Uncomplexed Form Research Articles

Salt-induced release of DNA from nucleosome core particles.

At elevated salt concentrations, the structure of chromatin is destabilized. This paper is concerned with the processes by which DNA is released from nucleosome core particles in free, uncomplexed form. Our experiments indicate that the DNA release reaction has distinctly different characteristics below and above approximately 0.75 M NaCl. Below this concentration of salt, release of the histones from the DNA is highly cooperative, so that no dissociation intermediates are even seen. Above this salt concentration, histone release is not so cooperative; H2A and H2B are released from the DNA more readily than are H3 and H4. This results in an apparently heterogeneous population of (H2A, H2B)-depleted intermediate species sedimenting at rates between that of free DNA and that of intact core particles. Dissociation of core particles at NaCl concentrations below 0.75 M is readily reversible. Reassociation of DNA and histones from higher salt concentrations is nearly quantitative if carried out by gradual decrease of salt concentration, but rapid dilution to low salt results in the formation of a fraction of metastable nucleosome multimers. To help organize our description of the DNA release process, we introduce a stability diagram for the core particle, defined with respect to the independent variables of salt concentration and particle concentration. We draw upon our own experimental work and also upon the work of several other laboratories. We distinguish five major regions in this diagram.

Structure of the ion channel peptide antibiotic gramicidin A

The crystal structure of the uncomplexed orthorhombic form of gramicidin A has been determined at 0.86 A resolution. The polypeptide crystallizes from ethanol as a left-handed, double-stranded, antiparallel beta 5.6-helical dimer that is 31 A long and an average of 4.8 A in diameter. The uncomplexed channel does not contain ions or solvent molecules, and its diameter is not uniform but varies from a minimum of 3.85 A to a maximum of 5.47 A. There are three empty cavities in the channel that have a diameter exceeding 5.25 A and appear to be large enough to accommodate water molecules or potassium ions in a chemically reasonable coordination environment. The observed crystal structure does not offer any obvious clues as to why an antiparallel beta 5.6-helix cannot function as an ion channel in lipid bilayers.

Three-Dimensional Structure at 0.86 Å of the Uncomplexed form of the Transmembrane Ion Channel Peptide Gramicidin A

The crystal structure of the uncomplexed orthorhombic form of gramicidin A has been determined at 120 K and at 0.86 angstrom resolution. The pentadecapeptide crystallizes as a left-handed antiparallel double-stranded helical dimer with 5.6 amino acid residues per turn. The helix has an overall length of 31 angstroms and an average inner channel diameter of 4.80 angstroms. The channel of this crystalline form is void of ions or solvent molecules. The channel diameter varies from a minimum of 3.85 angstroms to a maximum of 5.47 angstroms and contains three pockets where the cross-channel contacts are 5.25 angstroms or greater. The range of variation seen for the phi and psi torsion angles of the backbone of the helix suggests that these potential ion binding sites can be induced to travel the length of the channel in a peristaltic manner by cooperatively varying these angles. The indole rings of the eight tryptophan residues of the dimer are overlapped in three separate regions on the outer surface of the helix when viewed down the barrel of the channel. This arrangement would permit long-chained lipid molecules to nest parallel to the outer channel surface between these protruding tryptophan regions and act like molecular splines to constrain helical twist deformations of the channel.

Crystal and molecular structures of the complex of α-chymotrypsin with its inhibitor Turkey ovomucoid third domain at 1.8 Å resolution

The molecular structure of the complex between bovine pancreatic α-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 Å (1 Å = 0.1 nm) resolution shell [ I ≥ σ( I)]. The reactive site loop of OMTKY3, from Lys131 to Arg211 (I indicates inhibitor), is highly complementary to the surface of α-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 Å with 21 residues of the enzyme. A short contact (2.95 Å) from O γ of Ser195 to the carbonylcarbon atom of the scissile bond between Leu181 and Glu191 is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P′ 3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the α-chymotrypsin portion of this complex with the several structures of α and γ-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common α-carbon atoms averages 0.38 Å). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central α-helix and flanking small 3-stranded β-sheet) that have α-carbon atoms fitting to within 1.0 Å (root-mean-square deviation of 0.45 Å) whereas the residues of the reactive-site loop differ in position by up to 1.9 Å (C α of Leu181). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and α-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds.

Chymotrypsin inhibitor 2 (CI-2), a serine proteinase inhibitor from barley seeds, has been crystallized and its three-dimensional structure determined at 2.0-A resolution by the molecular replacement method. The structure has been refined by restrained-parameter least-squares methods to a crystallographic R factor (= sigma parallel Fo magnitude of-Fo parallel/sigma magnitude of Fo) o of 0.198. CI-2 is a member of the potato inhibitor 1 family. It lacks the characteristic stabilizing disulfide bonds of most other members of serine proteinase inhibitor families. The body of CI-2 shows few conformational changes between the free inhibitor and the previously reported structure of CI-2 in complex with subtilisin Novo [McPhalen, C.A., Svendsen, I., Jonassen, I., & James, M.N.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7242-7246]. However, the reactive site loop has some significant conformational differences between the free inhibitor and its complexed form. The residues in this segment of polypeptide exhibit relatively large thermal motion parameters and some disorder in the uncomplexed form of the inhibitor. The reactive site bond is between Met-59I and Glu-60I in the consecutive sequential numbering of CI-2 (Met-60-Glu-61 according to the alignment of Svendsen et al. [Svendsen, I., Hejgaard, J., & Chavan, J.K. (1984) Carlsberg Res. Commun. 49, 493-502]). The network of hydrogen bonds and electrostatic interactions stabilizing the conformation of the reactive site loop is much less extensive in the free than in the complexed inhibitor.

Localization and characterization of Sendai virus nonstructural C and C′ proteins by antibodies against synthetic peptides

Antibodies were raised in rabbits against two synthetic peptides, each 30 residues in length, one corresponding to the predicted common carboxyl termini of the nonstructural C and C' proteins of Sendai virus and the other to the unique amino terminus of the larger C protein. Each peptide was inoculated as a covalent complex with tetanus toxoid or in uncomplexed form. Only antibodies to the free carboxyl-terminal peptide precipitated both C and C' proteins made by in vitro translation of viral mRNA and reacted with the C protein from infected cells. These results confirm that the C and C' proteins are carboxyl-coterminal. Contrasting with the reported colocalization of intracellular measles virus C proteins with nucleocapsid inclusions, immunofluorescence studies revealed that Sendai virus C proteins were uniformly distributed in the cytoplasm whereas the viral P protein was present in inclusions that were mainly perinuclear. Since almost all P protein molecules are associated with viral nucleocapsids, these observations suggested that Sendai virus C protein molecules may be both nucleocapsid-associated and free in the cytoplasm. This interpretation was supported when the C and C' proteins were found in both nucleocapsid and free protein fractions of cell lysates. Anti-C antibodies did not inhibit viral RNA synthesis when added to an extract of infected cells. This result was consistent with the conclusion that the C proteins have no direct role in viral transcription, since virions lack C proteins but are transcriptionally active. Therefore, the functions of the C proteins remain undefined.

Complexation of crown ethers with neutral molecules. 2. Comparison of free macrocycles and their complexes with malononitrile in solution

Thermodynamic parameters for the complexation of a neutral guest (malononitrile) with crown ethers having ring sizes of 15 to 33 ring atoms and various functionalities were obtained. The results give useful information about the conformations of the free crown ethers and their interactions with solvent molecules. 18-Membered macrocycles form the most stable complexes with malononitrile. Crown ethers in which the uncomplexed form strong intraannular interactions are present form weaker complexes while macrocycles in which additional binding sites (e.g., C=O) are available for complexation form more stable complexes with malononitrile than expected. A compensating effect of T ..delta.. S/sup 0/ and ..delta.. H/sup 0/ of complexation was found for all complexes. The crystal structure of 2,6-pyrido-18-crown-6-CH/sub 2/(CN)/sub 2/ (1:2) was determined; it shows a hydrogen bond to the pyridyl nitrogen atom.

Interaction of Certain Heavy Metals with Lake Humic Acids

Abstract Present studies were carried out to investigate the interaction properties of humic acids with certain heavy metals—Ca, Zn, Fe and Mn by using their isotopes. Humic acids solubilized maximum portion of the calcium present (97% in case of Dal lake), of which more than 90% was present in uncomplexed form. With 59Fe results were different, solubilizing less than 30% of it and most of this solubilized portion was in complex form. Results are present in detail.

COMPARISON OF ALUMINUM SPECIATION IN SOIL SOLUTIONS EXTRACTED BY BATCH AND COLUMN METHODS

Soil solutions from an Orthic Ferro-Humic Podzol and a Typic Humisol were extracted using a soil to solution ratio of 1:10 by the batch and column procedures. The aluminum speciation in the extracts was determined by measuring three forms of dissolved aluminum (uncomplexed monomeric, total monomeric and total reactive). The two methods of extraction gave different results. For most of the mineral horizons and the moderately to well-decomposed organic horizons the batch method tended to extract more dissolved aluminum, particularly in uncomplexed monomeric form. The greatest differences between the two methods were for the lowest bulk density materials, the L and F horizons of the Podzol and the Of of the Humisol. The variations were due to channelling in the column and the inability of the shaker to assure good mixing between the soil and solution. It is concluded that the batch method is best suited for routine laboratory analyses. Key words: Soil solution chemistry, Podzols, Humisols

Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53.

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.

Ions and ionophores

It will be shown using typical examples that it is possible to account, by explicit computations, for the complexing preferences of ionophores for certain cations. In the case of valinomycine, the preference observed for complexing the alkali cations is in the order Rb + ∼> K + > Cs + ⪡ Na +, and can be accounted for by making an energy balance between the energies of binding of the cation to the ionophore, assumed to remain a rigid bracelet-like cage, and the desolvation energies of the cation: the calculated values of the complexation energies are in the order Na + > K + > Rb + > Cs +; the energies required to desolvate the ions decrease in the same order, but the numerical values are such as to bring the balance between the two quantities into the experimentally observed order. In the case of nonactine, a much more flexible molecule, the computations show that, in the observed preference K + > Na +, the complexation energies largely favor Na + and that the substraction of the desolvation energies does not suffice to reverse the order of the two cations. In that case the determining factor stems from the intramolecular repulsions between the oxygenated ligands inside the cavity, repulsions appreciably larger in the conformation of the Na + cavity than in that of the K + cavity, so that the total balance favors K +. An attempt to account for the preference NH + 4 > K + was made, assuming that the conformational changes between the two complexes can be neglected: there, the computed complexations energy largely favors NH + 4, the prefer- red cation, and the substraction of the desolvation energy does not modify the order. The optimized computed position for NH + 4 shows very interesting differences with respect to K +, confirming some inferences made on the basis of early experimental infrared measurements in solution. It will also be shown how the notions of molecular electrostatic potential, molecular electrostatic field, and atomic accessibilities can be useful in the domain of cation-ionophore interactions. Two examples will be given: a) the K +-complex of valinomycine in connection with its ability to fix an anion, and the recently observed K +picratevalinomycine complex; b) the uncomplexed form of valinomycine to propose arguments for a choice between two possible mechanisms recently suggested for initiating the ion capture. Finally the case of divalent cations will be considered.

Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

Crystallisation and preliminary crystallographic data of a porcine neurophysin I—Tyr-Phe-NH 2 complex

The hormones oxytocin and vasopressin are stored in the neurosecretory granules of the neurohypophysis bound to acidic 10 000 mol. wt proteins called neurophysins. Three major neurophysins can be extracted from the porcine pituitary. The amino acid sequences of forms I and III are very similar, form I being a truncated form of III with three residues removed from the C-terminus [I 3. The physiological role of neurophysins is poorly understood. They are secreted with the hormones into the bled-strew where they circulate in an uncomplexed form. ~tra~llularly, they may stabilise the hormones during intra-axonal transport and storage by preventing leakage, enzymatic degradation and reduction of the susceptible 1-6 disulphide bond. The protein also concentrates the hormones in an insoluble form until required and reduces osmotic problems in the storage compartment . Neurophysins represent a family of proteins that show a highly conserved central core sequence with marked variability at the aminoand c~boxyl-terra both within and between species. The chemical specificity for ligand binding has been porously studied and there is a large amount of kin&G information derived from spectroscopic and thermodynamic studies [2]. The structure analysis of neurophysin bound to a hormone peptide analogue should contribute to the understanding of many biochemical questions. This structure might provide a rare opportunity to define

NMR and Raman spectroscopic study of complex formation between 2-methyl-2-azabicyclo-[2,2,2] -octa-3-one and TiCl 4

From analysis of 1H and 13C NMR and Raman spectroscopic data, in CDCl 3 and 1,1,2,2-tetrachloroethane- d 2 solution, the lactam 2-methyl-2-azabicyclo-[2,2,2]-octa-3-one (L) was found to form complexes with TiCl 4 (M) of overall composition M 2L, ML and ML 2. Two forms of the ML 2 complex were observed by NMR. In all these complexes, Ti coordinates to the carbonyl oxygen, and the metal—carbonyl bond is of the same type. The complexes ML and ML 2 contain hexacoordinated Ti; in M 2L the coordination number is probably five. The dynamics of exchange of the lactam between the ML 2 complexes and the uncomplexed form have been characterized and structures are proposed for the four complexes.

Characterization of the Membrane-Bound Inorganic Pyrophosphatase in Rhodospirillum rubrum

The membrane-bound inorganic pyrophosphatase (EC 3.6.1.1) from Rhodospirillum rubrum has been investigated with the tools of enzyme kinetics, and with two amino acid reagents, N-ethyl-maleimide (MalNET) and 4-chloro-7-nitrobenzofurazan (Nbf-Cl). 1. The concentration of the true substrate, MgPPi, was varied with constant concentrations of free Mg2+ or PPi. It was observed that Mg2+ acted as an activator. 2. Heat inactivation of the enzyme at 62 degrees C was slowed down in the presence of Mg2+. 3. MalNET and Nbf-Cl bind to the enzyme, and inhibit its activity. The effect of both reagents is dependent on the temperature. 4. A model is proposed where the 1:1 complex of Mg2+:PPi acts as substrate and Mg2+ interacts directly with the enzyme as an activator. PPi can bind to the enzyme, but is not hydrolyzed in the uncomplexed form.

Differences Between the Anthranilate-5-phosphoribosylpyrophosphate Phosphoribosyltransferases of Salmonella typhimurium Strains LT2 and LT7

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.

Thermodynamics of 222 cryptates and the selective transfer of ions in the water–methanol system

Free energies of transfer from water to methanol have been obtained for 222 cryptand and 222 cryptates, and used to calculate partition coefficients for the transfer of cations from the uncomplexed form in water to the cryptated form in methanol; the 222 cryptand alters the cation transfer selectivity by factors of up to 107 by comparison with transfer of uncomplexed cation in water to uncomplexed cation in methanol.

Analysis of alkaloid mixtures by charge-transfer complexation.

Binary mixtures of weak and strong UV-absorbing alkaloids were analyzed by a charge-transfer spectrophotometric method, utilizing iodine in ethylene dichloride as the acceptor. In the uncomplexed form, the strong absorbing alkaloid (papaverine, quinine, ergotamine, or reserpine) was measured at a wavelength where there was no interference from weak absorbers (at 335, 332, 315, or 300 nm, respectively). The weak absorbing alkaloid (ephedrine, codeine, atropine, or homatropine methylbromide) was determined by computing its contribution to the total charge-transfer band at 295 nm where absorbance was linearly additive for mixtures. The greater increase in the original epsilon-values of the weak absorbers upon complexation with iodine relative to the corresponding increase in the epsilon-values of the strong absorbers led to good recoveries even at the low dose ratios of the weakly absorbing, and often more potent, alkaloids.

The phospholipid head-group orientation: Effect on hydration and electrical conductivity

The water adsorption isotherms have been obtained for egg phosphatidylethanolamine when it is complexed to egg phosphatidylcholine and cholesterol, respectively. In the presence of phosphatidylcholine, the phosphatidylethanolamine water binding is changed to a strong binding as compared to when the phospholipid is in its uncomplexed form. Cholesterol increases the water adsorbed by the phospholipid, however, it does not change the nature of the isotherm. Phosphatidylmonomethylethanolamine also exhibits a strong water binding. The electrical conductivity of these phospholipids has been measured concurrently with their hydration. Electrical activation energies have been obtained for the fully hydrated phospholipids and are a function of both the amount of water adsorbed and the orientation of the polar head-group. The results are discussed in terms of a model for water adsorption, previously put forth by the authors.

Conformations of peptides in solution by nuclear magnetic resonance spectroscopy. Part IV. Conformations of valinomycin determined from homoallylic proton coupling across peptide bonds

100 MHz Proton magnetic resonance measurements have been made on valinomycin in the complexed and uncomplexed form in polar [CD3OD, (CD3)2N·CDO] and non-polar (CDCl3) solutions. A small five-bond long-range coupling was observed between α-CH groups across the peptide bonds of the L-Lac-L-Val and D-Hylv-D-Val molecular fragments. Observation of 5J(HH) enables the L- and D-Val α-CH proton signals to be assigned conveniently. Analysis of 5J(HH) in terms of homoallylic coupling provides information on peptide conformational angles ϕ and ψ, i.e. for valinomycin ψ(L-Lac), ϕ(L-Val), ψ(D-Hylv), ϕ(D-Val). The results for both complexed and uncomplexed valinomycin are compared with those of recent crystal structure analyses and with conformational models of these systems in polar and non-polar solvents.