ALBERT

Notes: The two-state theory of protein denaturation, in which it is assumed that a protein exists as two species, native and denatured, is examined by three approaches in this paper. First, the point is made that denaturation of an isolated molecule involves a continuous shift in the probabilities of occurrence of many states generated by the partition function for the protein. It is argued that the maximum term approximation does not lead to a two-state mechanism of denaturation, and that the extent of cooperation implied by the two-state theory should give very much sharper transitions than are actually found in proteins. Second, the two-state theory is applied to the various model systems treated in this series of papers, and is found to be inadequate. Since the detailed behaviour of the models is known, it is possible to deduce the effect of the incorrect application of the two-state theory to a system that “denatures” by gradual unwinding. This exercise is useful when, thirdly, we examine experimental data that seem to depart radically from a two-state interpretation. We conclude that a mechanism of gradual unwinding is the most generally valid assumption, and that the two-state theory has no theoretical or experimental support.

Notes: Three types of band-forming centerpieces useful in band centrifugation in the analytical ultracentrifuge are described. The mode of filling and the advantages and disadvantages of each type are discussed.

Notes: The ability of periodate-oxidized amylose to form aggregates in aqueous solution was studied by sedimentation, light scattering, and viscosity analyses. Ultracentrifuge schlieren patterns show that aggregation can be appreciable at pH 3.0 and 1.0. The hydroxyl ion-catalyzed degradation of the oxidized amylose is faster at pH 3.0 than at pH 1.0. Viscosity and sedimentation analyses conducted at pH 3.0 show that a minimum in the degree of aggregation of the oxidized molecules is obtained at 15-25% oxidation. Solubulity studies and x-ray diffraction patterns on retrograded amylose show that maximum solubility of the retrograded amylose is obtained by oxidizing to the extent of 25-35%. It was therefore concluded that in the general range of 20-30% periodate oxidation, the oxidized amylose has a minimum ability to form intermolecular hydrogen bonds. Outside of this range, oxidized amylose readily forms aggregates in aqueous solutions.

Notes: Calorimetric measurements of the heat of the addition of the second strand of poly U to poly (A + 2U) to form the three-stranded poly (A + 2U) complex in 0.1M and 0.5M NaCl at 24 and 37°C. are reported. A value of ΔH = -3800 cal./mole of poly (A + 2U) formed was found to be fairly insensitive to the experimental conditions employed. The heat of the addition of the third strand to the preformed poly (A + U) helix is considerably less exothermic than the heat of reaction between poly A and poly U to form poly (A + U). The insensitivity of the heat of addition of the third strand (poly U) to changes in salt concentration and temperature lends qualitative support to the earlier hypothesis that the major portion of the variation of the ΔH of the poly A and poly U reaction with experimental conditions arises from differences in the conformation of poly A. Combining the information obtained in this study for the ΔH of the reaction forming poly (A + 2U) with data for the ΔH of the formation of poly (A + U) indicates that the conversion of poly (A + U) to poly (A + 2U) is opposed by an enthalpy change which increases with temperature. Extrapolation of these values to 52°C. where poly (A + U) is transformed to poly (A + 2U) in 0.5M NaCl leads to a value of ΔH = +3800 cal./mole (A + 2U) formed and ΔS = 11.5 cal./mole of (A + 2U) formed. It is concluded from the calorimetric data that the driving force for the poly (A + U) to poly (A + 2U) conversion reaction is the favorable entropy change.

Notes: The effect of salt type and concentration on the transformation of an oriented crystalline collagen tendon into a crosslinked network under conditions of equilibrium swelling was investigated. Our main observations are the following. The degree of swelling of crystalline tendons increases at low salt concentration Cs, and decreases at higher Cs for a wide variety of salts. The observation is not reconcilable with swelling taking place in interfibrillar spaces or structural voids. Within the tropocollagen units and at their ends, regions of reduced organization are postulated (as suggested by Bear and by Schmitt) which are able to interact with the diluent in the amorphous-like manner. At least four different factors should be considered in assessing the role of salt and salt concentration on the shrinkage temperature Ts under isoelectric conditions. They are: (1) specific effects, (2) diluent effects, (3) crosslinking effects, and (4) nonequilibrium effects. The diluent effects are correlated with the salting-in-salting-out power of the ions which was characterized in Part I of this series. Smaller amounts of diluents are generally available to the tendon when the salt has a higher salting-out power, and this corresponds to higher shrinkage temperatures, other conditions being the same. The crosslinking effect raises Ts due to a reduction of the diluent content and, probably for p-benzoquinone and formaldehyde, also to a reduction of the conformational entropy in the molten state. Nonequilibrium effects arise from the fact that shrinkage and recrystallization are kinetically hindered when the tendon is highly deswollen in strong salting-out solutions, or when the salt has a crosslinking power. The specific effect is the only effect which is not related to the amount of diluent present in the tendon. Its origin is less clear. For anions such as Cl- and SCN-, it is possibly related to an ability of the ion to prevent intersegmental hydrogen bonding and water carbonyl bridges. The competition of several of the above effects for a given salt solution makes possible various types of dependence of Ts upon Cs: Ts may either continuously decrease or continuously increase with increasing Cs, or it may go through a minimum. In absence of salt, the cooperative character of the transition at the pH at which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continuous decrease of Ts on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and Ts is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of Ts upon Cs is more complex than under isoelectric conditions. There is generally an increase of Ts with Cs which is equivalent to an increase of the denaturation temperature with Cs for helical polyelectrolytes in solution. At higher salt concentrations, however, Ts may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.

Notes: By using the Nemethy-Scheraga theory of water structure, a calculation was made of the energy changes for the rupture of a DH…A solute-solute hydrogen bond in water. A partition function was also obtained for the binding of water and other solutes to the DH and A groups in the special case where these are the NH and CO groups of a random coil polypeptide chain. In subsequent papers of this series, these calculated quantities will be used in a statistical mechanical treatment of the helix-coil transition for polypeptides in water.

Notes: A partition function is derived for a simple model of interacting helices in a short (20 residues) chain of poly-L-alanine. It is found that interhelical hydrophobic bonds effect a marked stabilization of helical forms, and give rise to a sharp transition of the type found in many proteins.

Notes: Crosslinked synthetic polypeptides of poly Glu51Lys33Tyr16 (mol. wt. 31,000) and of poly Glu52Lys33Tyr15 (No. 3) (mol. wt. 52,000) containing from one to six crosslinks per molecule have been prepared by use of FFDNB, WRK and ICD reagents.The monomeric fractions of these derivatives were isolated by Sephadex G-100 chromatography. The number of crosslinks per molecule of DNPene derivatives was determined by total hydrolysis of the derivatives, isolation of O, N∊-DNPene-Tyr-Lys bridges by paper chromatography and then spectrophotometric quantitation. The number of the amide-type crosslinks in Am derivatives was established by their deamination followed by total hydrolysis and quantitation of the remaining lysine residues. Crosslinked derivatives appear to have a more compact structure, as judged by their behavior on the Sephadex columns and by their intrinsic viscosities. They were further characterized in regard to their amino acid composition, average number of the crosslinks per molecule, nitrogen content, solubility, root-mean-square end-to-end distance, and their spectral properties. Their properties recommend them as useful models for the study of the tertiary structure of proteins in solution.

Notes: The introduction of intramolecular amide bonds into synthetic polypeptides produces molecules with organized spatial structure which are good models for the tertiary structure of proteins. Polarization of fluorescence measurements were used to study the internal structure and the overall rigidity of the intramolecularly crosslinked polypeptides. The graph of [(1/p) + (1/3)] against T/η changes from a straight line to a continuous curve: the temperature at which this change occurs and the slope of the straight line segment measure the stability of the internal structure of the molecule. The introduction of one to six crosslinks produces an organized internal structure that becomes more stable as the number of crosslinks increases. In contrast to the fluorescence measurements, the intrinsic viscosities, reflecting the overall hydrodynamic domain of the molecules, change (decrease) to the same extent whether one, four, or six intramolecular crosslinks are present. The overall rigidity of the polymers can be assessed by the rotational relaxation time ρh and the polarization at 10°C. p10. Both of these criteria show that the presence of six crosslinks significantly increases the rigidity, but one or four does not. The various hydrodynamic measurements may be fitted into a hierarchy of discrimination: intrinsic viscosity, sedimentation, and diffusion for size and shape; rotational relaxation time and polarization of fluorescence at a given temperature for overall molecular rigidity; and transition temperature and rate at which it is attained for internal molecular detail.

Notes: A theory for the dependence of the net thermodynamic solvation of a macroion in an electrolyte solution is presented. The solvation in moles salt/mole macroion is shown to approach - |Zp|/v in the limit of infinite dilution of salt and macroion. The solvation in moles water/mole macroion is shown to approach zero at zero water activity. Isopiestic determinations of the hydration of sodium deoxyribonucleate in NaCl, Na2SO4, and NaClO4 solutions indicate that short-range interactions of the NaDNA with solvent account for more than half of the observed solvation. The net hydration appears to be predominantly influenced only by water activity.

Notes: The cohesive energy densities (CED) of three amylose derivatives have been estimated from viscosity measurements in a range of solvents, by using methods proposed by Mangaraj and Bristow and Watson. The values assigned to the triacetate, (2.5) nitrate, and tricarbanilate are 92 ± 2, 90 ± 2, and 87 ± 2 cal./cc., respectively. The CED of unsubstituted amylose has been measured and found to be 154 ± 4 cal./cc. The results are discussed in relation to solvent power, structure, and possible correlation with the internal pressure of the polymer.

Notes: Viscosity measurements and calculated rotary diffusion constants show that collagen undergoes photopolymerization when irradiated with ultraviolet light of 2537 A. Fibril formation at the same time is inhibited. The results are correlated with the aromatic amino acid content of the dangling peptides protruding from the rigid portion of the macromolecule.

Notes: It is shown how the technique of fluorescence may be used to assess conclusions about conformation which are based on a statistical mechanical treatment of simple protein models. Specifically, the state of bonding and distance between two chromophores in a molecule depend on the overall conformation, which can be computed for model systems. On the assumption that the intensity and polarization of fluorescence are affected by exciton transfer between the chromophores, it is possible to compute the effect of conformation on fluorescence. It is demonstrated that the conformational changes computed in the preceding paper will give rise to marked changes in the intensity and polarization of fluorescence.

Notes: Intrinsic viscosity-temperature studies for isoelectric gelatin in KCl and KSCN aqueous solutions and equilibrium degree of swelling, ν-1, measurements for amorphous crosslinked rat tail tendons in the same solutions were carried out. On increasing salt concentration Cs, both [η] and ν-1 increase, go through a maximum, and then decrease at high Cs, KCl being more effective than KSCN in bringing about this decrease. The trend observed is similar to the variation of solubility of polypeptides and soluble proteins with Cs. By regarding a water-salt solution of a given Cs as a single diluent interacting with the protein modified by solvation and binding of ions, usual polymer solution theories were applied to the experimental results. Thus, quantities related to the entropy and enthalpy components of the excess chemical potential of the diluents were obtained. The data indicate that the initial increase of [η] and ν-1 with Cs results from the balance of an enthalpy component which, on increasing Cs, becomes less favorable to dilution and an entropy component which, conversely, becomes more favorable. The subsequent decrease of [η] and ν-1 with Cs is due to the prevailing of the enthalpy component. The maximum is reached at higher Cs for KSCN than for KCl, primarily because of the large increase in the entropy component operated by the former salt. The increase of the entropy parameter with Cs is associated to a breaking down of the coordinated water structure and to an alteration of the conformation of the macro molecules due to ion-dipole interaction and to ion absorption. The decrease in exothermicity of dilution with Cs indicates a reduced thermodynamic affinity of the diluent toward the polymer which probably results from alteration of the nature of both polymer and diluent.

Notes: The effect of the DNA-binding oligopeptide distamycin A on the B to A transition of DNA in ethanol/water solutions has been studied by means of CD. (The overbars indicate that it does not matter which particular form of the corresponding families is considered.) The results show that increasing the concentration of distamycin A reverses the A conformation (in 82% ethanol) to the B conformation due to its strong binding and stabilization of the latter. In accordance with previous data for pure aqueous solutions, a site size of 3.5 base pairs is obtained from the studies in water/ethanolic solutions. From the data on the B to A transition in the presence of distamycin A, we estimated the length of the cooperativity ν0 = 10 base pairs.The results demonstrate that the oligopeptide systems of distamycin, as well as those of netropsin, are effective stabilizers of the DNA B-conformation.

Notes: Tetrapeptides with proline in position 2, asparagine or leucine in position 3, and glycine in positions 1 and 4, with end groups free or blocked on the N-terminal side, were studied in their various ionic states in 2H2O and in Me2SO-d6 by 1H- and 13C-nmr. In order to clarify or refine some details, successive substitutions of the residues in these peptides with amino acids enriched to 85% in 13C, or to 85% 13C plus 97% 2H were carried out. The 1H and 13C chemical shifts as well as the 1H-1H, 13C-13C, and 13C-1H coupling constants and the signal intensities show strong similarity of behavior between the tetrapeptides of asparagine and leucine. The main conformational characteristics are (1) the almost total stabilization of the trans conformer in the type I β-turn structure when the peptide is in the zwitterion state dissolved in Me2SO. This is deduced from the 3JC3αH-N3H and the 3JC2′-H3α coupling constants, which both furnish a dihedral angle of φ3 = -90°, and from the positive value of the temperature coefficient of the glycine-4 amide protons, which suggests a type 4 → 1 hydrogen bond; (2) the evolution of cis and trans isomer fractions which change with the ionic state of the peptides in Me2SO, whereas they remain constant in aqueous solution; and (3) the conformation of the pyrrolidine ring as it follows the variations in cis:trans isomer populations together with the side-chain rotamer fractions of the residue in position 3. In the β-turn conformation the isomer cis is less abundant and the pyrrolidine ring is more flexible; this explains the perfect accommodation of the proline residue in position 2 of a bend. The interdependence of these phenomena where interactive forces play a predominant role underlines the importance of cooperative effects in the molecule. The results also suggest that the cis isomer of proline can adapt itself just as well as the trans isomer to position 2 of a type I β-turn.

Notes: An algorithm is presented for the Monte Carlo simulation of the decay of fluorescence polarization from segmentally flexible molecules. Based on the random walk model of Brownian motion, the treatment explicitly follows the stochastic changes in the diffusion coefficients as the molecule bends. It includes the effects of a linear restoring force opposing the bending and the effects of hydrodynamic coupling between the translational, rotational, and bending motions. One application is presented: the simulation of anisotropy decay curves for hinged rods. A variety of decay curves are obtained, including single- and multiexponential behavior, and the following conclusions are reached: (1) increasing the flexibility is usually, but not always, accompanied by a more rapid rate of depolarization; (2) reducing the size of the fluorescent subunit will usually, but not always, increase the rate of depolarization; and (3) the complex interplay between the effects of molecular shape, relative sizes of the subunits, restoring force, and orientation of the transition dipoles renders it unlikely that any simple method can be used to interpret anisotrophy data without simulation. In particular, it is not possible to determine the extent of bending by fitting the data with the two-exponential approximation used by some investigators in the past.

Notes: 13C-nmr spectra of red seaweed galactans, belonging to the agar and carrageenan groups or having the “intermediate” type of structure, were interpreted on the basis of 13C-nmr spectra of model compounds. Signal assignments have been made for most of the known extreme structures of such galactans. 13C-nmr spectroscopy was shown to be a rapid and convenient method of structural analysis, which permits one to determine the type of galactan structure, the absolute configurations of its constituents (galactose and 3,6-anhydrogalactose), and the positions of the sulfate and O-methyl groups in a polysaccharide molecule.

Notes: The dielectric response of human umbilical cord hyaluronic acid in various environments has been studied at microwave frquencies using a resonant microwave cavity as a probe. Both the real and imaginary parts of complex dielectric constant and the loss tangent for hyaluronate solutions are obtained by utilizing equations for perturbation of a resonant cavity. Dielectric changes at room temperature have been observed in aqueous solutions of hyaluronic acid as a function of concentration ranging from 0 to 350 mg/ml. The data indicate the existence of ordered phases in hyaluronate solutions at selective concentrations, that is, exhibiting lyotropic-type transitions. Hyaluronate solutions at 1.5 and 3 mg/ml concentrations have been studied at various pH in the range of 6-8 and at constant ionic strength 0.1. A temperature-dependent transition in hyaluronate solution of 120 mg/ml concentration has been observed at physiological temperature. It is shown that this temperature-dependent behavior can be related to the orientational polarizability term in the Debye theory of polar molecules in liquids.

Notes: In 1974, Zimm described a theory which predicts that the sedimentation coefficient of high-molecular-weight DNA will decrease as the rotor speed of measurement increases. In 1979, this theory was revised, and the new formula predicts speed-dependence effects that are substantially smaller than the predictions of the original version. This report describes the results of subjecting both the original and the revised versions of the theory to quantitative tests using a well-defined sucrose-gradient system and a DNA of known molecular weight (T4c DNA). T4c bacteriophage is a mutant, whose DNA contains the unmodified base cytosine, instead of the glucosylated hydroxymethylcytosine characteristic of the T-even bacteriophages, and has a molecular weight of 115 ± 3 × 106. The DNA of the wild-type phage (T4D+) was also used in some experiments.In addition to the quantitative tests, the experiments test for an effect first observed by Rubenstein and Leighton, which showed that the sedimentation coefficient measured for T2 DNA depended on the composition of the centrifuge tube used for the measurement (tube composition effect). It can be inferred from this observation that an interaction occurs between particle and tube wall during sedimentation, and this leads to a reduction in sedimentation velocity independent of the reduction in S described by Zimm's theory.The results show that in the range of 25,000-50,000 rpm, the original but theoretically incorrect form of the theory quite accurately describes the sedimentation behavior of both T4c and T4D+ DNA, although T4D+ was a special case in some respects. The revised (corrected) form of the theory predicts much less of a speed-dependence effect than that actually observed. The discrepancy between corrected theory and observation suggests that other factors (perhaps arising from the use of the swinging bucket rotor geometry) are causing the additional observed reduction in S20,w. However, the experiments show that the tube composition effect does not seem to be one of these.

Notes: The fixation of cis (NH3)2Cl2Pt(II) to poly(I)·poly(C) leads to the formation of two complexed species. One involves coordination to a single base (accounting for about 70% of the total platinum bound over the rb range 0.07-0.25) and the other to two bases which are not adjacent to each other but may be on the same strand and separated by a loop. Reaction of the platinum compound with poly(I) gives in addition to the above two species a minor one (about 15%, independent of rb over the range 0.05-0.30) in which the platinum is bound to two adjacent bases. The availability of such coordination reduces the dominance of the 1:1 species, which, however, remains the major one (ca. 55%).

Notes: The complex of CH3Hg(II) with the accessible cysteines of glyceraldehyde-3-phosphate dehydrogenase (GAPD, EC 1.2.1.12) from rabbit muscle has been studied by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The wavelength dependence of the phosphorescence decay kinetics has also been measured. Comparison of CH3Hg(II)-GAPD with GAPD by these methods shows that a specific optically resolved tryptophan site of GAPD is perturbed by the interaction with a nearby mercury atom. The perturbation on the luminescence and ODMR properties is typical of an external heavy-atom effect. Based on the x-ray diffraction structure of the lobster enzyme, it is proposed that the heavy-atom effect results from the interaction of tryptophan-310 with CH3Hg(II) bound to cysteine-281 in the rabbit muscle enzyme.

Notes: The energy of interaction of a spermine molecule with the A- and B-forms of DNA has been calculated, assuming that the molecule of spermine is fixed in the narrow groove of the DNA helix with the formation of hydrogen bonds between the amino groups of spermine and the phosphate groups of DNA. The atom-atom potentials method was used. Optimal structures for the A-DNA-spermine and B-DNA-spermine complexes are suggested. It is shown that, in agreement with the experimental data, the interaction of the spermine molecule with the A-DNA is energetically more favorable than that with the B-DNA. Two main factors are responsible for this: (1) the distance between neighboring phosphates of the chain in A-DNA (which is about 1 Å less than that in B-DNA) corresponds better to the distance between the amino groups of the propyl part of spermine; and (2) the orientation of phosphate groups in A-DNA inside the groove is preferable for complex formation with spermine to the outside groove arrangement of the phosphates in B-DNA. These conclusions are further confirmed by the calculations for DNA-propane diamine complexes.

Notes: The crystal structure of a synthetic analog of valinomycin, cyclo[-(D-Ile-Lac-Ile-D-Hyi)3-] (C60H102N6O18), has been determined by x-ray diffraction procedures. The crystals are orthorhombic, space group P212121, with cell parameters a = 11.516, b = 15.705, c = 39.310 Å, and Z = 4. The atomic coordinates for the C, N, O atoms were refined in the anisotropic thermal motion approximation and for the H atoms in the isotropic approximation. Values of standard (R) and weighted (Rw) reliability factors after refinement are 0.073 and 0.056, respectively. The structure is completely asymmetric. The cyclic molecular backbone is stabilized by six intramolecular hydrogen bonds N—H…O=C, five bonds being of the 4→1 type and one being of the 5→1 type. The side chains are located on the molecular periphery. The conformational state of isoleucinomycin in the crystal is intermediate between the corresponding crystalline states of valinomycin and meso-valinomycin. The observed conformation suggests that complexation could proceed via entry of the ion at the face possessing the L-Lac residues, the less crowded face.

Notes: Possible conformations of lacto-N-tetraose, lacto-N-neotetraose, related disaccharides, and other milk oligosaccharides have been studied by an energy-minimization procedure using empirical potential functions. Lacto-N-tetraose favors a “curved” conformation, while lacto-N-neotetraose favors an approximately “straight” conformation. These two conformations differ mainly in the position of the terminal galactose residue with respect to the rest of the molecule. This difference explains the greater strength of lacto-N-neotetraose compared with lacto-N-tetraose in its ability to inhibit the cross-reaction of blood group P1 fractions with Type XIV pneumococcal antipolysaccharide. Although the favored conformation of lacto-N-tetraose (inactive) agrees with the model proposed by the earlier workers, that for lacto-N-neotetraose (active) differs. The favored conformations for the disaccharides galactose-β(1-4)-N-acetylglucosamine, galactose-β(1-3)-N-acetylglucosamine, and lactose are similar in overall shape, differing only in the nature and orientation of the side groups. This explains their nearly equal inhibitory activity. These theoretical models also explain the increased activity of lacto-N-fucopentaose I over that of lacto-N-tetraose and the relative activities of the substituted lactoses. The present studies suggest that it is the overall shape of the molecule which is important for activity, rather than the terminal β(1-4)-linked galactose residue alone.

Notes: 13C spin-lattice relaxation times of poly(L-lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the 13Cα and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix-coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the Cα-Hα vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ≫ 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.

Notes: The solid-state conformation of copolymers of β-benzyl-L-aspartate [L-Asp(OBzl)] with L-leucine (L-Leu), L-alanine (L-Ala), L-valine (L-Val), γ-benzyl-L-glutamate [L-Glu(OBzl)], or ∊-carbobenzoxy-L-lysine (Cbz-L-Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700-250 cm-1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L-Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L-Asp(OBzl)] copolymers was observed in the following composition ranges: L-Leu, 0-15 mol %; L-Ala, 0-32 mol %; L-Val, 0-8 mol %; L-Glu(OBzl), 3-10 mol %; and Cbz-L-Lys, 0-9 mol %.

Notes: Dry purified ligamentum nuchae elastin has been investigated for physical aging. The samples were quenched from a temperature (197°C) close to the softening point to a number of measuring temperatures ranging from -20 to +180°C. At each temperature, the small-strain torsional creep properties were determined at a number of elapsed intervals after the quench. Aging effects were found over the whole temperature range, and the creep and aging behavior of elastin turned out to be very similar to that of synthetic polymers.

Notes: Covalently closed circular DNA can exist in different configurations known as circular, toroidal, and interwound. Changes among these forms can be made in several ways, including the insertion of dye molecules between adjacent base pairs, which tends to untwist the double-helical structure. The aim of this paper is to discuss these configurations, and the changes among them, in the context of classical elastomechanics. The concepts of twisting, linkage and writhing are explained. Simple experiments on a twisted linear-elastic rod are described, and it is shown that although the circular and interwound forms may be modeled in this way, the toroidal form does not occur, being mechanically unstable. Theoretical energy calculations by Levitt on bent and twisted DNA show that DNA exhibits a particular kind of nonlinear elasticity in which there is an unusual coupling between bending and twisting. The aim of the paper is to show qualitatively that this special kind of elasticity can stabilize the toroidal form of closed circular DNA.

Notes: Light-scattering, viscosity, and sedimentation experiments on aqueous solutions of k-carrageenan show that this sulfated polygalactose is an expanded flexible random coil. This expansion is due to long-range interactions that are predominantly electrostatic. Extrapolation of viscosity data to infinite ionic strength provided values for the intrinsic viscosity which were subjected to the Stockmayer-Fixman analysis, giving a value for the Mark-Houwink coefficient under theta-conditions, Kθ, of 0.27. The characteristic ratio, C∞, under these conditions is 7.8, and the conformation factor σ is 2. In a solution of 0.118 ionic strength, where a Mark-Houwink exponent aη of 0.86 is found, the radii of gyration calculated from viscosity data are lower than those found from the angular dependence of scattered light. On the other hand, the radius of gyration found from the sedimentation rate agrees well with the light-scattering radius. The relations between molecular parameters are corrected for the poly-dispersity of the sample.

Notes: An approximate analytic expression for the translational friction coefficient of a toroid modeled as a continuous shell of frictional elements is derived using the Kirkwood approximation. The accuracy of this expression was determined by comparing the friction coefficients predicted by it to those predicted by extrapolated shell-model calculations using the modified Oseen tensor. To show that these calculations do indeed yield the correct friction coefficients, actual translational friction coefficients were determined by observing settling rates of macroscopic model rings or toroids in a high-viscosity silicone fluid. Our conclusion is that the approximate expression yields friction coefficients that are about 1.5-3% low for finite rings. For thin rings, a comparison is also made with the exact result of Yamakawa and Yamaki [J. Chem. Phys. 57, 1572 (1972); 58, 2049 (1973)] for the translational friction of plane polygonal rings. This comparison shows that the approximate expression yields results which are low by 2-3% unless the rings are extremely thin, in which case the error is larger. In the limit of an infinitely thin ring the approximate expression reduces to the Kirkwood result [J. Polym. Sci. 12, 1 (1954)], which is low by 8.3%. We discuss briefly how this work may be useful in determining the structure of DNA compacted by various solvent-electrolyte systems and polyamines.

Notes: Internal Brownian motions of clean φ29 and λ-DNAs have been studied using photon-correlation techniques at both visible (λ0 = 632.8 nm) and uv (λ0 = 363.8 nm) wavelengths. The present dynamic light scattering data, which extend to K2 = 19 × 1010 cm-2, can in every case be satisfactorily simulated by a Rouse-Zimm model polymer with an appropriate choice of the three model parameters. The effects of pH, salt concentration, single-strand breaks, and molecular weight on those model parameters have also been investigated. Intact clean DNAs exhibit surprisingly little variation with pH from 7.85 to 10.25, with salt concentration from 0.01 NaCl to 5.4M NH4Cl, or with molecular weight or GC content. The single-strand breaks have no effect at pH 9.46, but produce dramatic changes in the model parameters at pH 10.0 and 10.25, indicating the introduction of titratable joints at those pHs. The failure of either single-strand breaks or a large change in GC content to alter the model parameters in the neutral pH range is a strong indication that local denaturation is not required for those flexions and torsions that dominate the relaxation of fluctuations in the scattered light. The Langevin relaxation time for the slowest internal mode of a particular Rouse-Zimm model derived from the dynamic light scattering data is compared with pertinent literature data extrapolated to the same molecular weight. The present algorithm for determining model parameters from the light-scattering Dapp vs K2 curve actually yields a Langevin time in fairly good agreement with the literature value. For unknown reasons the light-scattering D0 values generally exceed those obtained from the molecular weight and sedimentation coefficient by about 20%.

Notes: Raman spectra of polyribouridylic acid excited in the uv region, from 363 to 290 nm, are reported. The conformational changes of the polymer from random coil to ordered structure with stacked bases at high and low temperature, respectively, are reflected by important changes in the Raman line intensities; this Raman hypochromism is itself a function of the excitation wavelength - its profile has been determined and shows negative values in the region of 290 nm (near resonance), i.e., hypochromism becomes hyperchromism. Thus the knowledge of the hypochromism excitation profile is important in following order-disorder transition of a polymer using resonance Raman spectroscopy. Theoretical attempts are proposed for explanation, involving not only the relative variations of the molar extinction coefficient on the order-disorder transition of the polymer, but also the damping factors of the vibronic levels. The theoretical curve is found to fit adequately the experimental data over the excitation range, using only the frequency of the O-O transition of uracil and a vibronic linewidth of 2200 cm-1.

Notes: The preferential interaction of sodium poly(α-L-glutamate) and poly(α-L-glutamic acid) with the solvent components in water/2-chloroethanol mixtures has been determined using density-increment measurements. The degree of preferential interaction was deduced from the density increments at constant molality of 2-chloroethanol and at constant chemical potential of 2-chloroethanol. Sodium poly(α-L-glutamate) and poly(α-L-glutamic acid) are both preferentially hydrated in the whole range of solvent composition. A dehydration process occurs during the 2-chloroethanol-induced coil-to-helix transition of sodium poly(α-L-glutamate). This dehydration process was attributed to the release of some moles of water from the neighborhood of the peptide bond during the nucleation of the helix. After the conformational transition, sodium poly(α-L-glutamate) is solvated by one 2-chloroethanol molecule. The location of water and 2-chloroethanol molecules in the different parts of the residue (more polar and less polar portions) is also discussed.

Notes: Complex formation between tryptamine and mononucleotides and dinucleoside phosphates containing adenine and/or cytosine has been studied at five pD's ranging from 1.1 to 7.4 by proton magnetic resonance spectroscopy. Chemical shifts of base ring protons and the ribose anomeric proton in the nucleotides and indole ring protons in tryptamine were monitored and their changes with pD and intermolecular interactions interpreted qualitatively. Stacked complexes were found to exist at all pD's in the range studied. Complex geometries differ depending on pD. An electrostatic interaction between the tryptamine amino group and the nucleotide phosphate group contributes to complex formation above pD 4 but is not strong enough to shift the dinucleoside phosphate equilibrium towards the unstacked conformer.

Notes: Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around φ = -135° ± 5° and ψ = 75° ± 5°, and (3) the αL conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C5 (extended) conformation is second lowest in energy. When the peptide bond (ωi) is in the cis configuration the ith residue is energetically forbidden in the range φ = 0° to 180° and ψ = -180° to +180°. Conformations of low energy for ωi = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the αR conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His2]TRH. These conformers differ only in the φ and ψ values at the (N-Me)His2 residue. Among the different low-energy conformers found for each of the ENK analogs [D-Ala2,(N-Me)Phe4, Met5]ENK amide and [D-Ala2,(N-Me)Met5]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu7]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D-Ala6,(M-Me)Leu7]LHRH is considered.

Notes: Based on steric and electrostatic considerations, the prerequisites for binding to DNA via the intercalation mechanism are proposed. Steric contour energy curves are presented to demonstrate the region inaccessible to an intercalant. They are calculated with a 6-n (n = 14) potential. This method is a soft potential analog of an excluded-volume approach. Electrostatic contours on the steric surface illustrate the relatively positive and negative regions of the binding site. The principal intercalation sites, predicted to fit into B-DNA via a tetramer-duplex unit, and the unconstrained dimer-duplex units, obtained in crystal structures, are examined. These contours illustrate the requirements of size, conformation, and net atomic charges necessary for intercalation and optimum binding. Based on the limited space available for intercalation by the presence of the backbone and the maximum base-pair separation of 8.25 Å, an Essential Metabolite Exclusion Hypothesis is presented.

Notes: A study of the near-uv CD spectrum of lysozyme was carried out in the presence and absence of the inhibitor tri-N-acetylglucosamine, and theoretical chiroptical calculations based on the tetragonal crystal structure of the enzyme and the enzyme-inhibitor complex were performed. The results of these calculations indicate that the near-uv CD spectrum of lysozyme can be adequately explained in terms of negative rotatory strengths arising from the tryptophan 1La (293-300 nm) and the disulfide n-σ* bands (250 rm), and positive rotatory strength contributions from the tryptophan 1Lb bands (291 nm) and the tyrosine 1Lb bands (275 nm). Contributions to the rotatory strength of each band were approximated in terms of specific interactions between chromophores. It was found that the rotatory strength of most of the near-uv transitions arises primarily from coupling interactions involving other side-chain chromophores and amide groups which are in close proximity. Changes which are observed in the lysozyme CD spectrum on binding of tri-N-acetylglucosamine may be explained in terms of changes in the rotatory strength which result from interactions of the 1La transitions of the active-site tryptophans with the acetamide groups of the inhibitor. The reasonable agreement which is found between the experimental and calculated rotatory strengths implies that the crystal conformation of lysozyme must resemble the solution conformation.

Notes: The synthesis of the following oligo- and co-oligopeptides by the liquid-phase method is described: (L-Met)15 (I), [L-Glu(OBzl)]20 (II), (L-Val)8-Gly (IV), (L-Ile)8-Gly (V), (L-Ile)4-Gly-(L-Ile)4 (VI), (L-Ile)4-Pro-(L-Ile)4 (VII), (L-Met)5-L-Pro-(L-Met)5 (VIII), [L-Glu(OBzl)]7-L-Pro-[L-Glu(OBzl)]7 (IX). The oligomers are covalently bound to bifunctional polyethylene glycol (PEG) and monofunctional PEG-M of Mr 5 × 103-2 × 104. Analytical controls were carried out after each step of synthesis in order to ensure quantitative coupling yields. All products could be obtained in high purity as indicated by amino acid analysis, thin-layer chromatography and chiroptical methods. The solubility of the oligomers was strongly enhanced by the presence of the C-terminal PEG group, enabling conformational investigations in a variety of solvents. A significant relationship between conformation and physicochemical properties of the oligopeptides was observed. Oligomers with tendencies to adopt α-helical (I, II) or unordered structures (VI-IX) showed no pronounced change in solubility or coupling kinetics during chain elongation, whereas the onset of a β-structure (IV, V) was paralleled by a drastic decrease in solubility and reactivity of the terminal amino groups. Most notably, the insertion of a proline or glycine in the middle of a β-forming peptide chain (VI, VII) resulted in a considerable increase in solubility compared to the corresponding homo-oligomers. The impact of the conformational properties of a peptide chain on strategic considerations of peptide synthesis in solution is delineated.

Notes: The conformational analysis of the CD spectrum is reported for the synthetic and membrane-modifying nonadecapeptide analog of alamethicin N-t-Boc-(Aib-L-Ala)5-Gly-Ala-Aib-Pro-Ala-Aib-Aib-Glu(OBzl)- Gln-OMe. The CD data are evaluated according to three different methods and are discussed with respect to those obtained from natural alamethicin and suitable models such as N-t-Boc-(Aib-L-Ala)7-OPOE, fragments of the synthetic nonadecapeptide, and the hexadecapeptide N-t-Boc-(Aib-L-Ala)5-Pro-Ala-Aib-Aib-Glu(OBzl)-Gln-OMe. The synthetic nonadecapeptide with the longer helical region exhibits membrane activities comparable to those of alamethicin, whereas the hexadecapeptide with the shorter helix is inactive.

Notes: This paper deals with the light scattering from particles rotating in a flow with a transverse velocity gradient. It is theoretically substantiated and experimentally proved that the scattered light spectrum contains reliable information of the particle configuration and dimensions. The proposed technique may prove also very promising for the analysis of particle polydispersion.

Notes: The synthesis of poly(ε-L-lysine) is described. This is a poly(ε-amino acid) in which the ε-amino group of lysine is condensed with the α-carboxyl group to produce a chain backbone that is a variant of the usual one seen in proteins and the side chain is the α-amino group. Conformational studies of poly(ε-L-lysine) and its t-butyloxycarbonyl derivative suggest the likelihood of a chain order that is formally similar to the antiparallel pleated-sheet conformation of proteins.

Notes: We have studied the kinetics of oscillatory tensile forces in collagen membranes. These forces were generated by sinusoidal electric fields applied across the membrane. Both the magnitude and phase of the measured force changed with frequency over a three-decade range. The membrane-separated electrolyte baths had different ionic strength but identical non-isoelectric pH. Changes in intramembrane ionic strength due to the electric field were calculated over the same frequency range via an electrodiffusion model that was generalized to include convection and electrokinetic coupling. A comparison of the experimental and theoretical phases and amplitudes versus frequency suggests that electrodiffusion is the dominant rate-limiting process in this electromechanochemical transduction. These results are relevant to electrostatic interactions in connective tissues and to membrane-based filtration devices in which membrane permeability may be actively varied and controlled by an applied electric field.

Notes: Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔCp0 at 30°C. Limiting partial molal heat capacities ΔCp20 have been determined by combining ΔCp0 with Cp2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group —CONH— was semiquantitatively estimated.

Notes: Integral enthalpies of solution at low concentrations of several amino acids and peptides in 2 and 6M urea solutions have been determined at 25 and 35°C. These data have been used to derive the enthalpies of transfer (at 25 and 35°C) and heat capacities of transfer (at 30°C) of these amino acids and peptides from water to aqueous urea solutions. Furthermore, the enthalpies of transfer and heat capacities of transfer per CH2 group and per peptide group —CONH— have also been estimated. These results show that while the enthalpies and heat capacities of transfer per CH2 group are positive and negative, respectively, the reverse is true for —CONH— group. The implications of these results in the mechanism of the denaturation of proteins by urea are discussed.

Notes: The CD spectra of cUpUp, cCpCp, and cGpGp derived from DCC-catalyzed polymerization of the relevant protected ribonucleoside 3′-phosphates are described. Similar studies on Up, U 〉 p, and cUp, as well as cUpUpUp and cUpUpUpUp, are presented. The spectral properties of the cyclic oligomers are compared with those of the corresponding linear oligomers with terminal 3′-phosphates so as to demonstrate that disruption of normal right-handed base stacking is considerable in these RNA loops.

Notes: The polymerization of 1,2-anhydro-3,4,6-tri-O-benzyl-β-D-mannopyranose proceeds in the presence of Lewis acids, cationic coordination catalysts, and strong bases. Debenzylation of the products yields oligomeric saccharides or low polymers. Polymerization in toluene by means of potassium alkoxide complexed with crown ethers leads to essentially stereoregular (1 → 2)-α-D-mannopyranan. The original derivatives have been characterized by optical rotation, viscosity, molecular weight, gel permeation chromatography, and spectrometry. The free polysaccharides have been characterized by optical rotation, molecular weight, and 1H- and 13C-nmr spectrometry and compared to yeast mannan hydrolysate oligomers.

Notes: Molecular mechanics calculations have been used to determine the preferred physical association sites of the known alkylating agent dimethyl aziridinium ion (Az+) and a CH3+ prototype test probe with B-form, tetrameric DNA sequences. Electrostatic interactions are most important in determining these preferential physical association sites. In turn, the intermolecular energy minima depend on the charge distribution assigned to the DNA sequence. However, for three reported DNA charge distributions, only two distinct sets of energy minima were obtained for the CH3+-like ion interacting with (G-C)4, (A-T)4, and [(G-C)·(A-T)]2 deoxyribonucleic acids. These minima correspond to physical association geometries in which the CH3+-like ion is near known alkylation sites. The results of the Az+ … [(G-C)·(A-T)]2 interaction are virtually identical to those found for the CH3+-like ion. Aqueous solvation energetics have little effect on the physical association of Az+ with [(G-C)·(A-T)]2.

Notes: A series of molecular orbital calculations, using MINDO/3 and CNDO/2L methods, have been used to characterize the chemical reaction of protonated aziridine with DNA nucleophilic base sites. The N-7 atom of guanine is found to be the preferred alkylation site only when the O-6 atom of guanine is involved in base-pair hydrogen bonding. Otherwise O-6 is the predicted major site of alkylation. This indirectly suggests that protonated aziridine alkylation processes involve base-paired DNA structures, since N-7 guanine is the observed major site of alkylation. Alkylation of N-3 adenine is predicted to be more favorable than chemical attack of the N-7 adenine position. Both of these sites, however, are predicted to be less reactive than N-7 of guanine. These chemical reactivity studies resolve alkylation specifically not achieved in the DNA-alkylator physical association calculations reported in the preceding paper.

Notes: Infrared dichroic studies and deuterium exchange measurements of somatostatin and some of its analogs incorporated in uniaxially oriented polyoxyethylene are described. Band positions and dichroic ratios in the N-H stretching and amide I and II regions are similar to those of flexible and nonordered peptides like valinomycin and poly[Glu(ONa)]. This information, together with fast deuterium exchange rates, suggests that somatostatin exists in a flexible nonordered conformation in polyoxyethylene. One analog, di-S3,14-acetamidomethyl dihydrosomatostatin, was found to exist in both nonordered and β-like conformations.

Notes: The Monte Carlo simulation of the structure of the 16 ordered and disordered waters in the unit cell of crystals of the cyclic peptide cyclo(-L-Ala-L-Pro-D-Phe)2 is reported. The water structure has been characterized in terms of the statistically averaged positions of the individual molecules, their root-mean-square movements about these positions, the probability of finding a water in a given spatial position in the crystal (probability maps), and examination of individual configurations of the system. In this way a picture is obtained of the water structure, including water orientations (hydrogen positions), the hydrogen-bonding network, and fluctuations in these structural features, to a degree hitherto unavailable either from experimental or theoretical studies. In addition, the variation in water structure in various peptide environments was studied and correlated with the energetics of the individual water molecules. Variations in the crystalline environment of different water molecules lead to energy differences of as much as 4-5 kcal/mol in their average energies. Similarly, differences are observed in the water-peptide and water-water components of the energy. Two different water potentials were tested. The results were compared with experimental data in terms of mean positions, root-mean-square movements, and the Fourier transform of the simulated water structure. The agreement factor (R factor) calculated from the theoretical water probability distribution was 18.8% compared to the x-ray value of 14.5%, and the value of 28% when the water is omitted.

Notes: Conformational energy calculations on the N-acetyl-N′-methylamides of dehydroalanine and N-methyldehydroalanine indicate that their conformational behavior is very different from that of the corresponding saturated compounds. The conformational data in the literature from x-ray and nmr investigations on peptides containing α,β-unsaturated residues are discussed on the basis of these theoretical results.

Notes: Amphipatic block copolymers (OβEb) with a hydrophilic saccharide block and a hydrophobic polypeptide block were synthesized. In these copolymers the saccharide block is the glyco-amino acid Oβ from ovomucoid and the peptide block (Eb) is a poly(γ-benzyl-L-glutamate) block. Copolymers OβEb exhibit, in the solid state and in Me2SO concentrated solutions, mesomorphic lamellar structures where the polypeptide chains are in an α-helical conformation. Depending on the molecular weight of the polypeptide block, three types of lamellar structures are obtained, and they differ by the mode of organization of the polypeptide chains in their lamellae and by the T or Y conformation of the saccharide block.

Notes: The formation of complex between the Cu2+ ion and poly(α-L-glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv-visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu2+, R, of 32, 16, and 8 and in the pH range 4-7. The results showed that more than 90% of Cu2+ ions bind to the poly(Glu) at pH 〉 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)-Cu(II) complexes fall into three classes in the pH range 4-7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix-coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)-Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.

Notes: The variable-temperature proton nmr spectra of the oligoribonucleotides in the series CpApX and the series ApGpX, X = A, G, C, U, together with the parent dimers CpA and ApG have been measured. A complete analysis of all the nonexchangeable base proton resonances and ribose H-1′ proton resonances was made. The presence of trends in the shielding abilities of the various bases at both the nearest-neighbor and next-nearest-neighbor positions were identified. The observed shieldings could be used to predict the chemical shifts of protons in related systems. Based on the empirical results from ribodinucleoside monophosphates, the temperature-dependent behavior of the J1′2′ coupling constants of the triribonucleotides suggested that the compounds in the CpApX series stacked from the 5′-end to the 3′-end, while those in the ApGpX series stacked from the 3′-end to the 5′-end.

Notes: Closed random Gaussian polygonal chains of N (6 〈 N 〈 150) bonds of equal length b and thickness d have been generated on a computer. The knot type, the writhing number w, the radius of gyration, and the average of the inverse of the distance between two apices have been determined for each chain. For all the studied knot types - 0, 31, 41, 51, and 52 - the probability density of finding a given w is Gaussian. The Gaussian is centered about 0 for the amphichiral knots. Therefore, for long circular DNAs, the contribution to the supercoiling energy, which depends on w only, may be considered as purely entropic and may be expressed as ARTw2/N, in agreement with previous semiempirical considerations. The parameter A increases with chain thickness, it decreases as N gets larger but rapidly reaches a plateau. Comparison with experimental data from the literature would suggest that the ratio of the writhing to the constraint increases with ionic strength. The ratio of sedimentation constant of the supercoiled DNA to the sedimentation constant of the nicked DNA varies as N1/4 (w/N)2, and therefore depends on the writhing density and on the length of the DNA.

Notes: A band-centrifugation method for the analysis of an assembly reaction of a simple virus from its RNA and protein is described. The experiment was carried out by sedimenting a band of viral RNA through a solution of depolymerized coat protein. The resulting radial distribution of the reaction products, followed as a function of time, was analyzed by a computer simulation of the series of reaction. This method is based on a numerical solution of the continuity equation for the sedimentation-diffusion process [Claverie, J.-M., Dreux, H. & Cohen, R. (1975) Biopolymers 14, 1685-1700; Cohen, R. & Claverie, J.-M. (1975) Biopolymers 14, 1701-1716]. A numerical method for the simulation of the chemical reaction is derived. From the simulated reaction series, equilibrium constants emerge for the successive addition of protein subunits to the growing nucleoprotein particle.The method is applied to the assembly of alfalfa mosaic virus. If the reaction between RNA and protein is carried out in 0.32M CsCl, pH 7.0, two stages during particle growth are resolved, each characterized by an equilibrium constant K. The determined values for K range from 5 × 105 to 3 × 106 l. mol-1. The existence of these two stages may have a structural implication in the assembly, as they likely represent an elongation and a termination stage. If the reaction is carried out under more favorable conditions (0.25M CsCl, pH 7.0), a kinetic constant of at least 105 l. mol-1 sec-1 is derived for each reaction step. Under these conditions the assembly appears to be completed within 1 min, which is too fast to detect distinct stages by band sedimentation.

Notes: Purified insoluble elastin samples labeled with [1-13C]valine, [1-13C]alanine, and [1-13C]-lysine were prepared from chick aorta in culture. The molecular mobility at the labeled sites was investigated using 13C-1H magnetic double-resonance spectroscopy. Linewidths, T1, and nuclear Overhauser effect (NOE) values of the labeled carbons alone were obtained from dipolar decoupled difference spectra. Analysis of these parameters together with signal intensity measurements showed that essentially all the valyl residues, ca. 75% of the alanyl residues, and ca. 60% of the lysyl residues were characterized by rapid backbone motions having τ = 65 nsec. Resonances due to the remaining alanyl and lysyl residues were detected in cross-polarization experiments, which enhance the signals of motionally restricted carbons. Since lysyl and alanyl residues are found in the crosslink regions of elastin, whereas valyl residues are not, we conclude that crosslinks rather than secondary structures in the extensible region of the protein are the main source of motional restrictions in the protein. Elastin chain mobility was monitored by linewidth measurements over the range -90 to +70°C. When the swelling solvent (0.15M NaCl) was fixed at 0.6 g/g of elastin, a rapid monotonic reduction in chain mobility was observed as the temperature was lowered from 50 to 5°C. Liquidlike mobility was completely lost at 5°C. In contrast, the same sample in contact with excess solvent retained its liquidlike molecular mobility until -13°C, where it abruptly became rigid. The molecular mobility of this sample was temperature insensitive in the physiologically interesting range, 20-40°C, as a consequence of the opposing influences of temperature and swelling. Taken together these nmr data indicate that under physiological conditions, elastin is a network of mobile chains whose motions are strongly influenced by protein-solvent interactions.

Notes: Preferential solvation of the glucans amylose, pullulan, and dextran in binary dimethyl-sulfoxide/water (DMSO/H2O) solvent mixtures has been measured using gel-permeation chromatography. The preferential solvation behavior of the three glucans in DMSO/H2O solvent mixtures is indistinguishable in the experiments reported. In solvent mixtures with mol ratio DMSO/H2O less than 1:2, all three glucans are solvated preferentially by H2O. The maximum extent of preferential solvation by H2O is about 2.5 mol H2O/mol of glucose residues. When the DMSO/H2O mol ratio exceeds 1:2, DMSO solvates the glucans preferentially to a maximum extent of about 1 mol DMSO/mol of glucose residues. An interpretation of the change in preferential solvation with mixed solvent composition is suggested in terms of the known characteristics of the binary solvent system, and the relationship of preferential solvation, reported here, to the absolute solvation of the glucan chains is discussed.

Notes: The dc electrical conductivity of large single crystals of hen egg-white lysozyme has been measured. The samples were grown from aqueous solution and dried in air with silica gel. The temperature dependence of the conductivity obeyed the relation σ = σ0 exp(- ΔE/kT), with ΔE = 1.2 eV. The ΔE value agreed with most of the previous results for various proteins in the form of lyophilized powder. On the other hand, log σ0, being between 7 and 11, was much larger than the previously reported values and differed among the samples. An irreversible decrease in σ0, without affecting ΔE, was observed on heating the samples above 85°C. It was shown that the set of results can be explained if the charge carriers responsible for the observed conduction are regarded as protons, originating from residual water molecules. Photoresponse of the samples to uv radiation below 305-315 nm was also observed. Reproducible and reliable results were obtained relatively easily in the present experiments, which is thought to be the main advantage of using single-crystalline samples.

Notes: A general formalism, which includes translation-rotation coupling, is proposed for calculating translational and rotational transport properties, as well as intrinsic viscosities, of rigid macromolecules with an arbitrary shape. This formalism is based on Brenner's theory of translational-rotational dynamics and on methods for the calculation of hydrodynamic properties that have been already presented, and can be regarded as a generalization of the one proposed by Nakajima and Wada. The calculated transport properties depend on the origin as predicted by Brenner's theory, but in a disagreement with him, the center of resistance and the center of diffusion do not coincide. As one can define several hydrodynamic centers, which in practice turn out to be located at different points, the influence of the choice of the center on the calculated transport properties is discussed. An analysis of the translation-rotation coupling effects in translational diffusion reveals that they arise exclusively from hydrodynamic interactions and are rather small in some cases of interest. Finally, we present a study of the rotational diffusion of rigid bent rods with a fixed length-to-diameter ratio. The diffusion coefficients obtained can be useful to estimate changes with respect to a straight rod.

Notes: The molecular weight distribution of poly-γ-benzyl-L-glutamate prepared by N-carboxy anhydride (NCA) polymerization with the use of di-n-butylamine, diisopropylamine, and sodium methoxide as initiators was investigated. In every case, narrow distributions were found. Moreover the results allow us to conclude that the same polymerization mechanism is operative with each of the above initiators. The experimental distribution curves show fairly good agreement with the theoretical distributions expected for a polymerization process without termination.

Notes: The influence of counterion radius on the polyelectrolyte behavior of poly-L-cysteate in aqueous solution under different conditions was investigated. A linear relationship was found between a critical concentration in solution of the synthetic charged poly-peptide and the crystallographic radius of different counterions, indicating that the stability of this macromolecule is inversely proportional to the size of the cations.

Notes: X-ray diffraction patterns from dilute solutions of poly-3,5-dibromo-L-tyrosine in dimethylformamide and equivalent concentrations of 2,6-dibromophenol in the same solvent are compared. The intensity difference as a function of scattering angle is indicative of the arrangement of residues in the polymer. A novel averaging procedure is used to compute the intensity patterns arising from regular arrays of bromine atoms. Interbromine spacings measured on a Drieding model of a right-handed α-helix with side groups restricted to rigid positions of maximum stagger give moderate agreement with experiment. Very good agreement with observations is obtained by allowing for a small oscillation of phenyl rings which effectively cancels out the contribution of large distances to the intensity curve.

Notes: Aqueous solutions of ribonuclease and human serum albumin were subjected to periods of controlled heating and the nuclear magnetic spin-lattice relaxation times, T1, of the water protons measured. The heat treatment causes an initial increase in the relaxation times of the water protons. These T1 variations indicate that a configurational change, compatible with a disordering of the protein structure occurs, which involves an increase in various modes of internal mobility. The T1 measurements also indicate a close, ordered association of the water molecules and the protein.

Notes: The melting characteristics of unfractionated yeast s-RNA in neutral aqueous solution have been examined by high-resolution proton magnetic resonance spectroscopy. These studies indicate that the bases of s-RNA become rotationally activated at lower temperatures than their associated ribose units. These observations, as well as the recently determined base sequence of an alanine-specific s-RNA, suggest that base complementarity may not be the primary determinant in the stabilization of the secondary structure of s-RNA. In addition, evidence for exchange of C8-protons of purine bases of s-RNA with D2O was obtained at temperatures above 70°C.

Notes: Anomalous optical rotatory dispersion (ORD) is induced in symmetric dyes when bound in metachromatic complexes with chondroitin sulfates (ChS) and kerato sulfate (KS) from mammalian and shark origin as well as with heparin (H) providing inference that secondary structures exist in these biopolymers. The induced rotation occurs in the absorption band of the bound dye and differs in shape and/or spectral position from the classical Cotton effect seen with single optically active chromophores. The AO:ChS complex has an induced ORD curve similar to that of the exciton band of a helical polymer. Strength of the induced rotations with methylene blue increases with the degree of metachromasy of the 1:1 complex. The sense of the MB Cotton effects may depend upon the conformations of the repeating dimeric units, since MB complexes with ChS (alternating 1-3,1-4 β-glycosides) give (+) effects while MB:H (1-4 α-glycosides) have (-) effects in the γ band region. Preliminary evidence for the occurrence of Cotton effects with the mucopolysaccharides themselves in the ultraviolet region was obtained. The sign of the Cotton effect for ChS-C is (-), trough about 45.9 kK., crossover about 48.8 kK. whereas that for H is (+), peak about 50.4 kK.

Notes: Poly-N5-(3-hydroxypropyl)-L-glutamine (PHPG) samples of molecular weights from 20,000 to 140,000 were synthesized by the action of 3-amino-1-propanol on poly-γ-benzyl-L-glutamate. PHPG is freely soluble in water and in a variety of organic solvents and was shown to be devoid of functional groups ionizing in the pH range 2-12. From hydrodynamic data (viscosity, sedimentation, diffusion) and optical rotatory measurements (ORD in the range 289-650 mμ, circular dichroism and Cotton effects in the range 210-280 mμ) on samples of different molecular weight it was concluded that PHPG is largely helical in methanol, ethylene glycol, formamide, dimethylform-amide, n-butylamine, and acetic acid, and randomly coiled in dichloroacetic acid and formic acid. In water the polymer is partly helical, the degree of helicity increasing with decreasing temperature and increasing molecular weight. The following solutes were found to decrease the helicity in the order indicated: NaOH 〉 guanidinium chloride 〉 urea 〉 HCl 〉 LiBr 〉 NaBr 〉 NaCl 〉 H2O. Detergents do not destroy the helical conformation. Helix-coil transition curves were obtained for the solvent pairs: methanol-water, acetic acid-water, and chloroform-dichloroacetic acid. Thermal transitions were observed in water, water-methanol (70:30 v/v), and dichloroacetic acid-ethylene dichloride (22:78 v/v), the latter being of the “inverted” type. Heats of helix formation were calculated and their significance is discussed.

Notes: Polyadenylic acid is degraded into mononucleotides and low molecular weight oligonucleotides in a 20 hr. period at 64°C. by the action of Mn (II), Co (II), Ni (II), and Cu (II) ions, and in a 2 hr. period by Zn (II) ions. The latter also degrade poly C, poly U, and RNA at approximately the same rate, but poly I is degraded very much more slowly. No such difference in reaction rates can be observed in the alkaline degradation of polynucleotides. The sluggishness of the reaction with poly I is not due to any highly ordered structure of the zinc-poly I complex, but probably to the weakening of the zinc-phosphate bond through simultaneous binding of the zinc to the inosine base. Evidence for such a hypothesis is derived from the inhibition of the zinc degradation of poly A by inosinic acid or poly I. No drastic difference in the nucleotide composition of the undegraded residue can be observed in the degradation of RNA by zinc and by alkali.

Notes: Evidence is presented that polyriboadenylic acid (Poly A) is a single-strand flexible helix of stacked bases at neutral pH and room temperature. As the temperature is raised the stacking decreases, but there is still perceptible order at 80°C. The thermal transition from the double-strand helix (which exists at low pH) to the single-strand helix has been followed by optical rotatory dispersion, optical absorption, and viscosity measurements.

Notes: The binding curves of various aminoacridines on calf thymus DNA have been determined by a spectrophotometric method. The dominant role of electrostatic forces in the interaction has been confirmed by the effect of ionic strength. Side chain and ring substituents in the aminoacridines do not inhibit binding, since this decreases in the order: acranil 〉 neomonacrin 〉 atebrin 〉 9-aminoacridine ∼ proflavine 〉 9-amino-1,2,3,4-tetrahydroacridine (THA). The last named shows the effect of diminishing the flat area of the rings in the aminoacridines. The shape of the binding curves of proflavine, 9-aminoacridine, and THA on thermally denatured DNA show that about 30-50% more binding sites are available to these cations on denatured DNA than on native DNA, but that the binding constants are the same. These observations are discussed in relation to the intercalation and other models for the interaction. It is concluded that exact and complete intercalation is not a necessary condition of strong binding and that other, less regular, models, in which the positive ring nitrogens of the acridines are close to the DNA phosphates and the acridine rings partially interact with the DNA base rings, are probably more consistent with the effect of denaturation of DNA on the binding curves which are characteristic of the interaction in solution.

Notes: In an attempt to clarify certain questions of the configuration of amylose in aqueous solution, carboxymethyl groups were introduced into corn amylose (degree of substitution ca. 0.8) and certain properties of the derivative (CMA) studied. The intrinsic viscosity exhibits a strong increase between pH 2 and 6 but remains constant at higher pH. A pH 6 and above, CMA exhibits typical polyelectrolyte behavior. CMA does not form a complex with iodine at pH 6 or above but does form a violet-colored complex at low pH. The absorption maximum of the CMA complex lies at a somewhat lower wavelength (550 mμ) than that of the complex formed by the parent amylose. The reduced viscosity and specific optical rotation of the CMA-iodine complex remains constant up to a concentration of 1.2 mg. iodine/100 mg. CMA, then both properties decrease with further increases of iodine content. All of the above results and hydrogen ion titration behavior are in agreement with the hypothesis that CMA exists as a coil of short helical segments in acid solution and that these helical segments break down on ionization of the carboxyl groups. The specific optical rotation of CMA increases between pH 2 and 6, remains constant between pH 6 and 12, and decreases above pH 12. This later result shows that the similar decrease in rotation seen in amylose cannot be attributed, at least entirely, to a helix-coil transition. There must be another effect on optical rotation, direct or indirect, of the ionization of hydroxyl groups.

Notes: A mathematical method has been developed, together with a digital computer program for its evaluation, for the determination of possible conformations of a polypeptide chain for any given amino acid sequence. Allowed conformations were required to satisfy predetermined criteria regarding the locations of potential energy minima for rotation around single bonds, steric restrictions arising from van der Waals contacts between atoms, and the formation of a closed polypeptide loop by means of a peptide linkage or a disulfide bridge. Requirements for specific noncovalent interactions can also be considered. Various assumptions regarding the selection of the minima for rotational potentials and of the van der Waals contact distances are discussed. According to the assumptions adopted in the paper, 21 conformations are allowed for a glycine residue which is part of a dipeptide. These conformations are assumed to lie at minima of the rotational potential function, and satisfy simultaneously the steric requirements. Dipeptides containing alanine or other residues are restricted to 7 such conformations. As an example of the applicability of the method, the computation was performed for the smallest loop containing a disulfide bridge in the ribonuclease molecule, i.e., the cyclic octapeptide formed by residues 65 to 72. Only 15 allowed conformations were found for the loop as a whole. Parameters characterizing these allowed conformations are tabulated. Approximate calculations indicate the importance of steric restrictions and of the presence of side chains in reducing the conformational freedom of this peptide. Further potentialities of the method are discussed.

Notes: A combinatorial formulation of the partition function is developed for short polypeptide chains, where summations can be evaluated. From the complete combinatorial expression it is possible to assess the validity of any approximations which are made in evaluating the partition function. It is shown that it is an excellent approximation to consider the hydrogen-bonded portion of a short (20 residues) chain as consisting of a single helical sequence of any size, with all sizes of helical sequences being about equally probable at the transition temperature. Another approximation, involving a linear dependence of the energy and entropy on the number of hydrogen bonds, helps to clarify the nature of the intermediate states in the denaturation process. Expressions are developed which incorporate the effect of hydrophobic bonds on the stability of the α-helix, and the binding of water to the random coil. It is concluded that hydrophobic bonds in helical sequences can greatly stabilize the helix, but that similar bonds in the random coil can largely offset this stabilization.

Notes: The Lifson-Roig matrix theory of the helix-coil transition in polyglycine is extended to situations where side-chain interactions (hydrophobic bonds) are present both in the helix and in the random coil, as discussed for short chains in paper II of this series. It is shown that the conditional probabilities of occurrence of any number and size of hydrophobic pockets in the random coil can be adequately described by a 2 × 2 matrix. This is combined with the Lifson-Roig 3 × 3 matrix to produce a 4 × 4 matrix which represents all possible combinations of any amount and size sequence of α-helix with random coil containing all possible types of hydrophobic pockets in molecules of any given chain length.

Notes: The method of sequence-generating functions is applied to long polypeptide chains to describe various types of hydrophobic bonding in the random coil. These results are then combined with a similar treatment of the α-helix in order to discuss the helix-coil transition in single helices and in molecules whose helical segments interact by side-chain hydrophobic bonding. Numerical calculations, based on the equations derived in this and preceding papers, are presented to show the relative probabilities of occurrence in the random coil of neighbor-neighbor hydrophobic bonds and pockets of hydrophobic bonding, and the relative probabilities of occurrence of the various states in a system of interacting helices. A discussion is also presented of the dependence of the helix-coil nucleation and growth parameters on solvent and side chain.

Notes: The partition function of paper II for short polypetide chains is incorporated here into one for a simple model for side-chain hydrogen bonds between helices. It is thus possible to calculate the fraction of amide hydrogen bonds, the fraction of side-chain hydrogen bonds, and the number of bound protons as a function of temperature and pH. Illustrative data are presented for the probabilities of occurrence of intermediate structures and for the influence of the overall conformation on the observed pK's of ionizable groups.

Notes: The theory of adsorption of semistiff chains on a planar surface developed by the authors previously has been used to consider the helix-coil transition in single-stranded macromolecule interacting with an adsorbent plane. The cases of nonselective interaction when the adsorption energy is independent of the unit conformation (a) and selective interaction with only helical (b) or coiled (c) sequences active in adsorption were investigated. In case (b) the existence of secondary structure favors chain bonding to the surface. This leads to the increase in the stability of the helical state and complete polypeptide chain spiralization. The profile of the conformational helix-coil transition acquires an asymmetrical shape inherent to the second-order phase transition.In case (c) the bonding of a partially helical chain to the surface is similar to the adsorption of Gaussian coils and is accompanied by the destruction of secondary structure, this destruction being appreciable even if the helical state in space was favorable.

Notes: Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double-strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10-5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single-strand breaks and a smaller number of double-strand breaks, also increased the initial rate. However, this increase was due only to the double-strand breaks, that is, single-strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 105 erg/mm2, suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.

Notes: A combination of the DeVoe and Kirkwood polarizability concepts is developed to calculate CD spectra of nucleic acid monomers. The method is perfectly general and applies to any system where the constituents have absorption properties which are widely separated in terms of frequency. The theory is applied to calculate the CD spectra of adenosine and 2′-deoxyadenosine conformers. Bond polarizabilities are evaluated for the ribosyl moiety of adenosine, as a function of glycosidic rotational angles and polarizability anisotropies. It is found that a wide range of C-C and C-O bond polarizabilities give similar CD results. Isotropic atom polarizabilities are also evaluated. It is found that the CD results using these polarizabilities do not differ significantly from those obtained with bond polarizabilities. The CD spectra of adenosine and 2′-deoxyadenosine are calculated for three x-ray diffraction determined geometries: A-form RNA, B-form DNA, and C-form DNA. The results indicate that the monomer CD spectra are strongly dependent on the precise geometry and appear to be of importance in understanding the spectra of oligomers and polymers. The deoxyadenosine conformers are found to have calculated CD spectra which are less intense than those of the ribosyl conformers. These results indicate that the measured differences between the CD magnitudes of ribo- and deoxyriboadenosine are due to the presence or absence of the 2′-hydroxyl. Weighted averaged adenosine CD spectra are calculated with the aid of probability distributions from conformational energy calculations. The results suggest a new method for obtaining empirical monomer parameters for use in optical calculations. The calculations in this paper indicate for the first time that DeVoe theory, in combination with the Kirkwood theory, provides a useful method for the calculation of the CD spectra of nonpolymeric molecules.