A comparative x-ray study of a nucleoprotamine and DNA complexes with polylysine and polyarginine

The high-humidity X-ray pattern of oriented fibers prepared from salmon spermheads strongly resembles that of DNA in the B form. However, the nucleoprotamine pattern has a more intense first layer line and increased lateral unit-cell dimensions. Complexes of DNA with poly-L -lysine and poly-L -arginine were prepared and photographed at various relative humidities. The most crystalline patterns were obtained at 92% and also indicate DNA in the B form. However, whereas polylysine–DNA, like the spermheads, has a primitive hexagonal cell, polyarginine–DNA, like NaDNA, has three molecules in the unit cell. Polylysine–DNA, but not polyarginine–DNA, also resembles spermheads in having a strong first layer line. All three complexes show increasing intermolecular distance with increasing humidity, but with sharp maxima when photographed in water, which indicates cross-linking between the molecules. Lowering the humidity causes the polylysine–DNA, but not polyarginine–DNA, to change conformation from the B to the C form. The structural implications of these results are discussed in the light of model-building studies and a comparison of calculated and observed X-ray intensities.     

The interaction of 8-anilino-1-naphthalenesulfonate with polylysine and polyarginine

Comparative studies on the interaction of 8-anilino-1-naphthalenesulfonate (ANS) with polylysine and polyarginine have been made by equilibrium dialysis and fluorescence or circular dichroism measurements, to investigate the structural characteristics of the polypeptides. The results are summarized as follows: (i) ANS binds to either of the polypeptides primarily by electrostatic interaction while hydrophobic interaction partially facilitates the dye binding; both interactions are stronger in the polyarginine-dye binding than the polylysine-dye binding. (ii) The fluorescence of ANS is more intensified when the dye binds to polyarginine than to polylysine regardless of the value of r (number of bound dye per amino-acid residue) of polypeptide-dye complexes, although the intensification depends on the r value and becomes maximum at r = 0.25–0.35 for both cases. (iii) The binding of ANS to each polypeptide is cooperative at r < 0.4. (iv) The circular dichroism is more efficiently induced in the spectral region of ANS by binding to polyarginine than to polylysine. From these results, it was concluded that, compared to polylysine, polyarginine suffers some structural change by ANS binding into a more compact molecular configuration having some regularity with a lower dielectric environment.     

Interaction of DNA with poly(L-Lys-L-Ala-Gly) and poly(L-Lys-L-Ala-L-Pro). Circular dichroism and thermal denaturation studies

Complexes of DNA with polypeptides composed of Lys, Ala, and Gly in both a sequential order, poly(L-lysine-L-alanine-glycine), and a statistical distribution, poly(L-lysine36-L-alanine28-glycine), were prepared using gradient dialysis. These polypeptide-DNA complexes were studied using ultraviolet absorption (UV) and circular dichroism (CD) to probe the conformation, binding, and melting behavior of DNA in the complex. Complexes with the sequential polypeptide showed no structural change in the DNA; however, the complexes with the random polypeptide yield CD spectra similar to phi DNA [Maniatis, T., Venable, Jr., J.S., and Lerman, L.S. (1974), J. Mol. Biol. 84, 37]. A second sequential polypeptide, poly(L-Lys-L-Ala-L-Pro)n, -DNA complex was also studied. It was found to exhibit pronounced structural changes as a function of ionic strength and poly-peptide-DNA ratio, more similar to the random sequence that the ordered sequence of the Lys, Ala, Gly polymer. Thus the importance of the composition and amino acid sequence in polypeptides which bind to DNA, even in such simple systems, is demonstrated. Evidence from thermal denaturation, employing simultaneous monitoring of CD and UV changes, supports a model in which specific polypeptides cause condensation of the DNA in the complex into an asymmetric tertiary structure. The relevance of these model systems to chromatin is discussed.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

Formation of psi (+) and psi (-) DNA

DNA molecules can be organized into ordered aggregates of opposite handedness by complexation with polylysine and other polypeptides; we have investigated the conditions under which ψ(+) and ψ(?) structures are produced with double-helical synthetic polynucleotides. Both poly(dGdC)·poly(dGdC) and poly(dAdT)·poly(dAdT) readily form ψ(?) structures with polylysine, although the method of preparation can alter the CD spectra. The GC copolymer, which is more susceptible to conversion into A or Z conformers, forms ψ(+) structures with lysine–alanine copolypeptides more readily than the AT copolymer. Nucleotide base modifications that favor the Z structure, such as bromination and methylation, also favor ψ(+) formation, and the Co(NH₃)₆Cl₃ reagent that readily induces the Z structure also leads to ψ(+). Thus, the production of the ψ(+) structure seems to be frequently correlated with susceptibility to A or Z formation, although there are some cases in which the B conformer also leads to ψ(+). Polyethylene glycol generally produces a ψ(?) structure; the differentiation between ψ(+) and ψ(?) structures seems to require electrically charged polymers.     

Interactions of basic polypeptides and proteins with calmodulin.

Low concentrations (less than 10 microgram/ml) of a number of highly basic polypeptides inhibit the calmodulin-stimulated cyclic nucleotide phosphodiesterase. Inhibitory compounds include synthetic polypeptides [polylysine (D and L) and polyarginine] and basic proteins (protamine, histones H1, H2A, H2B, H3 and H4 and myelin basic protein). Polylysine of mol.wt. about 2000 or higher was inhibitory, but pentalysine did not inhibit. Other basic proteins and compounds did not inhibit, including bradykinin, spermine and putrescine. In mixtures of calmodulin and basic protein, complexes were formed whether Ca2+ was present or not. This was true for polylysine, myelin basic protein and histone H2B. These interactions suggest that the inhibition of the phosphodiesterase is due to interaction of these basic proteins with calmodulin. The wide variety of basic polypeptides and proteins that affect the calmodulin stimulation of phosphodiesterase indicates that these interactions are not specific.     

Different effects of polylysine and polyarginine on the transition to a condensed state of DNA in polyethyleneglycol/salt solution.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol/salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol/salt solution of the DNA - polylysine complex were indistinguishable from those of free DNA while the DNA-polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol/salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol/salt solution, polyarginine binding has an effect of inhibiting the transition.     

Activity of foot-and-mouth disease virus RNA-dependent RNA polymerase in vitro: inhibition by polyamines and poly(amino acid)s

Replication of foot-and-mouth disease virus RNA in vitro is inhibited by high concentrations of the following polyamines in decreasing order of effectiveness: putrescine, spermine, and cadaverine. The basic poly(amino acids), polylysine, polyornithine, and polyarginine, as well as the basic protein salmine, are several orders of magnitude more inhibitory than polyamines. The interaction between polyornithine and foot-and-mouth disease virus RNA and its inhibition of replicase activity are related to the ability of basic polypeptides to bind to the RNA template. The neutral polymer, polysarcosine, and the polyanions, polyglutamic acid and heparin sulfate, do not inhibit replication; however, both polyanions relieve the inhibition by polyamines and polyamino acids. The mode of inhibition by polyamines and poly(amino acids) and the antagonism by heparin is discussed.     

Investigation of polylysine-dipalmitoylphosphatidylglycerol interactions in model membranes

The effect of poly(L-lysine) on dipalmitoylphosphatidylglycerol bilayers has been studied by Raman and infrared spectroscopies, small-angle X-ray diffraction, and carboxyfluorescein escape experiments. The polypeptide is shown to induce a stabilization of the bilayer detected by the increase of interchain vibrational coupling and a slight decrease of the overall disorder. In addition, long polylysine (Mr 150,000) induces a positive shift of the gel to fluid transition temperature and, at lipid to lysine molar ratios greater than 1, a lateral phase separation within the bilayer. Raman and infrared spectra indicate modifications at the head group level. In contrast, short polylysine (Mr 4,000) leads to a decrease of the lipid thermotropic transition temperature, and no modification of the polar head group and no phase separation could be observed. These differences between short and long polypeptides are correlated with the conformation the polypeptide adopts upon binding to the lipid, which favors the formation of alpha-helices in the case of long polypeptides (Mr greater than or equal to 14,000). The X-ray data suggest that the basic polypeptide acts as a bridge between neighboring bilayers, thus causing their aggregation and dehydration.     

Structural advantage of dendritic poly(L-lysine) for gene delivery into cells

This study aimed to investigate the relationships between structures of gene carrier molecules and their activities for gene delivery into cells. We compared 2 types of poly(L-lysine) as carriers, that is, dendritic poly(L-lysine) (KG6) and linear poly(L-lysine) (PLL). KG6 formed a neutral DNA complex, and its DNA compaction level was weaker than that of PLL. The amount of DNA binding and uptake into cells mediated by PLL was 4-fold higher than that with KG6. However, KG6-mediated gene expression was 100-fold higher than that by PLL. Since pK(a) values of terminal amines of KG6 were lowered even though small amounts of DNA were internalized into cells, sufficient DNA amounts for effective gene expression escaped to the cytosol due to the proton sponge effect in the endosome. In addition, weakly compacted DNA with KG6 was advantageous in accessing RNA polymerase in the cell nucleus. On the other hand, PLL did not show the proton sponge effect in the endosome and resulted in strong compaction of DNA. Even though large DNA amounts were internalized into cells, most of the DNA would not take part in gene expression systems in the nucleus. Amount of induced cytokine production after intravenous injection of DNA complexes with KG6 and PLL was low, and was similar to the case when DNA was injected alone. Therefore, no significant difference in effects on cytokine production was observed between KG6 and PLL.     

Solution conformation of poly(L-lysyl-L-glutamic acid) and poly(L-lysyl-L-glutamine)

Solution conformation of poly(L-lysyl-L-glutamic acid) (PLGU) and poly(L-lysyl-L-glutamine) (PLGN) was studied in water as a function of pH, added salt, detergents, methanol and trifluoroethanol (TFE). Both the polypeptides exhibit no ordered conformation in the pH range 1.5-12.5; salts and detergents did not have any marked effect. Replacement of side chain carboxyl by an amide group did not help in inducing PLGN to adopt a helical conformation even at pH as high as 12.0, unlike poly(L-lysine). The helicogenic solvents, methanol and TFE, induce formation of weak helices in PLGU as well as in PLGN. It is not unlikely that H-bonding between the side chains leads to stabilizing an unordered conformations.     

Interactions between model proteins and deoxyribonucleic acids.

Interactions between DNA and model proteins, poly(L-Lys(m)L-Ala(n)), where m + n = 100%, have been investigated using thermal denaturation and circular dichroism (CD). All complexes of DNA with these proteins precipitate in a small range of input ratios, protein to DNA, with the midpoints of all precipitation curves close to a 1:1 ratio of lysine to phosphate. The melting temperature of model protein-bound DNA regions decreases slightly as the alanine content of the model protein is increased, which can be explained as a result of insufficient charge neutralization of phosphates by lysine residues in the model proteins. In the free state, these model proteins possess varying amounts of alpha helix, random coil, or a mixture of these two, depending upon the relative lysine/alanine content. When bound to DNA, the CD of the complex shows a substantial increase in alpha-helical structure for those model proteins with 40-60% alanine, while there is no significant change in alpha-helical structure when the percent alanine is either substantially higher or lower (i.e., 81 or 19% alanine). Only those complexes formed with model proteins having 40-60% alanine undergo a drastic transition from a B-type CD to an A-type in the presence of intermediate ionic strength (0.2 M NaCl, for example). Poly(Lys19Ala81)-DNA complexes show a slight transition toward A-type CD at 0.4 M NaCl or higher. Apparently other factors, in addition to alanine and alpha-helical content, must be responsible for this B leads to A transition. At the other extreme of lysine/alanine ratio, with high lysine content, poly(Lys81Ala19) or polylysine, the presence of NaCl produces a B leads to psi transition. The possible significance of these differences in response to the binding of these model proteins is discussed.     

Immunoglobulin E decapeptide-induced 5-hydroxytryptamine release from rat peritoneal mast cells. Comparison with corticotropin-(1-24)-peptide, polyarginine, polylysine and antigen.

A synthetic basic decapeptide from the C4 domain of human immunoglobulin E, corticotropin-(1-24)-peptide, polyarginine and polylysine stimulated up to 90% net release of 5-hydroxytryptamine from mast cells in rat peritoneal-cell suspensions. Concentration-response curves to all four polypeptides were parallel. Polyarginine and polylysine (EC50 congruent to 0.05 microM) were approximately 100-fold more potent than immunoglobulin E decapeptide and corticotropin-(1-24)-peptide (EC50 congruent to 5 microM). Polypeptide-induced release was complete within 5-10s. Immunoglobulin-E-decapeptide-induced 5-hydroxytryptamine release was additive to that induced by low concentrations of polyarginine, but non-additive to that induced by high concentrations of polyarginine. In contrast, release induced by antigen was additive along the whole length of the concentration-response curve to polyarginine. Benzalkonium chloride inhibited immunoglobulin-E-decapeptide- and polyarginine-induced 5-hydroxytryptamine release but enhanced release induced by immunological stimulation.     

Investigation of DNA structural changes by infrared spectroscopy

DNA-oriented samples of various origins were studied under different conditions of humidiity and sodium chloride content by means of infrared spectroscopy. (1) Oriented DNA (M. Lysodeikticus, E. coli, calf thymus and salmon sperm) films at 3–4% sodium chloride yield polarized spectra which show drastic changes at relative humidities (r.h.) between 94% and 0% indicative of conformational changes: B form → a form → disordered form The measurements of the infrared dichroism at frequencies of about 1230 cm^?1 and at about 1090 cm^?1 allow one to determine the orientation of the phosphate group, whereas the measurements at 1710 cm^?1 characterize the base orientation. At humidities higher than 90% r.h. (B form) the bisector of OPO forms an angle of 70° relative to the helix axis, whereas at lower humidities, between 75% and 50% r.h. (A form) a rotation to about 45° is observed. Simultaneously, the 0—0 line of phosphate group changes its orientation from 55° to 65° to the helix when B → A transition takes place. The results are in general agreement with that of X-ray diffraction and allow one to determine the orientation of the phosphate group with greater precision. (2) The B–A conformational change is not observed for satellite DNA, isolated from Cancer pagurus, of which the guanine + cytosine content is below 5%. As a function of decreasing humidities, one observes the transition: B form → disordered form A diagram of conformational changes of DNA's as a function of base composition and of r.h., suggests that B–A transition will occur for DNA of relatively higher G + C content, whereas for high (A + T) content, base sequence may be of importance. The B–A transition is prevented in DNA at a relatively high or very low sodium chloride content.     

Interaction between poly(L-lysine48, L-histidine52) and DNA.

Poly(Lys⁴⁸, His⁵²), a random copolypeptide of L -lysine (48%) and L -histidine (52%), was used as a model protein for investigating the effects of protonation on the imidazole group of histidines on protein binding to DNA. The complexes formed between poly(Lys⁴⁸, His⁵²) and DNA were examined using absorbance, circular dichroism (CD), and thermal denaturation. Although increasing pH reduces the charges on histidine side chains in the model protein, the protein still binds the DNA with approximately one positive charge per negative charge in protein-bound regions. Nevertheless, CD and melting properties of poly(Lys⁴⁸, His⁵²)-DNA complexes still depend upon the solution pH which determines the protonation state of imidazole group of histidine side chains. At pH 7.0, the complexes show two characteristic melting bands with a t_m (46–51°C) for free base pairs and a t′_m (94°C) for protein-bound base pairs. The t′_m of the complexes is reduced to 90°C at pH 9.2, although at this pH there is still one lysine per phosphate in protein-bound regions. Presumably, the presence of deprotonated histidine residues destabilizes the native structure of protein-bound DNA. The binding of this model protein to DNA causes a red shift of the crossover point and both a red shift and a reduction of the positive CD band of DNA near 275 nm. This phenomenon is similar to that caused by polylysine binding. These effects, however, are greatly diminished when histidine side chains in the model protein are deprotonated. The structure of already formed poly(Lys⁴⁸, His⁵²)·DNA complexes can be perturbed by changing the solution pH. However, the results suggest a readjustment of the complex to accommodate charge interactions rather than a full dissociation of the complex followed by reassociation between the model protein and DNA.     

Dictyostelium myosin II heavy-chain kinase A is activated by heparin, DNA and acidic phospholipids and inhibited by polylysine, polyarginine and histones.

Dictyostelium myosin II heavy-chain kinase A (MHCK A) is activated by autophosphorylation. Heparin and DNA, as well as vesicles composed of phosphatidylserine or phosphatidylinositol, were found to increase the initial rate of MHCK A autophosphorylation 5-10-fold in a Ca(2+)-independent manner. The negatively charged molecules also increased the activity of the autophosphorylated MHCK A by about 2-fold. In contrast, positively charged polypeptides such as poly(D-lysine), poly(L-lysine), poly(L-arginine) and histones strongly inhibited (IC50 of 0.5 micrograms/ml) the activity of the active, autophosphorylated MHCK A. Similar levels of inhibition, on a weight basis, were observed for poly(L-lysine) fractions with molecular weights from 3800 to 150,000-300,000. The inhibition was competitive with respect to peptide substrate and mixed with respect to ATP. At much higher concentrations poly(L-lysine) also inhibited the ability of MHCK A to autophosphorylate. It is proposed that negatively charged compounds and autophosphorylation increase the activity of MHCK A by weakening the interaction between the catalytic domain and a positively charged autoinhibitory domain.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

Four-stranded DNA helices: conformational analysis of regular poly(dT).poly(dA).poly(dA).poly(dT) helices with various types of base binding

The paper presents results obtained in conformational analysis of homopolymeric four-stranded poly(dT).poly(dA).poly(dA).poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.