Showing papers on "Conformational change published in 1987"

Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement

Abstract: Glucocorticoids, a class of steroid hormones, associate specifically with intracellular receptors, facilitating a conformational change that converts the receptor in vitro to a DNA-binding protein1 and in vivo to a nuclear species2 that activates a class of transcriptional enhancers termed glucocorticoid response elements3,4 (GREs). The DNA sequences recognized specifically by the hormone–receptor complex correspond directly to those required for GRE enhancement4,5. The structural transition that accompanies steroid binding, 'receptor transformation', has been monitored by changes in receptor chromatographic properties6, accessibility to monoclonal antibodies7, association with other receptor subunits or with heterologous proteins8,9, add aqueous two-phase partition coefficient10,11. However, the significance of the structural change for the biological activity of the receptor is not understood. We have used cloned rat glucocorticoid-receptor coding sequences12 to produce and characterize a novel class of receptor mutants that elicit GRE enhancer function in transfected cells even in the absence of hormone. The constitutive activity of those receptor derivatives, together with mapping studies that distinguish between the DNA- and hormone-binding domains of the receptor, imply that the conformational change corresponding to receptor transformation may simply unmask pre-existing functional domains for DNA binding, enhancer activation, or both.

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin.

Abstract: At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.

Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III.

Abstract: The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Proline-induced constraints in alpha-helices.

Abstract: The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala4-Pro-NHMe and of Ac-Ala7-Pro-Ala7-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ϕ and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.

Sliding-layer conformational change limited by the quaternary structure of plant RuBisCO

Abstract: RuBisCO, D-ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39), converts carbon dioxide to sugar in the first step of photosynthesis. In plants and some bacteria, this enzyme has an L8S8 structure, where L is the large catalytic subunit and S is the small subunit of unknown function. The molecule resembles a keg 105 A along the 4-fold axis and 132 A in diameter at the widest point of the keg. Here we describe the quaternary structure of RuBisCO from N. tabacum, the first L8S8 type known from an X-ray crystallographic study at near-atomic resolution (3 A). The structure shows that all eight L subunits are elongated along the 4-fold axis so that the molecule cannot be simply described as layers of subunits, as it had been from studies by electron microscopy. The structure, with its elongated and interdigitated L subunits, is evidence against a large, sliding-layer conformational change in plant RuBisCO, as proposed recently in Nature for the same enzyme from Alcaligenes eutrophus.

Conformational-changes in the oligonucleotide duplex d(gcgttgcg).d(cgcaacgc) induced by formation of a cis-syn thymine dimer - a two-dimensional nmr-study

Abstract: In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.

Viscosity dependence of ethidium-DNA intercalation kinetics.

Abstract: The kinetics of ethidium intercalation into double-stranded poly[d(G-C)] were investigated by use of repetitive pressure-jump chemical relaxation at 20 degrees C in low ionic strength (0.1 M NaCl) aqueous buffers containing either glycerol or methanol. The viscosity of the various solvents differed by more than an order of magnitude while other physical properties (e.g., dielectric constant) remained approximately constant. The single-reciprocal kinetic relaxation time (tau -1) increases linearly with DNA concentration. The observed association rate constant is lower in all organic-aqueous mixtures than in water and is inversely proportional to the viscosity. These results provide evidence for an additional step in the intercalation mechanism which is identified as an obligatory DNA conformational change preceding ethidium intercalation. From the data presented, the equilibrium constant of this local conformational change is approximately 10(-3), i.e., greatly favoring the structure incapable of intercalation. The corresponding kinetics were not directly determined; however, in order to be consistent with all of the data the forward and/or reverse rate constants of the conformational change must be larger than the rate of the intercalation reaction. Thus, it is proposed that the rate of the conformational change back to the nonintercalating B-DNA structure is greater than approximately 500 s-1, implying a rate of opening greater than approximately 0.5 s-1, in agreement with other hydrogen exchange and NMR data. The observed overall rate constant for the dissociation of ethidium is inversely proportional to the solvent density, possibly reflecting a dependence on the solvent free volume. The overall volume change of intercalation is less negative in the organic-aqueous solvent mixtures than in water.

Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.

Abstract: The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.

Spatial relationship between the nucleotide‐binding site, Lys‐61 and Cys‐374 in actin and a conformational change induced by myosin subfragment‐1 binding

Abstract: The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.

A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification.

Abstract: Accompanying the phototransformation of native 124-kilodalton (kDa) oat phytochrome from red-absorbing form (Pr) to far-red-absorbing form (Pfr), there is a photoreversible change in circular dichroism (CD) in the far-UV region indicative of a 3% increase in alpha-helical folding of apoprotein. To elucidate the conformational change involved in the phytochrome phototransformation, several monoclonal antibodies have been used as epitope-specific probes. Monoclonal antibody oat-25 suppressed the photoreversible CD spectral change using phytochrome with an A666/A280 as Pr of 1.13. Monoclonal antibodies oat-22, oat-13, and oat-31 did not significantly affect the CD spectral change of phytochrome. Oat-25 requires an epitope near the N-terminus of phytochrome. Oat-22, oat-13, and oat-31 recognize epitopes on the N-terminus, chromophore-containing half of phytochrome, albeit further removed from the N-terminus than that recognized by oat-25. Interestingly, oat-13 and oat-31 did, however, induce a time-dependent decrease in the far-UV CD, apparently due to aggregation of phytochrome (both Pr and Pfr forms). Monoclonal antibodies oat-26 and oat-28, which recognize epitopes on the C-terminus half of phytochrome, also did not suppress the photoreversible CD change, although oat-26 and oat-28 slightly inhibited it. The photoreversible CD spectral change can also be inhibited by sodium borohydride, which bleaches the chromophore by reducing it, and by tetranitromethane, which oxidizes the chromophore of phytochrome. Although explanations of these results based on indirect interactions between the chromophore and the N-terminus segment are possible, we propose that an additional alpha-helical folding of the Pfr form of the phytochrome may result from a photoreversible interaction between the Pfr form of the chromophore and the N-terminus segment.

Conformational change in a viral glycoprotein during maturation due to disulfide bond disruption.

Abstract: The fusion glycoprotein of Newcastle disease virus is synthesized as an inactive precursor, F0. During intracellular transport and maturation, F0 undergoes a conformational change resulting from the loss of intramolecular disulfide bonds. F0 is also cleaved to yield F1, F2, the active, membrane-fusing form of the protein. Two monoclonal antibodies were used to explore this conformational change and its relationship to cleavage. These antibodies failed to precipitate the pulse-labeled fusion protein but did precipitate the F0 and the F1, F2 forms of the "chase" fusion protein. Use of the inhibitors carbonylcyanide m-chlorophenylhydrazone and monensin showed that the fusion protein acquired the ability to react with the monoclonal antibodies after it left the rough endoplasmic reticulum but before it left the medial Golgi membranes and before it was cleaved. The acquisition of antigenicity correlates with the disruption of intramolecular disulfide bonds during transit through the cell. This correlation was directly confirmed. The pulse-labeled fusion protein could be recognized by both monoclonal antibodies if the protein was first reduced. The formation and disruption of intramolecular disulfide bonds as a posttranslational modification of glycoproteins is discussed.

The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

Abstract: The hemagglutinin of influenza virus (HA), an acid-activated membrane fusion protein, is synthesized in the endoplasmic reticulum and transported through the Golgi complex to the cell surface of infected cells as an uncleaved, fusion-incompetent precursor, HA0. The mature, proteolytically activated HA is known to undergo a rapid, irreversible, acid-induced conformational change which mediates membrane fusion and virus penetration. On the basis of antigenic modifications and the acquisition of trypsin susceptibility, we demonstrate here that HA0, while unable to cause fusion, is acid sensitive. It undergoes irreversible conformational changes quite similar to those of HA at mildly acidic pH (pH less than 6.0). The ectodomain of HA0 does not, however, acquire hydrophobic properties and the changes occur in a less concerted manner (the pH dependence is much broader and the rate of conversion slower). These differences are likely to account for the inability of acid-treated HA0 to trigger membrane fusion. It was shown, moreover, that HA0 acquired its acid-sensitive properties immediately following trimerization in the endoplasmic reticulum. Since HA0 did not convert to the acid form at any point during its intracellular transport, we concluded that the trans-Golgi compartment, known to be more acidic than the cytosol and involved in constitutive membrane transport, is not likely to have a pH less than 6.0.

A High-Resolution Solid-State 13C NMR Study of the Secondary Structure of Branched (1→3)-β-D-Glucans from Fungi: Evidence of Two Kinds of Conformers, Curdlan-Type Single-Helix and Laminaran-Type Triple-Helix Forms, as Manifested from the Conformation-Dependent 13C Chemical Shifts

Abstract: The high-resolution 13C NMR spectra of a variety of fungal branched (1→3)-β-D-glucans were recorded in a DMSO solution and a lyophilized solid in order to gain an insight into the primary and secondary structures in relation to their gel-forming property We found that all the five lyophilized branched (1→3)-β-D-glucans examined so far, except lentinan, exhibit the laminaran-type triple-helix form, as established by the close resemblance of the 13C NMR spectra among them On the contrary, lentinan taking the curdlan-type singlehelix conformation was readily converted to the triple-helix form by lyophilization after having been dissolved in a 8M urea solution (1M=1 mol dm−3) and dialysis against distilled water This finding should be compared with the similar conformational change of curdlan: the conversion by this procedure was at most 50%; annealing at 150°C, followed by slow cooling, was essential for a complete conversion This treatment did not induce the reverse conformational change from the triple

Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study.

Abstract: When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 +/- 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers.(ABSTRACT TRUNCATED AT 250 WORDS)

A High-Resolution Solid-State 13C NMR Study of the Secondary Structure of Linear (1→3)-β-D-Glucans: A Conformational Elucidation of Noncrystalline and Crystalline Forms by Means of Conformation-Dependent 13C Chemical Shifts

Abstract: The high-resolution solid-state 13C NMR spectra of annealed curdlan, a (1→3)-β-D-glucan, were recorded in order to obtain reference data on the 13C chemical shifts of the triple helix form, which would be useful for the conformational elucidation of two distinct noncrystalline forms, curdlan- and laminaran-types. It was found that the C-3 peak of crystalline annealed glucan is substantially displaced from that of noncrystalline starting powder, reflecting the conformational change. The laminaran-type conformation, which is found in either laminaran or a number of fungal branched glucans, was identical with the triple helix, judging from the similarity in the 13C NMR spectra, although the linewidths of the former are much larger than those of the latter. The curdlan-type form, on the other hand, was ascribed to the single helix form on the basis of the previous data on high-resolution 13C NMR. In addition, the 13C NMR spectra of crystalline paramylon showed a number of additional signals other than the pea...

Comparison of rates of cation release and of conformational change in dog kidney Na, K-ATPase.

Abstract: 1. It is now widely believed that the main rate-limiting step in the sodium-potassium pump (Na, K-ATPase) cycle is a conformational change between two forms of the dephosphoenzyme (E2 and E1) and that this change releases to the cell interior potassium ions occluded within the E2 form. 2. If this hypothesis is correct, and if occluded ions cannot be released directly from dephosphoenzyme in the E2 conformation, we should expect that, under any given conditions, the rate of release of the occluded ions would be identical with the rate of the conformational change. 3. Using the potassium congeners 86Rb, 137Cs and 204Tl, the rates of release of the occluded ions can be measured by a rapid ion-exchange technique. Using the fluorescence probes fluorescein isothiocyanate (FITC), eosin or 5-iodoacetamido-fluorescein (5-IAF), the rates of the conformational change can be measured by stopped-flow fluorimetry. 4. A comparison of the two rates in the absence of ATP showed that the rate of release of the occluded ions was usually somewhat faster than the rate of the fluorescence change. The discrepancy was probably caused by a very slow direct release of occluded ions from enzyme in the E2 form, but we cannot exclude the possibility that it is the result of systematic errors. In the presence of 5 microM-ATP, both rates were increased and there was no significant difference between them. 5. The results are compatible with the hypothesis that the same conformational change alters the fluorescence of the fluorescent probes and releases the occluded potassium congener ions.

The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes.

Abstract: The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

Spectroscopic characterization of thioredoxin covalently modified with monofunctional organoarsenical reagents.

Abstract: Thioredoxin upon reduction with mercaptoethylamine was subjected to covalent modification by the monofunctional organoarsenical reagents H2NPhAsO and HO(CH2)4AsCl2 The degree of modification was monitored by the percentage loss in free thiol content as measured by the reaction with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) The modification results in the formation of a stable 15-membered cyclic dithioarsenite ring that readily extrudes the arsenic moiety upon the addition of 2,3-dimercaptopropanol The conformational effects of this modification were monitored by steady-state fluorescence and circular dichroism On the basis of circular dichroic spectra, it appeared that the protein experiences no significant backbone conformational change from this modification The degree of conformational change was found to be within the range observed upon reduction of the oxidized thioredoxin Steady-state fluorescence revealed that the arsenicals caused strong quenching of the tryptophan fluorescence Stern-Volmer titrations revealed that the quenching was a function of both the nature of the organic group and its covalent attachment to the "spatially close" thiols The analysis of the spectroscopic results obtained with the arsenical reagents provides further insight into the nature of the conformational change that has been observed upon reduction of thioredoxin

Circular dichroic investigations of secondary structure in synthetic peptide inhibitors of cAMP-dependent protein kinase: a model for inhibitory potential.

Abstract: The structure of the inhibitory domain of the inhibitor protein of the cAMP-dependent protein kinase has been assessed by circular dichroism studies of synthetic inhibitory peptides. Using the inhibitory peptide PKI(5-22)amide (Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile22) [Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992] and shorter peptides of this sequence, it has been estimated that this parent peptide is composed of approximately 30% alpha-helix with the remainder being random coil with one beta-turn. The pseudosubstrate arginine cluster (Arg15-Arg19) is within the suggested region of random coil and beta-turn, representing one critical region of binding recognition by the protein kinase. The alpha-helix region proposed between Thr6 and Ile11 likewise contributes to the full biological potency and specificity of the inhibitor peptide and inhibitor protein. The removal of the two N-terminal threonines, for example, causes both a marked conformational change in the peptide and a diminishment by an order of magnitude of inhibitory activity. It is proposed that this alpha-helix region could serve one of several possibilities, including that it may provide a suitable constraint on the Tyr7 such that the hydroxyl is oriented in a position proximal to the pseudosubstrate domain, and/or may allow the optimal location of other protein kinase recognition signals. These data provide an initial description of some of the structural features of the inhibitor protein that could contribute to its high biological potency.

Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations.

Abstract: The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer. lambda-cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.

Role of C-terminal tail of long neurotoxins from snake venoms in molecular conformation and acetylcholine receptor binding: proton nuclear magnetic resonance and competition binding studies.

Abstract: The role of the "C-terminal tail" segment of long neurotoxins has been investigated. The C-terminal four to five residues of alpha-bungarotoxin and Laticauda colubrina b have been cleaved off by carboxypeptidase P. The effect of such deletion on the toxin conformation has been monitored in proton nuclear magnetic resonance spectra and circular dichroism spectra. The removal of the C-terminal residues primarily affects the chemical shifts of proton resonances of the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with tritium-labeled toxin alpha to Narke japonica acetylcholine receptor has revealed that cleavage of the C-terminal residues reduces the binding activity of alpha-bungarotoxin or Laticauda colubrina b to acetylcholine receptor. Thus it is likely that (the basic amino acid residues in) the C-terminal tail is directly involved in the binding of long neurotoxins to electric organ (and muscle) acetylcholine receptor.