Formation and stability of helical poly(N epsilon,N epsilon,N epsilon-trimethyl-L-lysine) in sodium perchlorate solution

Poly(trimethyl-L-lysine), [Lys(Me₃)]_n, is converted from random coil to α-helix at about 1/30 of the NaClO₄ concentration required by poly(L-lysine), (Lys)_n. NaClO₄ generates turbidity in [Lys(Me)₃]_n at concentrations above that required for helix formation, and decreases turbidity above lM NaClO₄. The turbidity runs parallel to enhanced, and then decreased, fluorescence of a dansyl label. Helix formation per se does not induce enhanced fluorescence. Increasing NaClO₄ concentration increases T_m linearly with log[NaClO₄] for both (Lys)_n and [Lys(Me₃)]_n until the denaturing effect of high NaClO₄ sets in. Increasing NaClO₄ also increases the breadth of the transition. Heating helical [Lys(Me₃)]_n or (Lys)_n does not produce a CD spectrum resembling that of “random-coil” (Lys)_n, except for [Lys(Me₃)]_n at relatively low NaClO₄ concentration.     

Highly branched poly(L-lysine)

This paper describes the synthesis of several novel water-soluble highly branched polypeptides. The synthesis starts with the ring-opening polymerization of epsilon-benzyloxycarbonyl-l-lysine N-carboxyanhydride (Z-Lys NCA) or epsilon-trifluoroacetyl-l-lysine N-carboxyanhydride (TFA-Lys NCA), followed by end functionalization of the peptide chain with N(alpha),N(epsilon)-di(9-fluorenylmethoxycarbonyl)-l-lysine (N(alpha),N(epsilon)-diFmoc Lys). Deprotection of the N(alpha),N(epsilon)-diFmoc Lys end group affords two new primary amine groups that can initiate the polymerization of a second generation of branches. Repetition of this ring-opening polymerization-end functionalization sequence affords highly branched poly(epsilon-benzyloxycarbonyl-l-lysine) (poly(Z-Lys)) and poly(epsilon-trifluoroacetyl-l-lysine) (poly(TFA-Lys)) in a small number of straightforward synthetic steps. Removal of the side-chain protective groups yields water-soluble and highly branched poly(l-lysine)s, which may be of potential interest for a variety of medical applications.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

Genetically encoding N(epsilon)-acetyllysine in recombinant proteins

N(epsilon)-acetylation of lysine (1) is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins containing N(epsilon)-acetyllysine (2) at defined sites. Here we demonstrate the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNA(CUA) pair. This strategy should find wide applications in defining the cellular role of this modification.     

A (1.5 + epsilon)-approximation algorithm for unsigned translocation distance

Genome rearrangement is an important area in computational biology and bioinformatics. The translocation operation is one of the popular operations for genome rearrangement. It was proved that computing the unsigned translocation distance is NP-hard. In this paper, we present a (1.5 + epsilon)-approximation algorithm for computing unsigned translocation distance which improves upon the best known 1.75-ratio. The running time of our algorithm is O(n2 + (4/epsilon)1.5 square root log(4/epsilon )2(4/epsilon), where n is the total number of genes in the genome.     

Formation of a left-handed RNA double helix: energetics of the A-Z transition of poly[r(G-C)] in concentrated NaClO4 solutions

Ultraviolet spectroscopic and nuclear magnetic resonance (NMR) studies have shown that poly[r(G-C)] in a solution of 4 M NaClO4 undergoes a transition to a left-handed Z-RNA helix upon raising the temperature to 60 degrees C [Hall, K., Cruz, P., Tinoco, I., Jr., Jovin, T. M., & van de Sande, J. H. (1984) Nature (London) 311, 584-586]. In the present report, the transition temperature of this particular order/order transition is shown to increase with decreasing NaClO4 concentration to about 110 degrees C, above which only the helix-to-random coil transition is detectable. The reversibility and cooperativity of the helix/helix conversion has facilitated the quantitative evaluation of the transition enthalpy by means of differential scanning microcalorimetry. In 5 M NaClO4, the transition temperature is 43 degrees C, the conversion enthalpy 4.2 kJ (1.0 kcal) per mole of base pair, and the corresponding entropy change 13 J (3.1 cal) deg-1. The van't Hoff enthalpy for the same process, determined from the temperature dependence of the optical transition, is 0.26 MJ (62 kcal) per mole of cooperative unit. The ratio of the two enthalpy values yields an apparent cooperative length for the A-Z transition of poly[r(G-C)] of approximately 60 base pairs, indicative of a concerted all-or-none process.     

Right-handed and left-handed helices of poly(dA-dC) X (dG-dT).

The secondary structures of poly(dA-dC) X (dG-dT) were studied using CD and IR spectroscopies. We give spectroscopic evidence of secondary structure transitions of poly(dA-dC) X (dG-dT) from a B to a Z-like helix, induced by transition metal ions (Ni2+) in presence of high concentrations of Cs+ and Na+. In the presence of Na+, the B in equilibrium Z transition occurs at any temperature, whereas premelting conditions are required in presence of Cs+. For these two alkali ions the Z-like form is only induced by Ni2+ ions through their specific interactions at N7 of purines, under conditions of low water activity due to the high alkali salt concentration. We also show that the CD spectrum obtained in presence of Cs+ ions and characterized by a negative band at 275 nm, cannot be interpreted in terms of Z-like left-handed helix but reflects a modified B right-handed helix.     

Spectroscopic studies of (m5dC-dG)3: thermal stability of B- and Z-forms.

The hexanucleoside pentaphosphate d(m5CpGpm5CpGpm5CpG) has been studied in solution by ultra-violet absorption, circular dichroism and 31P nuclear magnetic resonance under various experimental conditions. In 0.2 M NaClO4 at low temperature, an hexamer duplex is formed which has a B or B-like conformation. As the salt concentration is increased, a transition from a B-form to the Z-form occurs and is complete in 3 M NaClO4. In 3 M NaClO4, the behavior of the Z double helix is complex as a function of temperature. The variation of the circular dichroism at 295 nm is biphasic. A first transition occurs over a large range of temperature and corresponds to a conformational change due to a non-cooperative intramolecular process. Ultra-violet absorption and 31P nuclear magnetic resonance show that the new conformation arising from a distortion of the backbone is not similar to that observed in low salt conditions (B-form). At high hexanucleotide concentration, aggregates are formed. The second transition is cooperative and corresponds to the melting of a double stranded helix into single strands.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fluorescence study of the binding of poly(1,N6-ethenoadenylic acid) to Escherichia coli initiation factor 3

The binding of initiation Factor 3 (IF3) to poly (1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(epsilon A) due to the unstacking of the nucleotide bases. The poly(epsilon A) fluorescence enhancement titrates to an endpoint of 13 +/- 2 nucleotide residues per IF3. The maximum poly(epsilon A) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 mM NaCl concentration, the protein still binds to poly(epsilon A) at these salt concentrations as measured by sedimentation partition chromatography; the value of Kobs for the IF3-poly(epsilon A) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 mM NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(epsilon A) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(epsilon A) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(epsilon A) fluorescence.     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Aging Increases N epsilon -(Carboxymethyl)lysine and Caloric Restriction Decreases N epsilon -(Carboxyethyl)lysine and N epsilon -(Malondialdehyde)lysine in Rat Heart Mitochondrial Proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

15N-NMR spectroscopy. IV. Comparison of poly(L-lysine) and isopoly(L-lysine)

The natural abundance ¹⁵N-nmr spectroscopy has been used to characterize the isomeric polymers (L -Lys)_n and iso (L -Lys)_n in aqueous solution. Although the peptide nitrogens of the two polymers have nearly equivalent shifts at pH < 10, the amino nitrogens differ by 5–6 ppm at pH < 7 and provide an easy means of identification. Furthermore, the polymers are distinguishable by the pK_a of the amino group and the basicity of the peptide nitrogen. At pH 10.3 and 25°C, (Lys)_n exhibits line broadening and an upfield chemical shift of the peptide nitrogen, indicative of the coil → helix transition. The formation of 100% helix may produce a shift as large as 5 ppm, which probably makes ¹⁵N-nmr spectroscopy more suitable for studies of this transition.     

Effect of basic oligopeptides on the B-Z transition of poly(dG-dC). poly(dG-dC) in water-methanol solutions

The effect of basic oligopeptides (Lys-Ala-Ala)n (n = 1-5, 10) and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-dC).poly(dG-dC) in water-methanol solutions was investigated using CD and uv spectroscopy. In the absence of peptides, the concentration of methanol at the midpoint of the B-Z transition is 64% at 25 degrees C. The transition is temperature dependent and the B conformation is preferred at higher temperatures. All peptides tested shift the midpoint of the B-Z transition to lower concentrations of methanol. For shorter peptides this effect increases with an increasing number of monomeric units, showing the importance of the number of positive charges in the peptide molecule. Al conditions of low methanol content, the trimer and tetramer of the (Lys-Leu-Ala)n series have a greater effect on the B-Z transition than the corresponding oligomers of the (Lys-Ala-Ala)n series. This indicates an important influence of the presence of hydrophobic groups in the peptide side chains on the binding. In the presence of peptides, the B-Z transition is also temperature dependent and the B conformation is preferred at higher temperatures. The addition of peptides results in an increase of the transition midpoint and of the transition width. These parameters were used for the calculation of the transition enthalpy delta HB-Z in 65% methanol, which is -1.15 +/- 0.25 kcal/base pair. Since the van't Hoff enthalpy delta HVH calculated from the temperature dependence of the B-Z transition in the absence of peptides is -130 kcal/mol, the length of the cooperative unit is about 110 base pairs. The results suggest that the mechanism of Z-DNA induction is similar but not identical with that involved in the action of metal cations in aqueous solution.     

The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.