Showing papers on "Conformational change published in 1981"

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties

Abstract: Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Muller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Structural Dynamics of Yeast Hexokinase During Catalysis

Abstract: The binding of the substrate glucose to yeast hexokinase results in a substantial enzyme conformational change that is essential for catalysis and may be important for the enzyme's specificity, as well as the control of its activity. From high-resolution crystal structures of the monomeric enzyme crystallized both in the presence and in the absence of glucose, we find that glucose binds into the deep cleft that separates the molecule into two lobes and causes these two lobes to move together and close off the cleft. The structure of the hexokinase crystallized in the presence of xylose and ADP is being determined at low resolution. In this crystal form, the enzyme was thought to be in the conformation of the ternary complex. However, a low-resolution structure of this crystal form shows clearly that the enzyme is in the 'open' form and is not a ternary complex. Crystals of the A isozyme with glucose and ADP may be. Further, chemically sequenced tryptic peptides are being incorporated into the model obtained by crystallographic refinement at 2.1 A resolution. Completion of the sequence and the structure of the ternary complex should allow a detailed description of the enzymatic mechanism of this kinase and the role of substrate-induced conformational changes in catalysis and control.

Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase

Abstract: 1 Substrate analogue CoA derivatives were applied as inhibitors of citrate synthase. Substitution of the acyl-CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity. 2 Carboxymethyl-CoA, a structural analogue of enolic acetyl-CoA. Ks of the binary inhibitor-enzyme comples was high (230 μM) but that of the ternary inhibitor-oxaloacetate-enzyme complex was 0.07 μM. Both enzyme sub-units bond the inhibitor independently, also in the presence of oxaloacetate. 3 (3R,S)-3,4-Dicarboxy-3hydroxybutyl-CoA, an analogue of citryl-CoA, inhibited the overall reaction noncompetitively against acetyl-CoA and against oxaloacetate; it was a competitive inhibitor against the hydrolysis and cleavage reactions of (3S)-citryl-CoA. Kinetic data suggest that this inhibitor represents and intermediate analogue. 4 The results given above indicate conformational changes of the synthase during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a wellknown conformational change, is converted into a ligase. If both substrates are presents, the ligase is reconverted into the hydrolase upon formation of the intermediate, (3S)-citryl-CoA.

The First Step in the Polymerisation of Actin

Abstract: In the presence of certain cations (e.g. K+ or Mg2+) actin polymerizes. Below a certain concentration (the critical concentration) the monomer G-actin does not polymerize on the addition of K+ or Mg2+. However, the proteolysis experiments of Rich and Estes [J. Mol. Biol. 104, 777--792 (1976)] strongly suggest that cations induce a change in conformation of G-actin leading to a novel form of actin, G*-actin. This conformational change may be the first step in the polymerization of actin. We have studied G*-actin induced by K+, by difference spectroscopy. We show that G*-actin is a monomer and we confirm that the bound ATP is not cleaved. We also studied the G-actin in equilibrium with G*-actin equilibrium at 4 degrees C as a function of K+ or Mg2+ concentration. With KCl, the transformation can be accounted for as a screening effect. The effect of Mg2+ is more specific and the change in conformation of the G-actin could result from the binding of two or three Mg2+ ions/molecule. We suggest that the G-actin in equilibrium with G*-actin transformation results from the neutralization of a polyanionic region on the actin surface and that this region could be the highly negatively charged N terminus.

Thermodynamics of the binding of D-glucose to yeast hexokinase

Abstract: The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration. The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C. Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin. DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima. This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography [Bennett, W.S., & Steitz, T. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852]. In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative. The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths. This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity. It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.

Change of reactivity of lysine residues upon actin polymerization

Abstract: The reactivity of lysine residues of actin was measured by a surface labeling method--limited reductive methylation. After labeling, actin was subjected to CNBr and enzymatic cleavage, and all lysines were obtained either singly in a peptide or as a free residue. The specific activity of each lysine was taken as the measure of its reactivity. In actin denatured in 8 M urea, the reactivity of each lysine residue is approximately equal whereas those in G-actin fall into three categories: Lys-61 and Lys-113 are the most reactive ones; Lys-18, -213, -215, -314, and -358 are hardly reactive; the remainder, including Lys-50, -68, -84, -118, -191, -237, -283, -290, -325, -327, -335, and -372, are moderately reactive. The least reactive ones are probably buried in the native G-actin and all the others are most likely on the surface. Upon actin polymerization the reactivities of Lys-61, -68, -113, and -283 are significantly reduced while that of Lys-335 is strikingly enhanced. The decrease in reactivity could be readily explained if these residues were located in the monomer-monomer contact area although a polymerization-induced conformational change cannot be excluded. Such a conformational change may be invoked to explain the increase in the reactivity of Lys-335. Alternatively, the latter may be interacting with the bound ATP of G-actin, and the increased reactivity might be directly attributable to the loss of gamma-P for ATP accompanying polymerization.

Long‐term change in conformation of macromolecules in solution, 2. Poly(acrylamide‐co‐sodium acrylate)s

Abstract: The solution viscosity of aqueous poly(acrylamide-co-sodiumacrylate)s decreases with time in the scale of weeks. This unusual viscosity loss has been investigated by viscometry and by light scattering on high purity copolymer samples with different ratios of the components. — No viscosity loss can be observed in NaCl solution of high enough salt concentration. From the experiments it is concluded that a conformational change causes the viscosity decrease. No chain scission occurs. Light scattering measurements were used to directly confirm this hypothesis. — The viscosity loss may be caused by a conformational change of single molecules involving hydrogen bonds and can be interpreted as a transition from a partly stiffer, higher viscous structure to a more flexible one. As the driving force for the conformational change the entropy is discussed. The pronounced time dependence may be interpreted by a cooperative effect of loosening and combining of hydrogen bonds. — A similar behaviour has been observed earlier in aqueous poly(acrylamide) solutions. Thus, for some water soluble polymers one has to be aware of a time dependent parameter.

Anthramycin binding to deoxyribonucleic acid-mitomycin C complexes. Evidence for drug-induced deoxyribonucleic acid conformational change and cooperativity in mitomycin C binding.

Abstract: Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change.

Circular dichroism and magnetic circular dichroism of reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

Abstract: Studies of the circular dichroism (CD) and magnetic circular dichroism (MCD) of the dithionite-reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase (Av1) are reported. CD and MCD are measurable at room temperature across a wide spectral range, from the near-UV to the near-IR. The visible-near-UV CD is insignificantly affected by moderate variations in pH, temperature, ionic strength, and buffer, providing evidence against conformational change in the range studied. Mg2+ and ATP also cause no observable change in the visible-near-UV CD. Both CD and MCD in the visible-near-UV are unaffected by 30% inactivation by O2. However, the CD and MCD spectra of uncrystallized Av1 differ very significantly from those of crystallized Av1; in particular, the MCD spectrum is very sensitive to the presence of heme impurities. The identicality in both CD and MCD spectra of the reduced molybdenum-iron proteins from Azotobacter vinelandii and Klebsiella pneumoniae shows that these proteins contain metal clusters, identical in number, structure, and protein environment. While the absorption, CD, and MCD spectra of reduced Av1 are typical in many respects of simpler iron-sulfur proteins and are most similar to the [Fe4S4(SR)4]3- clusters found in reduced bacterial ferredoxins, significant differences exist. It is concluded, therefore, that the clusters present are not identical with those previously characterized, a conclusion earlier arrived at from electron paramagnetic resonance, Mossbauer, and EXAFS spectroscopies.

Conformational change of poly(L-lysine) by sodium octyl sulfate as studied by stopped-flow circular dichroism method.

Abstract: Circular dichroism (CD) studies were made on the conformational changes of poly(L-lysine) (PLL) induced by sodium octyl sulfate (SOS). The conformation of PLL has a negative double maximum characterizing the α-helix in 4.0–6.0×10−3 mol/dm3 SOS solutions. At a higher concentration of SOS, PLL takes the β-structure, the random coil remaining below it. These conformational changes were followed by measuring ellipticities at 192, 207, and 221 nm according to a CD stopped-flow method. The rate constant was ca. 2.7×10−1 s−1 for the coil-helix transition, the transition rate from coil to β-structure being high depending on SOS concentration. The β-structure was attained by adding a small amount of 1-octanol to the α-helical PLL in 5.0×10−3 mol/dm3 SOS. The effect of the surfactant on the triphasic conformation changes is discussed in terms of hydrophobic environment.

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1

Abstract: Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.

Selective N-bromosuccinimide oxidation of the nonfluorescent tryptophan-31 in the active center of thioredoxin from Escherichia coli.

Abstract: The two tryptophan residues (Trp-28 and Trp-31) of thioredoxin-S2 from Escherichia coli were selectively tritiated with trifluoroacetic [3H]acid. The 3H label was introduced to permit quantitative amino acid sequence analyses of the result of N-bromosuccinimide oxidation of tryptophan to oxindolylalanine. Addition of 3-fold molar excess of N-bromosuccinimide at pH 4 modifies a tryptophan in thioredoxin-S2 that is nonessential for enzyme activity with thioredoxin reductase and has a strongly quenched fluorescence in both oxidized and reduced thioredoxin. This residue was shown to be Trp-31 by amino acid sequence analyses of 3H-labeled chymotryptic peptides from the modified protein. The results demonstrate that the second tryptophan residue, Trp-28, signals a conformational change on reduction of the active-center disulfide to a dithiol by increasing its fluorescence quantum yield about 6-fold at pH 7. The differential reactivity of the tryptophan residues agrees with the known three-dimensional structure of thioredoxin-S2.

Enhanced intramolecular excimer formation of guest molecules included in the cavity of γ-cyclodextrin

Abstract: Whereas both α-and β-cyclodextrin significantly inhibit intramolecular excimer formation of bis-(4-biphenylmethyl)ammonium chloride, γ-cyclodextrin enhances it considerably, indicating its ability to induce a conformational change in a molecule by forming an inclusion complex.

Kinetics of formation of deoxyribonucleic acid cross-links by 4'-(aminomethyl)-4,5',8-trimethylpsoralen.

Abstract: If a mixture of T4 deoxyribonucleic acid (DNA) and 4'-(aminomethyl)-4,5',8-trimethylpsoralen is irradiated with two closely spaced pulses of long-wave UV laser light, the resulting cross-linking is dependent on the time delay between the pulses. As the delay lengthens to 1 mus, a rise in the number of cross-links is observed which follows first-order kinetics. This delay, the time required for most monoadducts to be able to absorb a second photon and thereupon form a cross-link, is interpreted in terms of a conformational change in the DNA at the psoralen intercalation site which may occur upon monoadduct formation.

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue.

Abstract: 1 The 3′-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act, as a photoaffinity label of (Na++ K+)-ATPase has been tested. 2 In the dark 3′-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na++ K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3 Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the α-subunit and an approximately 12000-Mr component. 4 Photolabeling of the α-subunit by N3-ATP irreversibly inactivates (Na++ K+)-ATPase. 5 Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6 Mg2+;, in concentrations required for photoinactivation, protects (Na++ K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7 It is assumed that a conformational change of the ATP-binding site of (Na++K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.