ALBERT

Notes: The template directed synthesis of poly[d(A-T)] from the nucleoside triphosphates in the presence of DNA polymerase I is carried out continuously in a stirred flow reactor for the first time. The initial objective is to test the kinetic stability of the established steady states at various flow rates. Graphical analysis predicts instable steady states for certain high flow rates. As a consequence of instabilities multiple steady states and steady-state hysteresis may occur. Steady-state hysteresis has now been found experimentally. For a different enzyme fraction of low exonuclease activity we found the steady-state absorbance at 260 nm to be almost invariant with flow rate at high enzyme concentrations even if the flow rate was increased by a large factor. We call this phenomenon kinetic buffering. Relaxation of a large flow perturbation approaches the steady state in a sigmoidal fashion. Concentration oscillations at 260 nm occurred in one experiment using an enzyme fraction of low exonuclease activity after perturbing the steady state by monomer (dATP). Advantages of the stirred flow reactor method over serial transfer are discussed.

Notes: The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1-24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.

Notes: An all-order classical coupled oscillator theory in which monomer band shapes are explicity taken into account is applied to the calculations of polynucleotide circular dichroism. Calculations are shown for ApA and oligoadenylic acid of varying chain lengths, in both RNA and B-DNA geometry, and the advantages of this theory are assessed. By introducing an effective dielectric constant, good agreement with measured spectra is obtained. Variations in monomer parameters are tested in an attempt to eliminate remaining discrepancies between calculated and measured spectra.

Notes: The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α-helices.

Notes: New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm-1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm-1. However, a low-frequency band at 25 cm-1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.

Notes: The adsorption of protein films on polystyrene latex spheres was studied by optical mixing spectroscopy. With this technique, we show that both the hydrodynamic thickness of protein films and their optical density can be measured. Thus, we found that films of the glycoproteins isolated from the human erythrocyte membrane were four times as thick as films of either human serum albumin or bovine serum albumin for about the same surface coverage. This result suggests an end-on orientation for the adsorbed glycoprotein molecules, which is consistent with the model proposed by others for the orientation of these molecules at the surface of the red blood cell itself.

Notes: Solvent binding to bovine serum albumin in 2-chloroethanol-water mixed solvents of different composition, measured previously by Inoue and Timasheff (Biopolymers (1972) 11, 737-43) is applied to a hydrodynamic study of the solvated protein.From sedimentation and diffusion data, the apparent molecular weight of the solvated protein particle can be calculated, provided an average partial specific volume, computed from the composition of the particle, is introduced in Svedberg's equation. The unsolvated molecular weight of the protein can than be calculated by subtraction of the bound solvent. Further data on the hydrodynamic particle (f/fmin and axial ratio of the equivalent ellipsoid) are readily calculated from these experiments, and reinforce the supposition that 2-chloroethanol is a strong helix-inducing solvent.

Notes: We have carried out a detailed study of ligand binding of ferrihemoglobins under various conditions. Our results show that n varies with time and that this variation is paralleled by changes in the spectrum of methemoglobin. This suggests some form of structural perturbation. The time-dependent value of n is discussed in terms of the observed spectral changes accompanying prolonged equilibration.

Notes: The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pKa values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pKa value of the imidazole ring in N-acetyl-L-histidine methylamide was assumed to represent the intrinsic (or unperturbed) pKa of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pKa values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO2- and NH3+ groups located at various positions. Some other factors affecting the pKa value of the imidazole ring are also discussed.

Notes: The hyperchromic and hypochromic changes in the intensity of the amide-I and amide-III lines of polypeptides and certain ring vibrations of the bases of polynucleotides are shown to be related to similar changes in the lower energy uv absorption bands. The selection rules strictly limit the pairs of excited electronic states that can contribute to the elements of the polarizability matrix. An energy-dependent term in this equation weights the contribution of the pairs of electronic transitions in favor of those involving the lower energy transitions. For both polypeptides and polynucleotides, there is a large hypochromic inensity change in the first π → π* exciton band upon the coil-to-helix transition. Through the selection rules, certain conformationally sensitive Raman lines are shown to derive their intensity predominantly from this band and hence also display hypochromism. Again, through an application of the selection rules, certain Raman lines can be demonstrated to depend predominantly for their intensity upon the n → π* transition, and consequently have the opposite hyperchromic intensity change upon the same conformational transition.

Notes: Transient electric birefringence experiments have been performed on well-characterized highly monodisperse samples of tobacco mosaic virus (TMV). Experiments and calculations are presented that show that the sensitivity and accuracy of the traditional transient electric birefringence technique can be enhanced by several orders of magnitude through the use of a low-powered laser and crystal polarizing optics, signal averaging, and digital data processing. Measurements of the field-free decay of the birefringence yield for the rotational diffusion coefficient, DR = 318 ± 3 sec-1. The length of the rodlike virion, calculated from the Broersma equation with this value of DR and a rod diameter of 15 nm, is 292.5 ± 1.0 nm. Measurements of the growth of the birefringence when an electric field is applied across the sample yield μ2/kT(α∥ - α⊥) = 0.47 ± 0.07, where μ is the permanent dipole moment (assumed parallel to the symmetry axis of the virus) and α∥ - α⊥ is the electrical polarizability anistropy of the virus. Using the value of α∥ - α⊥ measured by O'Konski et al., we then obtain μ = 2.5 × 10-14 esu cm.

Notes: The high-resolution nmr spectrum of baker's yeast tRNACUALeu, a recently sequenced non-denaturable tRNA, has been compared with the spectra of the native and denatured conformers of the closely related species tRNAUUGLeu. Because of the presence of many common base pairs in the different tRNA's, it is possible to assign most of the low-field resonances to specific secondary-structure base pairs. A comparison of the observed positions of the various resonances with those predicted by a semiempirical ring-current shift theory shows a root-mean-square deviation of 0.14, 0.11, and 0.12 ppm for tRNAUUGLeu (native), and tRNAUUGLeu (denatured), respectively. These results support the ring-current shift theory currently used to interpret the low-field nmr spectra of the tRNA molecules. Differences between the predicted and observed positions of some resonances provide new evidence for higher order effects such as shifts from second nearest neighbors, anomalous shifts exerted by G·U base pairs, and tertiary-structure effects. A model that was previously proposed for the denatured conformer of tRNAUUGLeu is also supported.

Notes: The molecular theory of the previous paper in this series is extended to determine the effect of divalent metal ions on helix stability relative to coil at fixed ionic strength and nucleotide phosphate concentration. Specification of the state of condensed counterions, as well as their concentration, is essential for the solution of this problem, and it is assumed that they translate freely within a thin cylindrical shell close to the polynucleotide. As a function of divalent counterion concentration mB2+ the relative stability of the helix is highly nonlinear. Although the overall trend is that the helix stability increases with addition of divalent metal ion, there is a narrow concentration range for which it decreases slightly. The behavior of the relative stability as a function of mB2+ is determined by the translational degrees of freedom of the counterions, both univalent and divalent, both condensed and uncondensed. Detailed comparison of the theory with data is not given here, but it is pointed out that the calculated values of the relative stability are consistent with the order of magnitude of the observed effect Mg2+ on the melting temperature.

Notes: X-ray diffraction studies have been made on the polytripeptide poly(L-prolyl-L-α-phenylglycyl-L-proline). Its structure has been found to be helical, with a poly(L-proline) II conformation, packed in an orthorhombic lattice, space group P21212, with a = 14.3 Å, b = 13.5 Å, and c = 9.4 Å.

Notes: The stability constant of the complex of tRNA with 50S subunits of ribosomes was compared in ordinary and heavy water. A considerable effect (about fourfold) was observed, showing the importance of hydrogen bonds in this interaction. In addition, the isotope effect of complementary polynucleotide interaction was measured for two examples. In the case of the binary complex of heptainosinic acid oligomers with poly(C) in the presence of 10-3 M MgCl2, the transfer from ordinary to heavy water gave an increase of the stability constant of about 5%. But in the case of a ternary complex of hexaadenylic acid with poly(U) under the same conditions, the stability constant in D2O increased threefold. The isotope effect depends strongly on magnesium ion concentration and is possibly due to some specific mechanism of magnesium ion complexing involving water molecules.

Notes: Monte-Carlo calculations of geometric and thermodynamic characteristics of the α-helix and the β-structure of polypeptides have been carried out. To describe a hydrogen bond both the Lippincott-Schroeder and Morse potentials were used. The internal rotation angles ϕ and ψ in the α-helix have been shown to fluctuate in the range of ±7°. The distribution functions on angles ϕ and ψ and on hydrogen bond lengths and angles in the α-helix have been computed and compared with those in myoglobin and lysozyme. Thermodynamic characteristics of the α-helix calculated in different approximations with the two forms of the hydrogen bond potentials have also been compared. The data obtained are close to the experimental values for polypeptides in neutral solution.Some geometric and thermodynamic characteristics of the regular parallel and antiparallel and irregular antiparallel β-structure have been found. In the β-structure the internal rotation angles vary within the interval ±15-20°. An increase in the cross and longitudinal dimensions of the β-structure only slightly influence both the geometric and thermodynamic characteristics.

Notes: The preparation of the co-oligopeptides of the series H-Gly-Phe-(Gly)n-Trp-Gly-OH (n = 0, 1, 2) and of other free peptides of glycine, L-tryptophan, and L-phenylalanine is reported. The syntheses have been carried out by conventional methods, using N-hydroxysuccinimide esters for the coupling steps. The ultraviolet absorption properties of the free peptides have been investigated in water. No hypo- or hyperchromicity was found for the aromatic chromophores, with the exception of H-Gly-Phe-Trp-OH, which shows a small but significant hypochromicity. The contribution of the peptide bond to the molar absorptivity in the far ultraviolet has been separated from that of the side chain plus the —COO- group by plotting the measured molar absorptivity ∊ of the farthest accessible uv maximum as a function of the number of peptide bonds (nA). The peptide bond contribution proved to be independent of nA in the range nA = 1-5, thus ruling out the onset of helical conformations in the longer chain peptides.

Notes: The properties of the electron distribution in amides, peptides, and carboxylic acids, obtained from ab-initio molecular orbital calculations using both minimal and extended basis sets have been studied. These properties are discussed in terms of some of the common assumptions made in empirical conformational calculations of biomolecules. In particular, population analyses of 15 compounds in these families were carried out with both the minimal and extended basis sets, and compared with results of CNDO/2 calculations. It is suggested that population analysis is a useful tool for recognizing patterns of charge distributions, and investigating the transferability of parameters of different functional groups. However, its use for providing partial charges for conformational analysis is a questionable procedure.A more detailed analysis of the charge distribution was carried out by calculating the spatial electron distribution in the four compounds, N-methylacetamide, acetic acid, diketopiperazine, and N-acetyl-N′-methylalanine. Both total electron-density maps and differencedensity maps are presented. The properties of the overall shape of the molecule and the atoms in the molecule, are discussed in terms of the former along with three-dimensional shape plots of the total density. The distortion accompanying molecular formation, resulting in such features as the lone pair orbital and “bonding deensities” is discussed in terms of the difference maps. Semiquantitative estimates of the bonding and orbital densities resulting from the integration of the densities are also presented. Finally, one of the novel features of the study is the presentation of three-dimensional surfaces of constant difference densities from which the shapes of the orbitals and bonding densities emerge.

Notes: The lack of the positive band at around 226 nm in the CD spectra of poly(prolyl-azetidine-2-carbonyl-proline) in trifluoroethanol and of poly(azetidine-2-carbonyl-prolyl-azetidine-2-carboxylic) acid in F3EtOH and water, the hyperchromism of the absorption maximum at about 202 nm, and the extremely small intensity of the Cβ-Pro, Cγ-Pro, and Cβ-Aze signals for the cis peptide bonds in the 13C nmr spectrum of poly(Pro-Aze-Pro) in F3EtOH indicate that both polyproline analogs exist as disordered chains in this solvent, the trans peptide group being maintained. The disordering of the chains is attributed to an increase in the accessible range of ψ due to the reduced dimensions of the square ring of L-azetidine-2-carboxylic acid residue relative to the pyrrolidine ring of proline and to strong interactions of the haloalcohol with the peptide groups of the chains.

Notes: Two methods for the characterization of protein molecular weights from their diffusion coefficients are discussed. These measurements can be made quickly and reliably at low concentrations using quasielastic light-scattering techniques. First, an empirical calibration of the diffusion coefficient at infinite dilution of denatured random coils against molecular weight is reported. The second method combines the measurement of D0 with the intrinsic viscosity [η]. This D0-[η] relationship proves to be very insensitive to polymers structure or solvent type. The data indicate that the ratio of the hydrodynamic radius measured by viscosity to the hydrodynamic radius measured by diffusion is about 15% smaller than that predicted by theoretical models. The nature of the molecular-weight average obtained for polydisperse systems is defined for a Schulz distribution. These hydrodynamic methods have also been used to demonstrate the presence of chain branching in the glycoprotein ovomucoid. In addition, a method is proposed by which the effective segment length and an excluded volume parameter for random coils may be evaluated for diffusion measurements.

Notes: The influence of one DNA region on the stability of an adjoining region (telestability) was examined. Melting curves of three block DNA's, d(C15A15)·d(T15G15), d(C20A15)·d(T15G20), and d(C20A10)·d(T10G20) were analyzed in terms of the nearest neighbor Ising model. Comparisons of predicted and experimental curves were made in 0.01 M and 0.1 M sodium ion solutions. The nearest neighbor formalism was also employed to analyze block DNA transition in the presence of actinomycin, a G·C specific molecule. The results show that nearest neighbor base-pair interaction cannot predict the melting curves of the block DNA's. Adjustments in theoretical parameters to account for phosphate repulsion assuming a B conformation throughout the DNA's do not alter this conclusion. Changes in the theoretical parameters, which provide good overall agreement, are consistent with a substantial stabilization of the A·T region nearest the G·C block. The melting temperature T A·T for the average A·T pari in d(C20A10)·d(T10G20), with 10 A·T pairs, appears to be 4°C greater than TA·T for d(C15A15)·d(T15G15) and d(C20A15)·d(T15G20), both with 15 A·T pairs. Actinomycin bound to the G·C end effectively stabilizes the A·T end by 9°C. These results indicate a long-range contribution to the interactions governing DNA stability. A possible mechanism for these interactions will be discussed.

Notes: Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L-glutamate) in dioxane have been carried out over a range of concentration 10-4-10-2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (≃ 10-3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration.Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L-glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.

Notes: Racemization in the synthesis of peptide intermediates and their polymers was investigated, using L-amino acid oxidase. The formation of N-hydroxysuccinimide esters from N-protected peptide acids yielded optically pure products in contrast to p-nitrophenyl and pentachlorophenyl active esters. The racemization in the polymerization step was found to be base sensitive. Partially racemized polymer can result from optically homogeneous monomer. Thus, the optical integrity of active monomer species carries no guarantee for that of the polymer.

Notes: Conformations containing all trans peptide bonds have previously been proposed for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S based on an evaluation of proton nuclear magnetic resonance parameters in a series of solvents. These gramicidin-S derivatives exhibit full biological activity despite the fact that the proposed solution conformations differ in backbone topology and relative orientation of the Phe and Orn side chains compared to gramicidin-S. The present authors discuss conformations for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S which incorporate cis peptide bonds at L-Orn-L-N-MeLeu, where the gramicidin-S backbone is essentially retained, and the relative orientation of the Pro, Orn, Val, and Phe side chains correspond to those observed for gramicidin-S. A novel hydrogen-bond arrangement involving one carbonyl group interacting with two peptide protons (1 ←4 and 1 ←5 types) is proposed to stabilize the backbone conformation in the gramicidin-S derivatives. A recent report on the cyclic heptapeptide antibiotic, Ilamycin B1, shows the presence of cis peptide bonds at N-CH3 amino acids, as well as the novel hydrogen-bond arrangement presented above.

Notes: The morphology of fibrin strongly depends on solvent medium, as shown by clotting experiments carried out in the presence of different salts. The clots were characterized by electron microscopy and spectrophotometric methods; the kinetics of gelation were determined.In the presence of electrolytes which strongly delay clotting, the strands are thin and few branching points are observed; opposite morphological changes are induced by salts which act as accelerating agents. On the basis of this correlation, and of previous data on the structure of fibrin, a kinetic model of the self-assembly process is outlined. It accounts well for the observed solvent effects on the morphology of the network.An important result emerging from our experiments is that the fibers undergo branching prior to gelation. Branching points arise from the defective growth of the fibers; a simple explanation of the occurrence of branching may be obtained by our self-assembly model.

Notes: The equilibrium between the cis and trans forms of X-Pro peptide bonds can readily be measured in the 13C nmr spectra. In the present paper we investigate how observation of this equilibrium could be used as an nmr probe for conformational studies of flexible polypeptide chains. The experiments include studies by 13C nmr of a series of linear oligopeptides containing different X-L-Pro peptide bonds, with X = Gly, L-Ala, L-Leu, L-Phe, D-Ala, D-Leu, and D-Phe. Overall the study confirms that X-Pro peptide bonds can generally be useful as 13C nmr probes reporting the formation of nonrandom conformation in flexible polypeptide chains. It was found that the cis-trans equilibrium of X-Pro is greatly affected by the side chain of X and the configuration of the α-carbon atom of X. On the basis of these observations some general rules are suggested for a practical applications of the X-Pro nmr probes in conformational studies of polypeptide chains.

Notes: Random copolymers of L-glutamic acid and ortho-nitrobenzyl-L-glutamate were synthesized with different percentages of nitrobenzyl-L-glutamate, from 3 to 22%. These copolymers were studied by circular dichroism (CD) and potentiometric titrations in aqueous solutions in order to determine the variation of the stability of the secondary structure when the concentration of nitrobenzylglutamate varies in the copolymer. We observed that the stability increases with the concentration of glutamate. Such a stabilization could be due to either side-chain interactions or interactions between the aromatic side chain with the backbone. An intermolecular aggregation was observed when the percentage of nitrobenzylglutamate was sufficently important, but such an aggregation may be avoided if a dioxane/water mixture is used as a solvent.

Notes: The kinetics of γ-benzyl-L-glutamate NCA's polymerizations in dioxane as solvent are discussed. The partial orders respective to [A], the NCA concentration and [I0], the initial initiator concentration are given; the rate constants are deduced and a mechanism is proposed to justify a rate of polymerization Vp = k[A][I0]2.The dependence of the rate constants on the conformation of the growing chains is demonstrated; the acceleration is attributed to the ordered structures favorable to hydrogen bonding. The kinetics of aging have also been examined and discussed; it is shown that they cannot modify the kinetics of polymerization.The DPn were obtained on the same samples before and after debenzylation; it is proved that at any concentration, DPn ≃ [A0]/[I0].The molecular-weight distributions were obtained by chromatography and a polydispersity lower than 1.3 was deduced for each sample. The trimodal distribution, appearing as soon as [A0]/[I0] is larger than 3, is attributed to the existence of the three structures σ, β, and α. The weight fraction of each of the structures was correlated to the kinetics of polymerization.

Notes: The crystal structure of native cellulose (Valonia) has been analyzed by electron diffraction. Possible models for the structure were refined by rigid-body least squares methods, which incorporated parameters defining the preferred orientation of the fibrils around their long axes in the cell wall lamellae. The structure was found to consist of an array of chains having the same sense (i.e., parallel), with packing parameters similar to those recently determined by X-ray diffraction. The eight-chain unit cell could be approximated adequately by a two-chain monoclinic unit cell with dimensions a = 8.18 Å, b = 7.84 Å, c = 10.38 Å (fiber axis), and γ = 97.04°; the space group is P21.

Notes: The coil-to-helix transition temperatures of hydrogen bearing and deuterated poly(γ-benzyl-L-glutamate) in 1,3-dichlorotetrafluoroacetone/H2O and/D2O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α-helical form of poly(γ-benzyl-L-glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ-benzyl-L-glutamate), while the polypeptide-active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β-benzyl-L-aspartate) reveals an anomaly with respect to the present analysis.

Notes: Transient electric birefringence measurements on poly(L-lysine hydrobromide) in methanol-water mixtures have been carried out at various solvent compositions in the vicinity of the helix-coil transition region (from 87 to 98 vol % methanol). Anomalous birefringence transients were observed between 90 and 95 vol % methanol above a threshold field strength. A distinct difference between the responses to weak and strong electric fields was noticed over a narrow range of the solvent composition. The effects of polymer concentration and temperature on the field-strength dependence of the birefringence were studied at a solvent composition of 90 vol % methanol where the anomalous transients appeared most clearly. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different concentrations and temperatures could be superimposed by shifting them horizontally along the abscissa. The threshold field strength which was determined from the shift factor decreased with decreasing concentration. The results provide further evidence that strong electric fields can cause a helix-coil transition in this system under favorable conditions.

Notes: Derivatives of tetrapeptide sequences considered likely to form β-turns were investigated by the study of their proton magnetic resonances in methanol and in water. Differential broadening of N - H resonances by an added nitroxyl was used to indicate the presence of the sequestered N - H proton expected in β-turn conformations. Transfer of magnetic saturation from solvent water protons to N - H protons was also examined. The evidence is consistent with significant contributions by β-turn-like backbones to the conformational averages in methanol of the sequences Gly-L-Pro-D-Val-Gly, D(or L)-Val-L-Pro-Gly-Gly, and Gly-L-Pro-L-Asn-Gly, but not the sequence Gly-D-Ala-L-Val-Gly. It is suggested that a Type I turn, Likely in Gly-L-Pro-L-Asn-Gly derivatives, is characterized by sequestered N - H protons of both the third and fourth residues. For all of the peptide derivatives, save possibly Ac-L-Val-L-Pro-Gly-Gly-NHNH2, contributions from folded structures in water are not detectable by line-broadening experiments. However, the transfer of saturation experiments may be interpreted as indicating some degree of chain folding in water.

Notes: In this paper we use probabilistic arguments to derive and discuss the spatial distribution of molecules which are undergoing electrophoresis or centrifugation while at the same time they are switching back and forth between two configurational states. An exact solution is obtained for arbitrary values of the diffusion coefficients of the two isomerizing states. The traditional analytic method of solving this problem, namely, by use of “master equations,” is not completely satisfactory because one can find only the Fourier transform of the solution rather than the solution itself. Our treatment yields the answer one would get if it were feasible to perform the inversion of the Fourier transform.

Notes: A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L-Ala-L-Leu-Gly)n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L-alanyl-L-leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L-alanyl-L-leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.

Notes: The thermal transitions of single-stranded polynucleotides are noncooperative. In contrast, Cu(II) cooperatively disorders the single-stranded helical structures of poly(A) and poly(C), as demonstrated by ORD and UV spectral changes as a function of the Cu2+ activity, and by a dramatic chain-length dependence of the spectral changes. Equilibrium dialysis binding studies indicate that the cooperative disordering is paralleled by a somewhat less cooperative binding process.The difference between the thermal- and Cu(II)-induced transition is explained by the following mechanism. (1) Cu(II) initially binds in a noncooperative fashion to phosphate. (2) The Cu(II) so bound forms a second bond to a nonadjacent base site on the same polymer strand or another strand. These intramolecular and intermolecular crosslinks to the bases are responsible for the disordering. (3) The initial crosslinks formed provide nuclei for the cooperative formation of additional crosslinks, producing cooperative spectral changes paralleled by cooperative binding.A comparison of the spectral and binding transitions indicates that there is appreciable noncooperative binding of copper to phosphate, which produces no spectral changes in the presence of added electrolyte. This comparison also indicates that each copper crosslink disorders several bases. The formation of intermolecular crosslinks is demonstrated by a polymer concentration dependence of the disordering. The formation of intramolecular crosslinks can be deduced from the fact that the “cooperative unit” required to explain the differences between the hexamer, which does not readily form intramolecular crosslinks, and the polymer is considerably larger than the cooperative unit determined from the polymer results.The poly(A) disordering transition is less symmetrical than that of poly(C), particularly at low polymer concentrations. These results, together with other phenomena, are explained by a greater flexibility of poly(A), which favors the formation of small intramolecular loops.

Notes: Potential energy calculations were employed to examine the effect of ribose 2′-O-methylation on the conformation of GpC. Minimum energy conformations and allowed conformational regions were calculated for 2′MeGpC and Gp2′MeC. The two lowest energy conformations of 2′MeGpC and Gp2′MeC are similar to those of GpC itself. The helical RNA conformation (sugar pucker-C(3′)-endo, ω′ and ω,g-g-, bases-anti) is the global minimum, and a helix-reversing conformation with ω′, ω in the vicinity of 20°, 80° is next in energy. However, subtle differences between the three molecules are noted. When the substitution is on the 5′ ribose (Gp2′MeC), the energy of the helical conformation is less than that of GpC, due to favorable interactions of the added methyl group. When the substitution is at the 3′ ribose (2′MeGpC) these stabilizing interactions are outweighed by steric restrictions, and the helical conformation is of higher energy than for GpC. Furthermore, the statistical weight of the 2′MeGpC g- g- helical region is substantially less than the corresponding weight for Gp2′MeC. In addition, 2′MeGpC′s methoxy group is conformationally restricted to a narrow range centered at 76°. This group has a broadly allowed region between 50 and 175° in Gp2′MeC. These differences occur because the appended methyl group in 2′MeGpC is located in the interior of the helix cylinder, as it would be in polynucleotide, while it hangs unimpeded in Gp2′MeC. These findings suggest that 2′-O-methylation has both stabilizing and destabilizing influences on the helical conformation of RNA. For 2′MeGpC the destabilizing steric hindrance imposed by the nature of the guanine base dominates.

Notes: The molecular conformations of the linear oligopeptides H-(L-Ala)n-L-Pro-OH, with n = 1,2 and 3, have been investigated. 13C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from 1H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.

Notes: A method is described for predicting and solving crystal structures of linear homopolysaccharides. The method is based on the refinement of the structure with respect to either stereochemical constraints or x-ray diffraction intensities. In the refinement process, all conformational and packing features of the molecule, such as bond lengths, bond angles, conformational angles, nonbonded contacts, hydrogen bonds, etc., can be allowed to vary until the structure reaches both a conformation and crystalline packing that are in minimum disagreement with the stereochemical restraints and the diffraction data. In this fashion, both packing and conformational features of the structure can be simultaneously refined, and not separately as has been the custom in the past. The refinement procedure is based on a method of constrained optimization which possesses improved characteristics of reaching a solution and avoiding false minima, in comparison with least squares methods. The procedure is, in addition, capable of easily finding molecules of solvent of crystallization. The method was applied to further refining the previously solved crystal structure of V-amylose. The results indicated that contrary to the previously found six-fold molecular symmetry in the P212121 space group, the V-amylose molecule exhibits only two-fold symmetry with the asymmetric unit consisting of three glucose residues in one-half turn of the helix. The three residues are nonequivalent principally due to unequal rotational positions of the hydroxymethyl groups. The crystal structure of V-amylose predicted from stereochemical refinement was identical in all details with that obtained from refining against X-ray data. The excellent agreement with the diffraction data was indicated by the crystallographic disagreement index R = 0.25.

Notes: In order to obtain information about the conformational features of a 2′-O-methylated polyribonucleotide at the nearest neighbor level, a detailed nuclear magnetic resonance study of AmpA was undertaken. AmpA was isolated from alkali hydrolysates of yeast RNA, and proton spectra were recorded at 100 MHz in the Fourier transform mode in D2O solutions, 0.01 M, pH 5.4 and 1.5 at 25°C. 31P spectra were recorded at 40.48 MHz. Complete, accurate sets of nmr parameters derived for each nucleotidyl unit by simulation iteration methods.The nmr data were translated into conformational parameters for all the bonds using procedures developed in earlier studies from these laboratories. It is shown that AmpA exists in aqueous solution with a flexible molecular framework, which shows preferences for certain orientations. The ribose rings exist as a 2E ⇄ 3E equilibrium with the  - pA ribose showing a bias for the 3E pucker. The C(4′) - C(5′) bonds of both nucleotidyl units show significant preference (75-80%) to exist in gg conformation. The dominant conformer (80%) about C(5′) - O(5′) of the 5′-nucleotidyl unit is g′g′. Even though an unambiguous determination of the orientation of the 3′-phosphate group cannot be made, tentative evidence shows that it preferentially occupies g+ domains [O(3′) - P trans to C(3′) - C(2′)] in which the H(3′)  - C(3′) - O(3′) - P(3′) dihedral angle is about 31°. There is reasonable evidence that the 2′-O-methyl preferentially occupies the domain in which the O(2′) - CH3 bond is trans to C(2′) - C(1′).Lowering of pH to 1.5, which results in protonation of both the adenine moieties, causes destacking of AmpA. Such destacking is accompanied by small, but real, perturbations in the conformations about most of the bonds in the backbone.A detailed comparison of the solution conformations of ApA and AmpA clearly shows that 2′-O-methylation strongly influences the conformational preference about the C(3′) - O(3′) bond of the 3′-nucleotidyl unit, in addition to inducing small changes in the overall ribophosphate backbone conformational equilibria. The effect of 2′-O-methylation is such that the C(3′) - O(3′) is forced to occupy preferentially the g+ domain rather than the normally preferred g- domain [O(3′) - P trans to C(3′) - C(4′)] in ApA. The data on ApA and AmpA further reveal that the extent of stacking interaction is less in AmpA compared to ApA.It is suggested that stacked species of AmpA exist as right-handed stacks where the magnitude of ω and ω′ about O(5′) - P and P - O(3′) is about 290°. The reason for the lesser degree of stacking in AmpA compared to ApA is intramolecular interaction between 2′-O-methyl and the flexible O(3′) - P - O(5′) bridge, the interaction causing some perturbation in the magnitudes of ω/ω′, causing destacking. The destacking will lead to an increase in χCN by a few degrees, causing an increase in 2E populations; the latter in turn will shift the 3′ phosphate group from g- to g+ domains. In short, a coupled series of conformational events is envisioned at the onset of destacking, made feasible by the interaction between the 2′-O-methyl group and the swivel O(3′) - P - O(5′) bridge.

Notes: The binding isotherms of sodium decyl sulfate to poly(L-ornithine), poly(D,L-ornithine), and poly(L-lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm-Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L-Lys)n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L-Orn)n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.

Notes: The synthesis of a cross-linked polypentapeptide (an insoluble product) by means of a cross-linking reaction between a lysine-containing (1%) polypentapeptide and a glutamic acid-containing (2%) polypentapeptide is described, following description of the synthesis and polymerization of the corresponding pentapeptide p-nitrophenyl esters. The pentapeptide p-nitrophenylesters - H-Val1-Pro2-Gly3-Val4-Gly5-ONp, H-Val1-Pro2-Gly3-Lys4(ε-Z)-Gly5-ONp, and H-Val1-Pro2-Gly3-Glu4-Gly5-ONp - prior to polymerizing and the polypentapeptides prior to cross-linking were analyzed by proton and carbon-13 magnetic resonance. The cross-linking reaction was achieved in the coacervate state by intermolecular primary amide bond formation. The coupling reagent was a water-soluble carbodiimide (1-cyclohexyl-3-(2-N-methylmorpholinoethyl)-carbodiimide-p-toluenesulfonate).

Notes: It is concluded on the basis of comparison of polyelectrolyte theory with published data that the mean phosphate spacing b along the contour axis of an unfolded polynucleotide single strand is in the range 3-4 Å (polyelectrolyte parameter ξ ≈ 2), regardless of temperature, base composition, or extent of stacking. This result is consistent with the low-angle X-ray scattering measurements of Gulik, Inoue, and Luzzati on poly(C). No conclusion may be drawn from this value of b concerning the structure of the chain skeleton or the spatial arrangement of the bases other than that the chain is far from an all-trans local conformation (for which b would be about 6-7 Å, the length of a nucleotide unit). The structural implications, or lack thereof, are discussed in detail in the following paper.

Notes: The technique of intensity correlation light-scattering spectroscopy has been used to accurately determine the extent of physical swelling of lysozyme, ribonuclease, and chymotrypsinogen produced by thermal denaturation. The change in hydrodynamic radius is deduced from direct measurements of the diffusion coefficient, carried out in the temperature range 20° to 70°C at various values of pH in the range 1.0 to 3.0 at ionic strengths of from 0.01 M to 0.2 M. An average radius increase of 18% is observed for lysozyme and ribonuclease, with an estimate of 26% for chymotrypsinogen. Analysis of the pH dependence of the transition temperature leads to the conclusion that the lysozyme charge increases by approximately +2e during unfolding. We have applied this value of the charge increase along with the 18% average radius increase to estimate the electrostatic contribution to the free-energy change for denaturation of lysozyme. The results are consistent with the experimental observation that the transition temperature is nearly independent of ionic strength.

Notes: The normal vibration frequencies of poly(L-alanine) and poly(L-alanylglycine) in the antiparallel chain-pleated sheet structure have been calculated, using the force field for polyglycine I from the previous paper (Biopolymers 15, 2439-2464) plus additional force constants for the methyl group. The agreement with observed ir and Raman bands is very good. This substantiates the excellent transferability of the force field, since polyglycine I was shown to have a rippled-sheet structure. The amide I and amide II mode splittings are very well accounted for by transition dipole coupling, showing that subtle structural differences are sensitively manifested through this mechanism.

Notes: Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D-glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the  - CH2OH group. The positions of the two vicinal cis  - OH groups are determined predominantly by interactions with either the  - CH2OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D-lactic acid side chain, the O - C - C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups.The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C=O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule.The preferred conformations of the disaccharides NAG-NAG, NAM-NAG and NAG-NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C3 of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH2OH (donor) of residue i + 1 and the C=O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.

Notes: A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å.The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect.The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3-5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M-1, respectively.The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties.The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.

Notes: Raman spectra of native and solubilized feather keratin have been obtained, and the amide I and amide III regions have been analyzed by band resolution techniques. The amide I region of the native form indicates that at least 64% of the protein has an antiparallel chain pleated sheet structure, the remainder being unordered. For the solubilized keratin all of the protein is in an unordered state. The amide III region is not as easily analyzed into component contributions. Normal vibration analyses on N-acetyl-L-alanine-N-methylamide support the conclusion that the amide III region is not as satisfactory as the amide I region in characterizing unordered structures. Even in the latter case caution must be used, since the observed amide I band is an average over the conformational distribution in the particular unordered system.

Notes: The optical birefringence induced in DNA solutions by both oscillating hydrodynamic fields (flow birefringence) and oscillating electric fields (Kerr effect) is measured over a wide frequency range. The observed frequency response of the birefrigence is compared with theories for rigid ellipsoidal particles and for Gaussian chains. DNA at 6 × 105 molecular weight is found to exhibit rigid particle hydrodynamic behavior, while DNA at 5 × 106 molecular weight behaves like a flexible chain. Characterization of the hydrodynamic relaxation spectra for the DNA's by oscillatory flow birefringence allows precise comparison between theory and the experimental Kerr effect response. The dielectric model for DNA contains both permanent and dispersionless induced dipole moments. The dielectric behavior of DNA has the character of a permanent dipole but with anomalous low-frequency dispersion in the Kerr effect. The existing theories do not adequately describe this dispersion. A fluctuation dipole mechanism with relaxation times comparable to those associated with the hydrodynamic motion could possibly demonstrate the observed polar behavior.

Notes: Conformational energies have been estimated for the tripeptide fragments L-Ala-N-methyl-L-Ala-L-Ala, L-Ala-L-Ala-N-methyl-L-Ala, L-Ala-Sar-L-Ala, and L-Ala-Gly-N-methyl-L-Ala. The peptide bonds connecting L-Ala and Gly with N-methyl-L-Ala and L-Ala with Sar were permitted to adopt the planar cis as well as the usual trans conformation. Contour maps of the conformational energies of the central residue in these tripeptide fragments are presented and compared to the conformational energy maps previously calculated for unmethylated L-Ala and Gly surrounded by residues which are also unmethylated. In generl it is observed that L-Ala and Gly residues that are either N-methylated in their conformational freedom relative to the same residues in an unmethylated polypeptide chain.

Notes: Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L-glutamic acid) mixed at two molar ratios of carboxyl group-to-dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine-orange dimers bound to the α-helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.

Notes: Sequential determination of glycyl residues (and in several cases different amino-acid residues) in tetra and branched peptides using the nmr technique is reported. The method is based on changes in the nmr spectra of (1) the peptide hydrogens of the different residues and (2) the methylene groups of the glycyl residues, as a result of increasing the rate of the base-catalyzed exchange reaction of the peptide hydrogens. Hence, the spectral changes are pH dependent. However, the exact pH dependence is a function of the location of the residue in the peptide molecule. Thus, it is possible to determine the sequence of the amino-acid residues by studying the changes in the spectra with pH.For peptide molecules of known sequences, the above method can be used for unequivocal assignment of the peptide hydrogen signals.

Notes: The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na+ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.

Notes: A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.

Notes: Connectors for CPK space-filling molecular models have been supplemented with pointed steel wires (φ = 1.59 mm) of appropriate lengths and positions to represent atomic bonds. For illustration purposes, the polypeptide backbone of glucagon was equipped with such wire-core connectors and given a possible conformation. The molecular model was packed into a cubic box and, using a clinical instrument, X-ray films were taken from three orthogonal directions. Atomic coordinates on the films were then corrected to real-space coordinates.This method for retrieving atomic coordinates from space-filling models of modium-sized polypeptides is easy and can be done wih equipement available everywhere. Coordinates obtained in this way should be useful as starting points for energy minimization, for the calculation of frictional coefficients, and in attempts to solve crystal structures by X-ray diffraction, using molecular replacement methodology.

Notes: Single-stranded polynucleotides are used as model systems for the investigation of conformational changes induced by electric fields. It is demonstrated that the single-strand helix-coil transition in poly(A), poly(dA), and poly(C) can be induced by application of high electric fields. The transition is measured by UV absorbance using polarized light at an angle of 54.8° with respect to the vector of the electric field and by electrodichroism. A linear increase of the absorbance, reflecting the helix-to-coil transition, is observed at increasing field strength. When ions are added to the polymer, electric fields do not induce conformation changes, unless a threshold value of the electric field strength E0 is exceeded. At field strengths above this threshold, the degree of transition is a linear function of the increase in field strength. The threshold values E0 show a linear increase with the logarithm of the ion concentration. Bivalent ions cause thresholds at much lower ion concentrations than mo-novalent ions. The shielding efficiency of ions is correlated to the binding affinity of these ions to the polymer.The conformation changes induced by the field and the existence of thresholds can be explained on the basis of dissociation field effects. Similar threshold effects may be expected for other macromolecules as well as for membrane structures and may be important in the regulation of bioelectricity.

Notes: Circular dichroism (CD) spectroscopy has been used to investigate the effects of changes in salt concentration and pH on the interactions between basic polypeptides and connective tissue glycosaminoglycans in dilute aqueous solution. The polypeptides undergo conformation-directing interactions in the presence of glycosaminoglycans, which are subject to transitions as the ionic strength and pH are varied. For poly(L-lysine), the conformational change due to interaction breaks down as the ionic strength (monovalent ions) is increased. Based on the ionic strength at which disruption occurs, the glycosaminoglycans can be placed in order of increasing strength of interaction: chondroitin 6-sulfate, hyaluronic acid, chondroitin 4-sulfate, heparin, and dermatan sulfate. Prior to the conformational transition, scattering effects are observed, indicating the development of larger aggregates. Each glycosaminoglycan induces α-helicity for poly(L-arginine), which does not break down as the ionic strength is increased, indicating a stronger interaction for this polypeptide. The pH-induced transitions are in the pH range 2.5-3.8 and are probably related to deionization of carboxyl groups. For poly(L-lysine) the conformational effect is disrupted at low pH. For poly(L-arginine), the transitions are not complete, but appear to correspond to an increase in scattering.

Notes: Poly(Lys(HBr)-Gly-Pro-Pro-Gly-Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε-(p-nitrobenzyloxycarbonyl)-Lys-Gly-Pro-Pro-Gly-Pro-p-nitrophenyl-ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N-dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (Mr = 5-17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side-chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation.A high molecular weight fraction (3 × Mr = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.

Notes: Helix-coil transition of poly(γ-methyl-L-glutamate), poly(γ-ethyl-L-glutamate), and poly(γ-benzyl-L-glutamate) has been studied in mixed solvents by calorimetry, polarimetry, and viscometry. The experimental data have allowed the evaluation of solvation enthalpy Δhb, equilibrium constant K for hydrogen bond formation between the active solvent component and CO and NH groups, and the cooperativity parameter σ. The conformational transition of polypeptides in solution in a mixed solvent containing enough active solvent to maintain the coiled conformation has been produced by dilution with the helix-supporting solvent for the measurements of enthalpy of transition Δhs. The average value for Δhs is 3550 ± 300 J/mol and is practically independent of the nature of the side chain for the dichloroacetic acid-ethylene dichloride solvent pair at 25°C. A noticeable concentration effect exists in the case of poly(γ-benzyl-L-glutamate). The helical conformation is less stable for poly(γ-ethyl-L-glutamate), and this is explained by a steric effect hindering the access of dichloroacetic acid to side chains. Constant K has been calculated using polarimetric data and also from values of Δhs obtained at different temperatures using the Bixon and Lifson theory on the one hand and that of Sayama and coworkers on the other hand. Values of σ for poly(γ-ethyl-L-glutamate) have been calculated according to both theories mentioned, and the results show that the two sets of values are quite similar. The constant σ depends on the nature of the active solvent, on temperature, and on the binary-solvent composition. These conclusions are confirmed by viscometric results. Values of Δhb calculated from constant K are 5230 J/mol when Bixon and Lifson theory is used and 5569 J/mol when the theory at Sayama and coworkers is used. In both cases the value for Δhb is much lower than that of an intramolecular hydrogen bond. Experimental results suggest that the solvation mechanism would proceed in a manner so that mechanisms described in both theories are involved.

Notes: A fluctuation theory for electrolyte solutions is developed based on the coupling between the equations of nonequilibrium thermodynamics and the Poisson equation. The resulting fluctuation theory is applied to the analysis of electrophoretic light scattering. It is shown that in a binary electrolyte solution (two ionic species), the Doppler shift is not determined by the electrical mobility of either ion, but depends instead on the rate of change of transference number with salt concentration. In addition the ionic relaxation time is shown to be proportional to the conductivity of the solution.

Notes: We have measured the binding equilibria of proflavin to T2L bacteriophage, in both “slow” and “fast” sedimenting forms, and to free T2L DNA. Measurements were carried out by difference spectroscopy at 430 nm at temperatures from 13 to 43°C and at pH 5.6 and 7.6. We found no significant difference in the binding parameters of the two phage forms. Also, the fraction of nucleotides available as binding sites for proflavin was the same for both free and intraphage DNA. However, the binding constant is about an order of magnitude lower for encapsulated than for free T2L DNA, due to the decreased exothermicity of the binding reaction within the phage head.

Notes: Using the formalism of nearest-neighbor Ising model and assuming that the allowed states for a monomeric unity of a polypeptide chain in solutions containing strong acids are E (helix), C (coil), and CS (solvent-bonded coil), the partition function of the system was deduced analytically. Equations were obtained which permitted the prediction of the characteristic thermodynamic behavior of the helix-coil transition under these conditions. These equations were used to examine critically the possible correlations between experimental data obtained using different techniques. Particular attention was devoted to quantities called “transition enthalpies,” obtained from the slope of the transition curves at the point where the helix fraction is one-half (ΔHoptexp), or for measurements of the heat of solution of the polymer over the total range of solvent composition (ΔHsolexp), or from heat capacity measurements taken at various temperatures (ΔHcalexp). Literature data of ΔHjexp(j = opt, sol, cal) for the system poly-γ-benzyl-L-glutamate in mixtures of dichloroacetic acid and 1,2-dichloroethane were carefully analyzed.

Notes: Interaction of toluidine blue with helical and randomly coiled poly(α,L-glutamic acid) was studied with absorption spectra, titration, and electric-field pulse measurements. The obtained values of various parameters for the helical form of poly(α,L-glutamic acid)differed from those for its coiled form. The difference of parameters in these two forms of poly(α,L-glutamic acid) was attributed to differences of the binding mechanism in both forms. Furthermore, the binding of toluidine blue to poly(α,L-glutamic acid) was considered to be due to hydrogen binding in the helical conformation and ionic interaction in the coiled conformation of the polymer.

Notes: The basic theoretical groundwork for the use of derivative binding isotherms in the analysis of ligand binding is presented. The derivative binding isotherm is defined as Γ (Y) = df/dy where f = fractional degree of saturation and y = natural logarithm of the free ligand concentration. Since Γ (y) is a positive function, which goes to zero as y → ±∞, the mean value of y, 〈y〉, and the second and third moments, μ2 and μ3 about 〈y〉 are well defined.For a macromolecular system consisting of N equivalent and independent binding sites, Γ (y) is a symmetrical bell-shaped function with one maximum. The maximum occurs when y = -ln Kassoc; μ2 = π2/3, and μ3 = 0. For multiple sets of independent binding sites, Γ (y) is a superposition of Γ-type functions. If the sets are sufficiently well separated in binding free energy, multiple extrema may be seen at positions corresponding to the logarithms of the dissociation constants for the individual sets. In any case, 〈y〉 is equal to the mean value of the logarithms of the dissociation constants for the sets; μ2 〉 π2/3 and equal to π2/3 plus the variance of the logarithms of the dissociation constants about their mean value; and μ3 is, except by coincidence, not equal to zero and equals the third moment of the distribution of logarithms of the dissociation constants about their mean value.Analysis of Γ(y) for the case of cooperative interactions within a set of binding sites was investigated by examining (1) the Hill model (whose mathematical representation is equivalent to that used to describe antibody heterogeneity except that in the latter case the parameter a, the Sips, constant, is constrained (0 〈 a ≤1);(2) a common model for cooperativity in which the cooperative free energy is a linear function of the fraction bound; and (3) a general representation of cooperative interactions within a set of sites in terms of φ(f), a smooth function that gives the interaction free energy in units of RT.For the Hill model (or Sips model) Γ(y) is a symmetrical function with one maximum at y = (-1)/a)lnK, μ2 = π2/3a2; and μ3 = 0. For the case in which the cooperative free energy is a linear function of f [φ(f) = cf], 〈y〉 = -ln K0 + (c/2); μ2 = (π2/3) + c[(c/12) + 1] where c 〉 -4; and μ3 = 0. General expressions for the moments in terms of φ(f) are derived. In general, μ2 〈 (π2/3) for positive cooperativity and μ2 〉 (π2/3) for negative for negative cooperativity. Γ(y) will be symmetrical if and only if the cooperative free energy is introduced symmetrically about f = 0.5.

Notes: Protamine·DNA complexes in film and in solution have been investigated by means of infrared linear dichroism, ultraviolet circular dichroism, and laser Raman spectroscopy. At high relative humidity and in solution both infrared linear dichroism and ultraviolet circular dichroism indicate the presence of a modified B form of DNA (designated as B* in our other papers27,28,46). This modified B form is characterized by a change of the orientation of the \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{OPO} $\end{document} bisector angle by about 4° with respect to the helical axis when compared to the B form of DNA. At decreasing relative humidities the same modified B form is maintained and no structural transitions B → A (or B → C) normally occurring in free DNA were observed. The absence of the A form in these complexes was also confirmed by laser Raman scattering studies of protamine·DNA complexes. On the basis of these results, a model of the protamine·DNA complex is proposed, which suggests that the presence of apolar amino-acid residues, and probably the folding of the polypeptide chain, is responsible for preventing the B-to-A transition; this occurs either by protecting the high-humidity modified B form against dehydration or by steric interference of this protein probably located in one of the DNA grooves.

Notes: Proton and phosphorus-31 nuclear spin-lattice relaxation times T1 and spin-spin relaxation times T2 have been measured on the single-stranded polyriboadenylic acid [poly(A)]-Mn2+ system in a neutral D2O solution in the temperature range 10°-90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T1 have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn2+ to H2, H8, H1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn2+ penetrates into the phosphorus nucleus, giving 31P hyperfine coupling of more than 106 Hz. Evidence of penetration of the electron spin into H8 and H2 is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn2+ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn2+ ion added is bound directly to two phosphate groups with a Mn2+-phosphorus distance of 3.3 Å, while a part of the Mn2+ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn2+ are coordinated by N7 and N3 (or N1), respectively. The motional freedom of poly(A) in the environment of the Mn2+ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn2+-free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn2+-free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H8, H2, H1′, and phosphorus, respectively. T2 of phosphorus is determined by the dissociation rate (k-1) of Mn2+ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn2+ from the adenine ring are also estimated from proton T2 values below 50°C.

Notes: A range of linear charge densities of the ordered and disordered forms of DNA or polynucleotides can be obtained experimentally by acid or alkaline titration, or by the investigation of unusual complexes involving protonated bases or three-stranded helices. The variation of melting temperatures with Na+ concentration for various of these systems is known and in some cases is complemented by structural and thermodynamic information. We have extended the condensation-screening theory of Manning [Biopolymers, 11, 937-955 (1972)] to these systems. The stabilizing and destabilizing effects of Na+ (condensation and screening, respectively) and be independently varied, and the theory is successful in predicting the qualitative (in some cases, quanittative) behaviour that is observed. Comparison of theory and experiment indicates that the axial phosphate distance b for single-stranded polynucleotides increases with increasing pH. Values of the critical parameter ξ are obtained for the various polynucleotide structures. These values are essential for an understanding of ionic effects on charged ligand-polynucleotide interactions.

Notes: A systematic study of the conformational states of the dinucleotide diadenosine 5′,5′-pyrophosphate (AppA), an analog of the coenzyme NAD+, has been made using semi-empirical energy calculations. Taking low-energy mononucleotide structures as starting conformations, energy minimizations have been performed. The most stable structures exhibit stacking interactions between the adenine bases; there are many different stacked states of similar energy; their stability is derived from nonbonded interactions primarily between the bases but also from base-sugar interactions. The most common form of stacking in the most stable structures was found to be antiparallel A-A helix. These findings are consistent with the experimental data, which suggest that AppA adopts predominantly a stacked state in solution, and this state incorporates a variety of stacked conformations.

Notes: Moment index displacement automatically corrects a number of significant nonrandom instrumental errors in fluorescence time-decay measurements. Three-component data, obtained by measuring the fluorescence decay of three different species mixed in the same solution, were used as a test sample. It was shown, as predicted by theory, that moment index displacement corrects three nonrandom instrumental errors: (1) the presence of scatter in the data; (2) time origin shifts between lamp and fluorescence data; and (3) lamp drift, or time-dependent changes in the shape of the excitation curve. The data clearly show that the use of the method of moments with moment index displacement to analyze fluorescence decay data is not a curve-fitting procedure. This procedure will accurately obtain decay parameters for multiple-exponential decays from certain badly distorted data, yielding a calculated curve very different from the actual data.

Notes: The relaxation behaviour of the sodium salt of poly (L-glutamic acid) in the solid state has been examined by means of dynamic mechanical spectroscopy. Bound water was found to exert a profound influence on the relaxation behaviour and on a bulk property, the rigidity. Certain features of the loss spectrum have been identified with the hydration-dependent β-to-α conformational transition. Thus two side-chain relaxations are observed below ambient temperature, one associated with the β from (β1β) and a second at a lower temperature associated with the α form (β1α). The greater rigidity of the α form below the relaxation temperature and the larger rigidity drop accompanying the β1α can be explained in terms of the structural differences of the two conformations.

Notes: It has been observed that the amount of water associated with casein micelles is markedly higher when measured by intrinsic viscosity than by water content of the pelleted casein. A possible explanation of this discrepancy is that water is squeezed out of the pellet by the ultracentrifugal field. We have calculated the potential magnitude of this effect by considering a simple model system: an elastic gel swollen by solvent and compressed by the centrifugal field. Equilibrium is reached when the sum of the ultracentrifugal, elastic, and mixing free energies is a minimum. The equilibrium degree of swelling is calculated as a function of rotor speed, thickness of the unsolvated gel material, and enthalpy of mixing of solvent and solute. Sizable compressions can occur for highly swollen gels, if the enthalpy of mixing is moderately positive. Casein micelles from intrinsic viscosity measurements have a “hydration” of about 3.7 g water/g protein, corresponding to a swelling ratio of 6.3 relative to the dry protein. The observed pellet hydration is 1.9 g/g. Under the conditions of the experiment, pelleting at 25,000 rpm is predicted to decrease the swelling ratio to 4.9, and the hydration to 2.7 g/g, about half the observed decrease, if the enthalpy of mixing is 0.5 RT/mol segment. These calculations may be relevent to the deterination by pelleting of the solvent content of other biopolymer gels.

Notes: By using the static correlations of fluctuations in the dihedral angles of the α-helices of polyglycine and poly(L-alanine) calculated previously, geometrical fluctuations of a section (consisting of up to 18 peptide units) of the α-helices of infinite length are calculated. These fluctuations are found to differ in some respects (e.g., the dependence of amplitudes on the length of section) from those of a circular rod made of homogeneous continuous material. However, the moduli of the mechanical strengths (tensile Young's modulus, bending Young's modulus, and the shear modulus) of a circular rod are calculated, whose geometrical fluctuations are approximately equal to the fluctuations of a section consisting of 18 peptide units. They are of the order of 1011 dyn/cm2. The tensile rigidity, flexural rigidity, and torsional rigidity are calculated to be 1.20 × 10-3 dyn, 2.46 × 10-19 dyn·cm2 and 1.79 × 10-19 dyn·cm2 for polyglycine, and 1.96 × 10-3 dyn, 4.05 × 10-19 dyn·cm2 and 3.28 × 10-19 dyn·cm2 for poly(L-alanine), respectively.

Notes: The binding process between sodium poly(L-glutamate) and trans-2,2′,2″,2‴-tetrapyridyl-Fe(III) complex ions in aqueous solution at pH around 7 has been studied by means of equilibrium dialysis and optical measurements. The binding isotherm indicates the occurrence of a cooperative process, whereby bound molecules facilitate the association of additional molecules. According to circular dichroism (CD) data, this effect is coupled with that which sees a conformational change in the charged polypeptide upon progessive binding of complex counterions. All these features are discussed in the light of the structural characteristics of the interacting species. A stereochemical model of the association “complex” is proposed.

Notes: The conformational properties of ferric complexes of poly(Nε-acetoacetyl-L-lysine), poly(Nδ-acetoacetyl-L-ornithine), and poly(Nγ-acetoacetyl-L-diaminobutyric acid) were investigated in 1:1 water/dioxane by CD techniques. Optical activity was found in the visible and in the uv absorption region of the polymeric complexes. The conformation of the peptide backbone was always that of a right-handed α-helix, and was found independent of the degree of complexation, at least up to a degree of binding of 20%. In the absorption region of the side-chain chromophores the optical activity is substantially affected by complex formation. In all three cases a splitting of the ligand π → π* transition centered at 257 nm is observed. These data suggest a stereospecific complex formation. From the signs of the splitting it also appears that the chirality of the poly(Nδ-acetoacetyl-L-ornithine) complex is opposite that of the other two polymers.