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.     

Conformation and properties of DNA–(Lys33, Leu67)100–(Orn)20 complex: A nucleohistone model

The synthesis and characterization of the block copolypeptide (Leu⁶⁷, Lys³³)₁₀₀Orn₂₀, a synthetic model of histone, are reported. In neutral aqueous solutions, 80% of the etheropolypeptide block assumes an α-helical conformation, whereas the polyornithine block is in a random-coil conformation. In the association complexes with DNA, melting and titration experiments, as well as CD results, indicate that the polyornithine block interacts with DNA, whereas at least 2/3 of the lysine residues of the (Leu, Lys) moiety are excluded from the direct binding with DNA. CD spectra of the association complexes reveal significant differences from those obtained with DNA–polyornithine and DNA–polylysine complexes but substantial similarities with CD spectra of native and reconstituted nucleohistones. In contrast to DNA–polyornithine complexes, the CD spectra of the ternary complexes, copolypeptide–DNA–ethidium bromide, indicate a strong reduction of the dye intercalation. The low-angle x-ray diffraction pattern, reminiscent of that of chromatin, reveals the presence of a superstructure in these complexes. The results obtained are discussed in connection with the expected structural features of the model.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.     

Salt-induced conformational change of random copolymers (Lys50Tyr50)n and (Lys50Phe50)n studied by 1H- and 13C-nuclear magnetic resonances. Aggregation behavior of helical segments

¹H- and ¹³C-nmr studies of conformational transitions of random amino acid copolymers containing aromatic residues (Lys⁵⁰Tyr⁵⁰)_n and (Lys⁵⁰Phe⁵⁰)_n in the presence of neutral salts were performed to serve as models of the aggregation behavior of polypeptides of biological significance. The ¹H and ¹³C signal intensities of Tyr and Phe residues decreased preferentially with increasing concentration of neutral salts such as NaCl and NaClO₄. This behavior contrasts with that of (Lys)_n in the presence of similar neutral salts, where the displacement of the ¹³C signal is clearly seen on transition from the random-coil to the helical conformation. On the basis of the previous conformational studies, the loss of the peak areas is ascribed to the presence of immobilized helical segments by hydrophobic interaction between aromatic side chains. The remaining resonances are due to the residual random-coil regions, since the values of nuclear Overhauser enhancements and chemical shifts are unchanged in the presence and absence of the neutral salts.     

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

While the molecular structures of angiotensin II (Ang II) type 1 (AT₁) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT₁ receptor, different positions of the AT₁ receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT₁ receptor, Tyr¹¹³, Tyr¹⁸⁴, Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT₁ receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr¹¹³ in the AT₁ receptor and the hydroxyl group of olmesartan, between Lys¹⁹⁹ and carboxyl or tetrazole groups, and between His²⁵⁶ or Gln²⁵⁷ and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ of AT₁ receptor. Lys¹⁹⁹ in the AT₁ receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys¹⁹⁹. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr¹¹³. On the other hand, the n-butyl group of irbesartan may bind to Tyr¹¹³. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT₁ receptor and for the formation of unique modes of binding.     

Polypeptides. LVI. Effect of lithium bromide and of temperature on the conformation of a copolymer of glutamic acid and lysine

By using optical rotatory dispersion measurements, the helix content of poly Glu⁵⁰Lys⁵⁰ has been investigated and compared with that of poly Glu²⁰Lys²⁰Ala⁶⁰ in aqueous solutions. Measurements were made at pH 3 and at pH 8 in various concentrations of lithium bromide. Various factors affecting helix stabilization are considered and their perturbation by lithium bromide is related to the shape of the observed transition curves. A residual helix content of 12% in 8M LiBr, based upon a b₀ of +100 for a fully random conformation, was observed for poly Glu⁵⁰Lys⁵⁰ at pH 3 and 8. The loss of helix content of poly Glu⁵⁰Lys⁵⁰ as a function of temperature is also reported. ΔH is approximately ?6.9 kcal./mole for the overall transition, compared to ?6.5 kcal./mole for poly Glu²⁰Lys²⁰Ala⁶⁰. The midpoint of the broad transition is near 40°C. at pH 3, but much lower, at ?10 to 0°C., at pH 8. These results are discussed in terms of the stabilizing factors for the partial helix content of the polypeptides.     

Interaction of amphipathic polypeptides with phospholipids: characterization of conformations and the CD of oriented beta-sheets

The effects of interaction with phospholipids on the conformation of the alternating copolymer, poly(Leu-Lys), and the random copolymer poly(Leu⁵⁰, Lys⁵⁰) have been investigated by CD and ir spectroscopy. Poly(Leu-Lys) undergoes a partial unordered → β-sheet transition in solution in the presence of lysolecithin. On addition of lysolecithin plus cholate, an unordered → α -helix transition is observed. In films deposited from these solutions, poly(Leu-Lys) adopts the anti-parallel β-sheet conformation, as in aqueous solutions at moderate ionic strength. Polarized ir spectra showed that the plane of the β-sheet in such films deviates from the plane of the film by no more than 14°. The random copolymer, poly(Leu⁵⁰, Lys⁵⁰), is α-helical in the presence of lysolecithin and lysolecithin plus cholate, regardless of whether the sample is a solution or a film. CD measurements on the poly(Leu-Lys) films provide information about the component of the CD tensor for light propagating normal to the plane of the β-sheet. These measurements show (1) a negative n → π* CD band (214 nm maximum) with higher intensity than the average CD for isotropic solution; and (2) a positive band in the π → π* region (195 nm maximum), which is weaker than that in the isotropic spectrum.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Abstract

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly(d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)₂₀, d(pA)₇, and d(pT)₇). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometrics depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 ± 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)) (with the first endpoints giving n = 4.1, 4.0, 4.0, and 4.1 ± 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non- interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol Struct, and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex.

We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A- and/or T-containing DNAs, Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20°C, appeared to interact with one another in much the same manner as in the free polymers at 50°C. Finally, we found that the protein tyrosyl CD band at 228.5 nm decreased 39-42% when the protein bound to poly[d(A)] or poly[d(T)], but this band decreased no more than 9% when the gene 5 protein bound to short A- or T-containing oligomers. Thus, at least one tyrosyl residue has a significantly altered optical activity only when the DNA substrate is long enough either to cause a transition to a different protein conformation or to allow additional protein-protein contacts between adjacent helical turns of the DNA-protein complex.     

Characterization of a newIr-GLT gene and its location in theI-region of theH-2 complex

The data presented in this paper characterize the immune responses in mice to the two related random terpolymers, poly(Glu⁵⁷Lys³⁸Tyr⁵) (GLT⁵) and poly (Glu⁵⁵Lys³⁴Tyr¹⁵) (GLT¹⁵), which are similar to the previously reported response against the terpolymer poly(Glu⁵⁸Lys³⁸Phe⁴) (GLØ). Responsiveness is linked to theH-2 ^d ,H-2 ^ja ,H-2 ^q andH-2 ^r haplotypes. The observed responsiveness of the recombinant strain B10.A(5R)tentatively maps theIr-GLT gene to the right of theIB subregion, i. e., in theIC subregion which codes for lymphocyte alloantigens. If confirmed this would be the firstIr gene to be mapped in theIC subregion.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Studies on interaction between poly(L-lysine58, L-phenylalanine42) and deoxyribonucleic acids.

A random copolymer of 58% L-lysine and 42% L-phenylalanine, poly(Lys58Phe42), was used as a model protein for studying the role of phenylalanine residues in protein-DNA interaction. Complexes between this copolypeptide and DNA, made by direct mixing, were studied by absorbance, circular dichroism (CD), fluorescence, and thermal denaturation. Complex formation results in an increase in absorbance, and an enhancement, red-shift, and broadening of phenylalanine fluorescence. The fluorescence enhancement is opposite to the quenching observed when a tyrosine copolypeptide is bound to DNA (R. M. Santella and H.J. Li (1974), Biopolymers 13, 1909). The positive CD band of DNA near 275 nm is reduced and red-shifted by the binding of the phenylalanine copolypeptide to a greater extent than by the tyrosine copolypeptide. Thermal denaturation of the complexes in 2.5 times 10(-4) M EDTA (pH 8.0) shows three characteristic melting bands. For complexes with calf thymus DNA, free base pairs melt at Tm,I (47-49 degrees) and copolypeptide-bound base pairs show two melting bands (Tm,II at 73-75 degrees, and Tm,III at 88 -90 degrees). Similar thermal denaturation results have been observed for complexes with Micrococcus luteus DNA. The fluorecence intensity of the complexes is greatly increased when the temperature is raised to the Tm,II region. In addition to fluorescence measurements, the effects of increasing temperature on absorption and CD spectra of the complexes were also studied. Stacking interaction between the phenylalanine chromophore and DNA bases, either partial or full intercalation, is implicated by the experimental results. Several mechanisms are proposed to describe the reaction between the copolypeptide and DNA, and thermal denaturation of the complex.     

Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10⁻⁸ M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys¹⁰⁶, Trp¹¹⁰, Tyr¹¹³, Leu¹¹⁴, Tyr¹²⁶, Pro¹³⁴, and Trp¹⁶² with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His¹²⁵, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNA^Phe, tRNA^Tyr, tRNA ₁ ^Met , tRNA^Asn, tRNA^Ala, tRNA^Leu, and tRNA^Lys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.We respectfully dedicate this paper to Prof. H. Bauer on the occasion of his 80th birthday.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys³³, Leu⁶⁷)₁₀₀-Orn₂₀, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 Å, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimentional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Conformation of sequential and random copolypeptides of lysine and alanine in sodium dodecyl sulfate solution

A series of sequential polypeptides, (Lys_i-Ala_j)_n, and random copolypeptides, (Lys^x, Ala^y)_n, were synthesized. The competitive effect of Ala, a helix former, and Lys, whose homopolymer has a β-form in neutral NaDodSO₄ solution, was determined by CD and absorption spectroscopy. All the polypeptides studied were unordered in neutral solution without the surfactant. Of the six sequential polypeptides only (Lys-Ala)_n adopted a stable β-form in NaDodSO₄ solution. Most striking is the difference between this polypeptide, (Lys₂-Ala₂)_n and (Lys^x, Ala^y)_n, even though they all have equimolar Lys and Ala. (Lys₂-Ala₂)_n was partially helical in 2.5–5 mM NaDodSO₄ but approached the unordered form in 50 mM NaDodSO₄, whereas (Lys⁵⁰, Ala⁵⁰)_n was completely helical in all NaDodSO₄ concentrations. Even Lysrich (Lys₂-Ala)_n and (Lys₃-Ala)_n formed a partial helix and a trace of the β-form, respectively, in low NaDodSO₄ concentrations; both reverted to the unordered form in high NaDodSO₄ concentrations. These results can be explained by Pauling-Corey's model for β-pleated sheets. Only (Lys-Ala)_n has all DodSO-bound Lys⁺ residues on one side and Ala residues on the other side of the polypeptide chain. They can nestle quiet efficiently in a β-sheet and between neighboring β-sheets. Our results further imply that random copolypeptides are not completely random; they comprise varying segments of (Lys_k-Ala_m), where k and m could vary from zero to a small integer.