shown to destabilize the tertiary structures in the three proteins. The three apoproteins were indicated to change in the conformation by heat from the native-like to the unfolded state. Degree of restoration of the native tertiary structure in 5 mM Tris-HCl and 0.1 mM EDTA at pH 7.2 and 25 degrees C by addition of Ca2+ was determined from change in CD ellipticity at 270 nm, and the apparent binding constant of Ca2+ was analyzed to be 2.5 X 10(8) (bovine), 3.0 X 10(8) (human), and 2.8 X 10(8) M-1 (goat). Also, value of the binding constant of Ca2+ to the native-like apoform was estimated from the apparent binding constant and equilibrium constant of the conformational change of the apoform. The binding properties of Mn2+, Mg2+, and Zn2+ to the bovine protein at neutral pH are also discussed.

Nuclear magnetic resonance studies on calmodulin: calcium-induced conformational change

Abstract: The 400-MHz 1H nuclear magnetic resonance (NMR) studies were carried out on the Ca2+-induced conformational change of calmodulins (CaM's) isolated from scallop testis and pig brain. The resonances were found to be classified approximately into three groups. The resonances of group I, which are perturbed by the binding of Ca2+ to the high-affinity sites, include those of tyrosine-138, epsilon-trimethyllysine-115, histidine-107, tyrosine-99, etc. The previous assignments for tyrosine- (Tyr) 138 [Seamon, K. B. (1980) Biochemistry 19, 207] were corrected. The resonances of group II, which are affected by the binding of Ca2+ to the low-affinity sites, include those of a phenylalanine (Phe), a high field shifted methyl, and a low field shifted alpha-methine. Group III (related to the binding of Ca2+ to both the high-and low-affinity sites) includes the resonances of a Phe, a high field shifted methyl, and threonine-143. It is concluded that sites III and IV are the high-affinity sites. The off-rate of Ca2+ from the high-affinity sites is slower than 50 s-1 while the off-rate from the low-affinity sites is faster than 600 s-1. In the Ca2+-free state, there exists a hydrophobic region containing three phenylalanine (probably Phe-89, Phe-92, and Phe-141), a valine, and an isoleucine in the vicinity of sites III and IV. Tyr-138 is distant from these amino acids. Upon binding of Ca2+ to the high-affinity sites, one of the Phe residues and the valine approach Tyr-138. Similar structural changes were observed between CaM and troponin C when Ca2+ ions are bound to the high-affinity sites. CaM changes in a somewhat different way from troponin C when Ca2+ ions are bound to the low-affinity sites.

Spectral studies on the calcium binding properties of bovine brain S-100b protein.

Abstract: The effect of Ca2+ binding on the circular dichroism (CD) and 270-MHz proton nuclear magnetic resonance (NMR) spectra of brain-specific S-100b calcium binding protein has been examined at two pH values, 8.5 and 7.5. At pH 8.5, S-100b protein binds two Ca2+ per monomer with Kd values of 6 x 10(-5) and 2 x 10(-4) M, whereas at pH 7.5, the protein binds only one Ca2+ per monomer with a Kd of 2 x 10(-4) M. The presence of K+ inhibits the binding of Ca2+ to the higher affinity site at pH 8.5, and the affinity for calcium is lowered to Kd = 8.5 x 10(-4) M. Mg2+ has no effect on protein conformation. In the absence of Ca2+, S-100b undergoes a conformational change when the protein is titrated from pH 8.6 to 6.0. Addition of Ca2+ perturbed the environment of tyrosine and phenylalanine residues as measured by ultraviolet difference spectroscopy and 1H NMR. CD melt experiments and far-ultraviolet CD studies at alkaline pH and NMR experiments suggest that the protein is more stable in the presence of Ca2+. The single tyrosine residue in the protein ionizes only after the protein is denatured by exposure to high pH.

Anion transport inhibitor binding to band 3 in red blood cell membranes.

Abstract: The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.

Complex formation in sonicated mixtures of β-lactoglobulin and phosphatidylcholine

Abstract: β-Lactoglobulin, the major whey protein of bovine milk, is secreted via the endomembrane system of the mammary gland. The primary structure of β-lactoglobulin shares certain characteristics with membrane proteins, although the soluble protein assumes a globular conformation. We have prepared complexes of β-lactoglobulin and phosphatidylcholines by dissolving both in a helix-forming solvent (chloroform methanol). The complex is stable when transferred to aqueous solutions and sonicated to form vesicles. Both ionic and hydrophobic interactions appear to be involved in complex formation. We have used spectroscopy (circular dichroism, fluorescence, and nuclear magnetic resonance) and electron microscopy to study these complexes. At pH 3.7, the small, single bilayer vesicles produced by sonication are protected against aggregation by the presence of the protein. As determined by circular dichroism, the proportion of α-helix in β-lactoglobulin is increased by complexation with phosphatidylcholine. Circular dichroism and fluorescence spectra show the involvement of at least 1 tryptophan residue in the conformational change. At pH 7.2, β-lactoglobulin-phosphatidylcholine vesicles form aggregates as observed by electron microscopy and31P nuclear magnetic resonance spectroscopy. These aggregated vesicles could be resuspended by raising the pH. The ability of the partially unfolded β-lactoglobulin to interact with lipids is believed to be important to its transport through the endomembrane system.

Changes in aminoacyl transfer ribonucleic acid conformation upon association with elongation factor Tu-guanosine 5'-triphosphate. fluorescence studies of ternary complex conformation and topology.

Abstract: The association of aminoacyl-tRNA (aa-tRNA) with elongation factor Tu.GTP to form an aa-tRNA.EF-Tu.GTP ternary complex was investigated by using two different fluorescent probes, both of which monitored structural changes near the juncture of the two arms of the L-shaped tRNA. Aminoacylation of tRNAPhe-F8, a functionally active analogue of tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base, did not cause a change in the fluorescence emission intensity. However, when EF-Tu.GTP bound to Phe-tRNAPhe-F8, the emission intensity increased by approximately 30%, depending upon the solvent conditions. About half of this increase in fluorescence was due to an increase in the molar absorptivity of the fluorescein dye. Ternary complex formation did not alter the rate of iodide ion quenching of the Phe-tRNAPhe-F8 fluorescence. Since solvent access to fluorescein was not reduced when EF-Tu.GTP was bound to Phe-tRNAPhe-F8, the fluorescence intensity change noted above was not caused by a direct interaction between fluorescein and EF-Tu. Instead, the binding of EF-Tu.GTP to the aa-tRNA resulted in a conformational change in the aa-tRNA near s4U-8. Ternary complex formation also altered the nature of the single strong binding site for ethidium in unfractionated and unmodified aa-tRNA. However, ethidium binding to its strong site was not blocked. These results indicate that only the acceptor-T psi C arm of aa-tRNA interacts directly with EF-Tu.GTP and that the anticodon-D arm is available for direct interaction with the ribosome during recognition. Our data also suggest that EF-Tu facilitates protein biosynthesis by ensuring that every aa-tRNA is in a particular (possibly the same) conformation prior to initiation of the recognition process at the ribosomal complex.

Non-enzymic activation of the covalent binding reaction of the complement protein C3.

Abstract: The covalent binding of [3H]glycerol to C3 by the transfer of the acyl group of the internal thioester of C3 to the hydroxy group of glycerol can be activated either proteolytically by trypsin or by various chaotropes and denaturants. The activation of binding by trypsin or KBr showed similar dependence on the concentration of glycerol, indicating a similar activation mechanism. It is therefore concluded that the conformational change of the protein is the critical step in the binding reaction, and that the conversion of C3 into C3b under physiological conditions is only a means to induce the conformational change. Guanidinium chloride induces the binding of glycerol to C3 at concentrations of about 1 M. On increasing the concentration of guanidinium chloride the extent of binding declines and is accompanied by an increase in the autolytic cleavage reaction [Sim & Sim (1981) Biochem. J. 193, 129-141]. The autolytic cleavage reaction is therefore not independently activated with respect to the binding reaction. Its occurrence, however, is structurally restricted under physiological or limited denaturing conditions and is permissible only when C3 is brought to a higher denaturation state.

Baker's Yeast Adenlate Kinase

Abstract: The diastereomers of adenosine 5′-O-(1-thiotriphosphate) ATPαS) and adenosine 5′-O-(2-thiotriphosphate) (ATPβS) were used to determine the stereochemical mode of coordination of Mg2+ to ATP when bound in the yeast adenylate kinase complex ATPαS isomer A is the preferred substrate either in the presence of Mg2+ or Cd2+; ratios of the rate of reaction at infinite concentration of substrate, V, for A to B isomers of ATPαS with Mg2+ were 457/514 = 89 and with Cd2+ 124/128 = 97 Thus there is no reversal of stereospecificity on substituting Cd2+ for Mg2+ and this supports the view that metal does not chelate with the α-P In contrast the V ratios of ATPβS(A) to ATPβS(B) with Mg2+ were 133/015 = 89 and with Cd2+ 018/51 = 0035 Thus this reversal of stereospecificity when Cd2+ is substituted for Mg2+ (in view of the fact that Mg2+ binds to O and Cd2+ to S) leads to the conclusion that a metal chelation must occur via the β-phosphate Since the enzyme prefers MgATPβS(A) and CdATPβS(B) (both isomers have Δ-screw sense) to MgATPβS(B) and CdATPβS(A) (both isomers have Δ-screw sense) and Mg2+ also coordinates with the γ-phosphate (by analogy with other adenylate kinase isozymes), the interpretation is that baker' yeast adenylate kinase binds MgATP as Δ, β, γ The fluorescence of the two residues of yeast adenylate kinase has been used to investigate conformational changes of the enzyme on binding its substrates Fluorescene is maximal at 334 nm (λexx 295 nm, pH 75) and is reduced 10% with no shift in λmax in the presence of ATP and reduced a further 15% on the addition of Mg2+ or other metal acitvators to E-ATP In contrast non-activating metals such as Ba2+ and Sr2+ increase quenching only slightly With ITP, UTP, CTP, GTP the quenching is 41%, 52%, 87% and 66% with or without Mg2+ Differential conformational changes of yeast adenylate kinase on binding MgATP, ATP, and other nucleotides are shown by (a) fluorescence changes, (b) previously reported kinetic and binding studied and (c) by the fact that the Km for AMP in the presence of MgATP is 17 times less than in the presence of MgGTP These differential conformational changes result in differing tendencies of AMP to bind to the enzyme Ultraviolet difference spectra obtained on binding of ATP to the enzyme (shoulder at 271 nm and peak at 276 nm) have been compared with the difference spectra of the interaction of ATP, adenosine and 1-methyl adensoine respectiively with methyl mercuric hydroxide and formaldehyde On the basis of these comparisons, the shoulder at 271 nm reflects the interaction of N(1) and the peak at 276 nm the interaction of tthe 6-amino group of ATP with the enyzme The following mechanism of reaction is proposed: upon binding MgATP the enzyme undergoes a conformational change which closes the active-site crevice by bringing the two lobes closer, expelling water from the crevice AMP is bound with higher affinity and the α-oxygen and the α-oxygen attacks the γ-phosphate of ATP in a nucleophilic reaction

Quantitative aspects of the receptor binding of cytokinin agonists and antagonists.

Abstract: Congeneric 4-anilino- and 4-(alkylamino)-2-methylpyrrolo[2,3-d]pyrimidines showed cytokinin and anticytokinin activities, depending on the structure of their 4-substituents, and the antagonistic nature of the latter was established kinetically. The effect of the substituent on these activities was analyzed quantitatively by using physicochemical parameters and regression analysis to give a single, common equation for both the agonists and antagonists. The results indicated that the maximum width of the N4 substituents is an important factor both for binding to the receptor, thus the extent of activity, and for the quality of activity, agonistic or antagonistic. The electron-withdrawing effect and hydrophobicity of the substituents further enhance binding and, thus, activity, irrespective of the quality of the activity. These results coincide with and/or provide evidence for the hypothesis that in hormonal action, agonist binding causes a conformational change of an otherwise inactive receptor to the active form and that antagonists are species that bind similarly to the receptor but do not cause the effective conformational change.

Lack of Z-DNA conformation in mitomycin-modified polynucleotides having inverted circular dichroism

Abstract: Poly(dG-dC) . poly(dG-dC) and Micrococcus lysodeikticus DNA were modified by exposure to reductively activated mitomycin C, an antitumor antibiotic. The resulting covalent drug-polynucleotide complexes displayed varying degrees of CD inversions, which are strikingly similar to the inverted spectrum observed with Z-DNA. The following criteria have been used to establish, however, that the inverted CD pattern seen in mitomycin C-polynucleotide complexes does not reflect a Z-DNA conformation. (i) The ethanol-induced transition of poly(dG-dC) . poly(dG-dC) from B to Z conformation is not facilitated but rather is inhibited by mitomycin C modification. This may be due to the presence of crosslinks, (ii) Radioimmunoassay indicated no competition for Z-DNA-specific antibody by any of the mitomycin C-modified polynucleotides, (iii) 31P NMR of the complexes yielded a single relatively narrow resonance, which is inconsistent with the dinucleotide repeat characteristic of Z-DNA. Alternative explanations for the inverted CD pattern include a drug-induced left-handed but non-Z conformational change or the superposition of an induced CD onto the CD of B-DNA due to drug-base electronic interactions. These results illustrate the need for caution in interpreting CD changes alone as an indication of Z-DNA conformation.

Proton‐NMR studies of the solution conformations of vitamin‐D‐induced bovine intestinal calcium‐binding protein

Abstract: 1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80s−1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.

Interaction of creatine kinase from monkey brain with substrate: analysis of kinetics and fluorescence polarization.

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13-fold and 4-fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the KD for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate-induced conformational change. Creatine kinase labeled with two molecules of 5′-iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle-type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme-MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 - (4 - maleimidylphenyl) - 7 - diethylamino - 4 - methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.