bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthèse de cellulose bactérienne à pertir de glycérol sélectivement deutérié en position 1, 2, ou 3: Etude par résonance magnétique nucléaire
Authors: Chumpitazi‐Hermoza Bernabé F. , Gagnaire Didier , Taravel François R.
Published: December 2004

Glycerol specifically deuterated at C‐1, C‐2, or C‐3 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum.The material obtained were converted into glucitol hexaacetate and analyzed by 250‐MHz nuclear magnetic resonance and mass spectrometry. The spectra indicated that the protons of the C‐3 position of the starting glycerol were incorporated as substituents of the C‐6 and C‐1 positions of the cellulose. Similarly, protons of the C‐2 and C‐5 positions of the cellulose came essentially from water and the protons bonded at the C‐3 and C‐4 positions of the cellulose from protons bonded to C‐1 of the starting glycerol.
Solvent accessibilities in the dimeric subunits of RNA and DNA
Authors: Ponnuswamy P. K. , Thiyagarajan P.
Published: December 2004

To obtain information on the solvent‐solute interactions in the dinucleoside monophosphates pertaining to the dimeric subunits of RNA and DNA, we have computed the accessibility of a water molecule to the oxygen atoms of the subunits following the method of Lee and Richards [J. Mol. Biol. 55, 379–400 (1971)]. The solute molecules (dimeric unit) is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the total surface accessible to the solvent molecule—and hence the solvent accessibility of various atoms of the solute molecule for different conformations—are computed. From the calculated atomic accessibilities, solvation maps in the (ω′,ω) space have been constructed, keeping ψ at 60°, 180°, and −60°. The C(3′)‐endo sugar system in the case of DNA subunit have been considered. The solvation maps describing the solvatability of single and groups of atoms give significant information on the backbone conformational domains that are preferred for solvent interaction, thus adding knowledge to the relative stability of the various possible conformations. The B‐DNA‐type conformer exposes three polar atoms—namely, PO1, O(3′), and O(1′)—to external solvent, whereas the A‐DNA‐ and C‐DNA‐type conformers expose only one polar atom—O(3′) and O(1′), respectively—to the solvent. The O(2′) atom of the furanose ring system in the RNA subunit could give added stability via solvent association or interunit hydrogen bonding with or without a bridging water. The superposition solvation maps describing the accessibility of a group of polar atoms help to interpret a good number of phosphodiester conformations observed in a energetically less favored conformational domains in the tRNAPhe crystal. Another intresting fact that results from this study is the prediction that the trans oriented of ω is the most favorable conformations of random‐coil polynucleotides in solution.
Conformational properties of the N‐terminal residues of S‐peptide. II. The guanidine hydrochloride–water–trifluoroethanol system
Authors: Filippi B. , Borin G. , Moretto V. , Marchiori F.
Published: December 2004

The Conformational properties of synthetic S‐peptide analogs, in which the residues in the N‐terminal sequence 1–6 were progressively deleted or replaced with amino acids of lower helical propensity, were studied by CD. Increasing the concentration of guanidine hydrochloride and decreasing the temperature were found to produce progressive destruction of ordered conformations, in the parallel with the increasing solubility of the peptide unit, while increasing the concentration of trifluoroethanol and decreasing the temperature produced the opposite effect. The maximum helicity determined in the these sets of experiments is found equal to or greater than that determined in the formation of the ribonuclease S′ complexes. With some peptides the maximum value of predicted helical conformation is reached, and the tendency of tertiary structure to reduce the maximum possible helicity is evident. We discuss the validity of the procedure by which conformational information, drawn from measurements in helicogenic solvents, is related to the state in native protein.
Solvent effect in the stereoselective polymerization of α‐amino acid N‐carboxyanhydride and some considerations on the mechanism of stereoregulation
Authors: Hashimoto Yutaka , Imanishi Yukio , Higashimura Toshinobu
Published: December 2004

In the Polymerization of phenylalanine N‐carboxyanhydride (NCA) in No2Oh initiated by MeNHBzl, L‐,D‐, and DL‐NCA As were polymerized at the same rate, and no stereoselectivity was observed. When the same experiment was carried out in HCONEt2, however, L‐ and D‐NCA were both polymerized at a rate which was about twice as large as that of DL‐NCA. In this case, the polymerization is stereoselective, ascribable to a preferable reaction between the optical enantiomorphs of the terminal residue of the growing chain and the NCA of the same chirality. On the other hand, the polymerization initiated by SarNMe2 and MeNH(CH2)2CONMe2 were stereoselective in NO2Ph and HCONEt2, but they were not stereoselective in m‐(MeO)2Ph. These findings indicate that the polymerizations initiated by a strong base in highly dipolar solvents are stereoselective. Apparently, the reaction between a chiral, cyclic terminal of growing chain and a chiral, cyclic activated NCA in the activated‐NCA mechanism is highly stereoselective. In addition, from a kinetic investigation on on the copolymerization between L‐ and D‐NCAs, the penultimate chiral centers were also suggested to contribute to the stereoselection. Stereoselection by the α‐helical conformation of the growing chain and by a chiral, linear terminal amine have been considered so far, and the contribution from the present type of stereoselection must have been overlooked.
Pepsin‐catalyzed peptide synthesis
Authors: Pellegrini Antonio , Luigilusis Pier
Published: December 2004

The use of pepsin as a catalyst for the synthesis of peptide bonds was investigated. It is shown that the enzyme enables the preparation of several protected dipeptides and tripeptides containing two adjacent aromatic residues of the type P‐Al‐Phe‐Y, P‐PHe‐Ar‐Y, or P‐AR‐Phe‐Y where P and Y are amino and carboxyl protecting groups, AL is an aliphatic amino acid residue, and Ar is an aromatic, amino acid residue. They yields are in the rang 25–97%. The high yields, combined with the enzyme's stereospecificity, permit the isolation of optically pure enantiomers from racemic mixtures. For example, when Z‐DL‐Ph‐OH is allowed to react with an excess of H‐L‐Phe‐NH2, the stereoisomer Z‐L‐Phe‐L‐Phe‐NH2 is obtained in practically quantitative yield. At the same time, the unreacted, optically pure Z‐D‐Phe‐OH can be recovered (Z = carbobenzyloxy, Phe = phenylalanine). The advantages and disadvantages of the enzymatic coupling procedure as a possible routine method for peptide synthesis are discussed.
Thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes in butanol/aqueous salt biphasic solutions
Authors: Hurst Robert E.
Published: December 2004

The thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes wee studied in order to gain further insight into the mechanisms responsible for the sensitivity of the relative solubility of these complexes in aqueous slat and butanol phases to small changes in slat concentration. The dependence of the partition coefficient was measured as a function of temperature at three different salt concentrations. Increasing the temperature was found to favor the form of the complex which was soluble n the aqueous phase. Although the transition could be induced by temperature changes, he transition occurred over a 20°C range in temperature. The transition from the aqueous phase to the butanol phase was strongly exothermic, with ΔH = −22.3 kcal/mol polymer. The value of ΔS was found to be dependent on the salt concentration, ranging from −72.7 e.u./mol polymer in 0.05125M NaCl to −77.1 e.u./mol polymer in 0.05375M MaCl. When placed on a disaccharide basis, the corresponding values are ΔH = −402 cal/mol and ΔS = −1.31 to −1.3 e.u./mol. The sharpness of he transition was found to be due t the similarity in magnitude of ΔH and TΔS, and on the dependence of the later upon the salt concentration.
Comparison of the helix–coil conformational changes of poly(deoxyadenylate‐deoxytymidylate) and poly(deoxyadenylate‐deoxythymidylate) induced by variations of hydrogen‐ion concentration
Authors: Hirai Toshihiro , Hamori Eugene
Published: December 2004

Double‐helical poly(dG‐dC) and poly(dA‐dT) are DNA analogs in which the interactions between the two strands of the helix are, respectively, either the stronger G/C type or the weaker A/T type along the entire length of macromolecules. Thus, these synthetic polynucleotides can be considered as representatives of the most stable and the least stable DNA. In the investigations presented here, potentiometric titrations and stopped‐flow kinetic experiments were carried out in order to compare the pH‐induced helix–coil conformations (10°C and 150mM [Na+]) the pH of the helix–coil transition (pHm) is 12.81 for poly(dG‐dC) and 11.76 for poly(dA‐dT). The unwinding of double‐helical poly(dG‐dC) initiated by a sudden change in pH was found to be a simple exponential process with rate constants in the range of 200–600 sec−1, depending on the final value of the pH jump. The intramolecular double‐helix formation of poly(dG‐dC) was studied by lowering the pH of the solutions from a value above pHm to that below pHm in dilute solutions (15.5 ug/ml [polymer]). Under these conditions, the observed rewinding reactions displayed a major and two exponential phases, all of which were independent of polymer concentration. From the comparison of the results of poly(dA‐dT) and poly(dG‐dT) would unwind faster than poly(dG‐dC). However, if the pH jumps are such that they present the same perturbation of these polymers relative to their pHm values, no significant differences exist between the rates of helix–coil conformation changes of poly(dA‐dT) and poly(dG‐dC).
Intensity‐fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process
Authors: Patkowski A. , Jen S. , Chu B.
Published: December 2004

We have measured the translational (DT) and rotational (DR) diffusion coefficients of bulk tRNA from baker's yeast during the thermal unfolding process by means of photon‐correlation spectroscopy. It should be noted that our estimate of the rotational diffusion coefficient represented, for the first time, measurements on a small macromolecule in solution by the photoelectron time‐of‐arrival technique with a delay‐time resolution of 1 nsec. The melting curves expressed in terms of δDT vs temperature were consistent with the literature data in revealing the melting steps and their dependence on NaCl concentration. Additionally, it was possible to prove the existence of an intermediate, more compact structure during the initial steps of the thermal unfolding process. We found that the temperature ranges over which this intermediate structure appears depend strongly on salt concentration. By utilizing both translational and rotational diffusion coefficients and Perrin's equations for ellipsoids of revolution, we have computed the values of the equivalent length and width of tRNA molecules in solution at four different temperatures for NaCl concentrations of 0.2, 0.5, and 1M. The approximate model of ellipsoids of revolution also permits us to obtain an estimate of the radius of gyration, which is in very good agreement with literature data measured by means of small‐angle x‐ray scattering. Furthermore, we have measured the shape and size changes of tRNA with varying NaCl concentrations at room temperatures (25°C). The molecule becomes smaller and more spherical when NaCl concentration increases. As a result of partial melting at 70°C, the macromolecule is surprisingly elongated with an approximate axial ratio of 8:1 and has dimensions of about 180/22Å. Such information on conformational changes by a simultaneous determination of rotational and translational diffusion coefficients illustrates the potential of this approach, not available by other methods.
Transient electric birefringence of T‐even bacteriophages. IV. T2L0 and T6 with extended tail fibers
Authors: Boontje W. , Greve J. , Blok Joh.
Published: December 2004

Transient electric birefringence measurements of the bacteriophages T2L0 and T6 were performed under such conditions that the tail fibers are extended. The data obtained are compared to previously reported data for T4B. For all T‐even phages the degree of extension of the tail fibers is a function of pH, ionic strength, and temperature. For T4B, much higher ionic strengths are needed than for T2L0 and T6 to accomplish complete tail‐fiber extension. The rotational diffusion coefficients of the phages with fully extended fibers are equal to 120 ± 3 sec−1, 132 ± 5 sec−1, 157 ± 4 sec−1 for T2L0, T4B, and T6, respectively. The respective optical anistropies are − (2.66 ± 0.05) × 10−4, and − (3.07 ± 0.15) × 10−4. The differences in the rotational diffusion coefficient and optical anisotropy arise because the conformation of the fully extended tail fibers is different for the three phages. The tail fibers of T2L0 project further into the solution (away from the head) than do those of T4B and T6. The apparent permanent dipole moments of T2L0 and T6 decrease with increasing ionic strength. This decrease is caused by the screening of the surface charges on the phage body by the counter‐ions in the solution. The biological relevance of this decrease is illustrated by the fact that the adsorption rate of T6 phages to E. coli B bacteria shows a similar dependence of ionic strength. Evidence is pressented that the tail fibers may move more or less independently of the phage body when an electric field is applied to the suspension.
Light scattering during the hysteresis effect in poly(A)·2poly(U)
Authors: Siano Donald B.
Published: December 2004

Light‐scattering studies on buffered aqueous solutions of the triple‐stranded polyribonucleic acid poly(A)·2poly(U) were carried out at neutral pH and during titration. At pH 7.1 and 22°C, a sample of commercially available polymer in 0.005M phosphate buffer gave a Zimm plot which yielded values for the weight‐average molecular weight, Mw, of 874,000 ± 1800 g/mol, a root‐mean‐square radius, ρ of 930 ± 22 Å, and a second viral coefficient of 0.51 ± 0.05 × 10 −3 cm3g−1 mol. The light‐scattering data were also analyzed by serval linear and nonlinear least‐squares programs which were devised to determine the model (e.g., rod, coil, or zigzag) which could best describe the shape of the molecule. It was found that a rodlike model, perhaps with a few bends, was in best overall agreement with the data. The assumption that the molecule is a thin rod leads to a value for the linear density of 206 g mol−1 Å−1 and a translation of 3.3 Å per residue. These values are also in close agreement with those expected for a triple‐stranded, thin, base‐stacked molecule. During titration from neutral pH with 0.1M HCl, the observed apparent molecular weight slowly increased until at about pH 3.5 a sudden, large increase (about 30‐fold) occurred. The root‐mean‐square radius, on the other hand, after an initial small decrease (of about 25%), also exhibited a large increase (about 4‐fold). Upon back titration with 0.1M NaOH, the molecular parameters did not retrace the original path, but instead exhibited hysteresis—the Mw and ρz are both larger on the basic branch than on the acid branch at a corresponding pH. A plot of long ρz against log(Mw) during the interval in which the high‐moelcular‐weight form was present (below pH 3.5 on the acid branch, and on the basic branch) gave a straight line with a slope of ⅓. This suggests that the aggregates were composed of some tens of rather open radom coils, presumably of poly(A)·poly(A), and that the hysteresis may be caused under conditions by the metastability of the entangled coils.
The conformation of the anticodon loop of yeast tRNAPhe in solution and on ribosomes
Authors: Odom O. W. , Craig B. B. , Hardesty B. A.
Published: December 2004

The Y‐base of yeast tRNAPhe was replaced by the fluorophores 1‐aminoanthracene or proflavine to yield derivatives which are active in all of the reactions of peptide elongation on reticulocyte ribosomes. The relatively long lifetime, higher quantum yield, and environmental sensitivity of 1‐aminoanthracene make it a particulary useful adjunct to the Y‐base in studying conformational changes in the anticodon region. The absorption and emission spectra of 1‐aminoanthracene in tRNA in solutions in which it is active in peptide synthesis indicate that the probe is in a hydrophobic environment, apparently provided by stacking with the adjacent bases in the anticodon loop. The proflavine derivative, tRNA, was employed in iodide quenching, D2O enhancement, and fluorescence depolarization experiments. The results indicate that the fluorophore in partially but not completely protected from the solvent. Anisotropy studies indicate that in solutions approximating those which support peptide synthesis on ribosomes, the probes have significant but restricted flexibility within the anticodon loop. Considered with nmr data and Y‐base fluorescence from crystals of tRNA, the results indicate that the solution and crystal structures of tRNAPhe are very similar. In turn, fluorescene from modified tRNAPhe bound to ribosomes is similar to that observed in solution. It is of special significance for future experiments involving nonradiative energy transfer that these probles adjacent to the anticodon retain independent flexibility when bound to ribosomes with poly(U). The tRNAPhe itself appears to be held rigidly on the ribosomes. It is concluded that within the limits dictated by the position and sensitivity of the probes used in this study, the mechanism of tRNAPhe binding to ribosomes and the movement of tRNA and mRNA during the translocation steps of peptide synthesis can be interpreted in terms of the well‐defined crystal structure of tRNAPhe.
The spatial distributions of randomly coiling polynucleotides
Authors: Yevich Rosemarie , Olson Wilma K.
Published: December 2004

Statistical mechanical averages of vectors and tensors characterizing the allowed configurations of randomly coiling polynucleotides have been calculated for chains of 20–210 repeating units. Specifically, the persistence vector p = 〈r〉 has been obtained as a function of chain length. Configurational averages of the Cartesian tensors formed from the displacement vector ρ = r–p have been computed up to and including the tensor of seventh rank. From these tensors the three‐dimensional spatial distributions of end‐to‐end vectors have been constructed to provide comprehensive pictures of the directional tendencies of the randomly coiling polynucleotide. The elements of the third and fourth moment tensors, however, give sufficient information to represent accurately the qualitative features of the spatial distributions. For long chains, more than 26 (64) repeating units, the spatial distributions assume spherically symmetric shapes that can be adequately characterized by one‐dimensional radial distribution functions. These radial distribution functions agree well with the radial distributions calculated from Monte Carlo samples containing more than 5000 chains. The constraints of fixed bond lengths, fixed bond angles, and hindered internal rotations severely distort the spatial distributions of short polynucleotide chains to mushroom‐shaped volumes. These skewed distributions provide information useful to the analysis of small, single‐stranded loops, bulges, and circles. The formation of these structures requires the termini of the polynucleotides to lie within specifically delineated volumes with respect to coordinate systems affixed to the first bonds of the chains. The extent to which these loop closure volumes overlap the three‐dimensional spatial distributions provides estimates of loop formation that are much more reliable than earlier studies based upon the radial distribution function.
Hydrophobic interactions of DNA with long‐chain amines
Authors: Chatterjee R. , Chattoraj D. K.
Published: December 2004

The binding ratio, Γa, for several long‐chain amines to calf‐thymus DNA was measured as function of the ligand concentration, C, using the equilibrium dialysis method. The different amines used in the binding experiments at constant temperature were dodecyl trimethyl ammonium bromide (DTAB), myristyl trimethyl ammonium bromide (MTAB), cetyl trimethyl ammonium bromide (CTAB), and cetyl pyridinium chloride (CPCL). The formation and dissociation of the saturated DNA–amine complex were reversible. The initial slope of the binding isotherm decreased sharply with the reduction of the electrostatic effect as a result of the increase of the ionic strength of the medium. A sharp inflexion region was noted in the binding isotherm where the ligands bound in significant numbers may undergo hydrophobic interactions with each other. Γa increased with C until a maximum value, Γam, was reached, beyond which binding slowly decreased with an increase of concentration. Both Γam and Γa increased significantly with the increase of the hydrocarbon chain length of a ligand. The free energy change ΔGm for each saturated DNA–amine complex was evaluated on the basis of a thermodynamic relation and the standard state for binding was defined. The average free energy change for the binding per CH2 group of the amine was found to be −1550 cal/mol. The difference between ΔGm for CTAB and CPCL was examined on the basis of the structural difference of their head groups. The binding isotherms for MTAB and CPCL were obtained from the binding data at 15, 30, and 45°C. The binding increased with increasing temperature. From the plot of ΔGm/T vs 1/T, the changes in enthalpy and entropy due to the binding were evaluated for MTAB and CPCL. The binding reactions in these two cases were driven primarily by the entropy change due to the hydrophobic interaction. Standard free energy changes ΔG0m for the unsaturated complexes were close to ΔGm for the saturated complexes. The binding isotherms also depended on the nature of the neutral salt of the medium. At a given salt concentration, the order of the binding of the inorganic salts was as follows: KCl > NaCl > LiCl > Na2SO4 > MgCl2. The effect of pH on binding was also examined. The importance of these results on the formation of the reconstituted and natural nucleohistone complexes is discussed.
Use of amide 1H‐nmr titration shifts for studies of polypeptide conformation
Authors: Bundi Arno , Wüthrich Kurt
Published: December 2004

This paper shows that backbone amide proton titration shifts in polypeptide chains are a very sensitive manifestation of intramolecular hydrogen bonding between carboxylate groups and backbone amide protons. The population of specific hydrogen‐bonded structures in the ensemble of species that constitutes the conformation of a flexible nonglobular linear peptide can be determined from the extent of the titration shifts. As an illustration, an investigation of the molecular conformation of the linear peptide H‐Gly‐Gly‐L‐Glu‐L‐Ala‐OH is described. The proposed use of amide proton titration shifts for investigating polypeptide conformation is based on 360‐MHz 1H‐nmr studies of selected linear oligopeptides in H2O solutions. It was found that only a very limited number of amide protons in a polypeptide chain show sizable intrinsic intration shifts arising from through‐bond interactions with ionizable groups. These are the amide proton of the C‐terminal amino acid residue, the amide protons of Asp and the residues following Asp, and possibly the amide proton of the residue next to the N‐terminus. Since the intrinsic titration shifts are upfield, the downfield titration shifts arising from conformation‐dependent through‐space interactions, in particular hydrogen bonding between the amide protons and carboxylate groups, can readily be identified.
Synthesis and conformation of a polyoxyethylene‐bound undecapeptide of the alamethicin helix and (2‐methylalanyl‐L‐alanine)1–7
Authors: Mayr W. , Oekonomopulos R. , Jung G.
Published: December 2004

The stepwise synthesis and conformational studies of the N‐terminal helical partial sequence of the membrane‐modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N‐t‐Boc‐L‐Pro‐Aib‐Ala‐Gln‐Aib‐Val‐Aib‐Gly‐OH and N‐Ac‐Aib‐L‐Pro‐Aib‐Ala‐Aib‐Ala‐Gln‐Aib‐Val‐Aib‐Gly‐OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α‐helix formation of the N‐protected peptide polymers beginning with the nonapeptide and of the N‐protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature‐ and solvent‐dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random‐coil conformation.

For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib‐L‐Ala)n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right‐handed α‐helix with increasing peptide length. This change is observed for the N‐protected peptides at a chain length of 8 residues and for the N‐protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene‐bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t‐Boc‐(Aib‐L‐Ala)n‐OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.
Quasielastic light scattering by biopolymers. III. Effect of ionic strength on internal dynamics of DNA
Authors: Schmitz Kenneth S.
Published: December 2004

Quasielastic light‐scattering data on DNA in 0.1, 0.5, and 1.0M NaCl, neutral pH, have been obtained in the scattering angle range 15 ⩽ θ ⩽ 50. The relaxation curves have been analyzed as a single exponential with a reciprocal relaxation time of the form 2DeffK2, where K is the fluctuation vector [proportional to sin(θ/2)] and Deff is the effective diffusion coefficient. The 1/τ vs sin2(θ/2) curves exhibit two regions that are proportional to sin2(θ/2), in accordance with the Lee‐Schurr and Lin‐Schurr theories for independent segment‐mean force and Rouse‐Zimm flexible polymers, respectively. In the region KR < 1, where R is a characteristic length, Deff is simply the translational diffusion coefficient, whereas Deff in the region KR > 1 represents the diffusion coefficient for a polymer segment. The data reported here indicate that the “transition” region between the two K2 extremes is strongly dependent on the ionic strength of the solvent. This observation, along with the salt variation of the segmental diffusion coefficient, suggests the internal dynamics of DNA are dependent on the ionic strength of the solvent. The relation of the present study to those of Lin and Schurr and of Caloin et al. is discussed.
Thermodynamics of mucopolysaccharide–dye binding. III. Thermodynamic and cooperativity parameters of acridine orange–heparin system
Authors: Menter Julian M. , Hurst Robert E. , Nakamura Nobuto , West Seymour S.
Published: December 2004

Binding isotherms for acridine orange (AO)–heparin systems can be evaluated solely on the basis of quantitative fluorescence spectroscopic measurements. The evaluation of thermodynamic parameters indicates that the interactions of AO with heparins from several animal sources are similar to each other in magnitude. Binding is highly exothermic (ΔH = −6 kcal mol−1) and is stabilized by dye–polymer and dye–dye (coopertive) interactions, as well as by entropic factors (ΔS = +7 e.u.). The predominant stabilizing factor appears to be the electrostatic attraction between the AO cation and the heparin polyanion, although the other factors are important as well. At 24°C the value of the cooperative binding constants for the various heparins range from 8.8 to 11.3 × 105M−1, corresponding to a free energy of −8 kcal mol−1. The degree of cooperativity, which is a direct measure of dye–dye interaction, varies with polymer:dye ratio; the theoretical basis for this variation remains to be elucidated. Electrophoretic data indicate that each heparin sample consists of a mixture of species, each with its own charge density. This precludes definitive interpretation of observed small differences in the values of the thermodynamic parameters among the various samples until each sample can be resolved into its components.
Application of distance geometry to protein tertiary structure calculations
Authors: Kuntz I. D. , Crippen G. M. , Kollman P. A.
Published: December 2004

A general approach to the problem of molecular conformation is advanced. We describe a formalism that permits experimental and theoretical information to be incorporated into a set of upper and lower bounds on intramolecular distances. Structures (conformations) meeting these bounds can be readily generated and compared with each other. To illustrate the use of the method, we have employed a simple “firehose” model for protein folding to predict the long‐range hydrophobic interactions in a small protein: pancreatic trypsin inhibitor. Models of this type lead to the proper hairpin turns and a reasonable set of long‐range contacts for this protein. Application of the distance geometry method then yields backbone conformations with errors of 4–8 Å compared to the native structure. We discuss both the merits and shortcomings of the firehose model and the relation between distance geometry and energy minimization techniques.
Co‐oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co‐oligopeptides of tryptophan and glycine
Authors: Skrabal Peter , Rizzo Vincenzo , Baici Antonio , Bangerter Felix , Luisi Pier Luigi
Published: December 2004

The 1H‐nmr spectra of co‐oligopeptides of tryptophan and glycine with structure H‐Gly‐Trp‐(Gly)n‐Trp‐Gly‐OH (n = 0–2) and those of several di‐ and tripeptides have been recorded at 360 MHz with CD3OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H‐(αL,βS)(α,β‐d2)Trp‐OH, H‐Trp‐(αL,βS)(α,β‐d2)Trp‐OH, and H‐Trp‐(δ1,ε2,ζ2,ζ3,η2‐d5)Trp‐OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ1 and (in two cases) χ2 torsion angles of the side chains has been obtained from the vicinal coupling constants 3J and from the long‐range coupling constants 4J. These data and an analysis of the chemical shifts of the Gly‐Cα protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N‐ and C‐side: ‐NH2 > –NHCO– > –CONH–> –COO−. This rule does not hold for the second Trp residue of di‐ and tripeptides containing the ‐Trp‐Trp‐ sequence, which has tentatively been attributed to steric effects.
The conformation and aggregation of bovine β‐casein A. II. Thermodynamics of thermal association and the effects of changes in polar and apolar interactions on micellization
Authors: Evans M. T. A. , Phillips M. C. , Jones M. N.
Published: December 2004

A sedimentation analysis has been used to determine the proportion of protein present as monomer and aggregate in 0.5 and 1.0 g/dl solutions of β‐casein A in pH 7 phosphate buffer over the temperature range 10–40°C. The amount and molecular weight of the aggregate increase with temperature; under the conditions used, the aggregation number (n) of β‐casein is given approximately by n = 0.6t + 2 with t in degrees centigrade. The concentration of β‐casein in monomeric and aggregated states at different temperatures is used to calculate the standard enthalpy of aggregation ΔH° (Van't Hoff) by assuming that β‐casein undergoes a cooperative, two‐state, micellization process; aggregation is an endothermic process and ΔH° = 66.0 ± 2.6 kJ mol−1. Combination of this ΔH° with the amount of protein calculated to dissociate when 1 g/dl solutions are diluted isothermally to 0.5 g/dl gives the heat of dilution at various temperatures. These calculated heats of dilution are compared with the experimental values obtained by carrying out the same dilutions in a microcalorimeter. The heat of dilution decreases linearly with β‐casein concentration, but the extrapolated zero‐concentration values of 65.8 ± 1.6 kJ mol−1 is the same as the Van't Hoff enthalpy. This agreement in the enthalpy values indicates that the micellization of β‐casein occurs cooperatively. The effect of modifying the hydrophobic/hydrophilic balance of the system on the micellization of β‐casein A has been investigated. The hydrophobic interaction between the protein molecules is decreased by removing the three C‐terminal residues (Ileu Ileu Val) with carboxypeptidase‐A. This modification drastically reduces the ability of the β‐casein molecule to form micelles. Substitution of 2H2O for H2O at constant temperature perturbs the monomer–micelle equilibrium in favor of micelles because of enhanced hydrophobic interactions in the former solvent. The results are consistent with β‐casein micellization involving a delicate balance of the hydrophobic forces favoring aggregation and electrostatic forces opposing it.
Experimental investigations on the backbone folding of proline‐containing model tripeptides
Authors: Boussard Guy , Marraud Michel , Aubry André
Published: December 2004

Some proline‐containing tripeptides with the general formulas R0CO‐L‐Pro‐X‐NHR3 (X = Gly,Sar,L‐Ala,D‐Ala) and R0CO‐X‐L‐Pro‐NHR3 (X = Gly,L‐Ala,D‐Ala) have been investigated in solution by ir and 1H‐nmr spectroscopies. Their favored conformational states depend mainly on both the primary structure and the chiral sequence of the molecules. In inert solvents the βII‐folding mode is the most favored conformation for the L‐Pro‐D‐Ala and L‐Pro‐Gly tripeptides, while the βII′‐turn is largely preferred by D‐Ala‐L‐Pro derivatives. Under the same conditions only about one‐third of the whole conformers of L‐Pro‐L‐Ala molecules adopts the βI‐folding mode. Semiopened C7C5 and C5C7 conformations are appreciably populated in the L‐Pro‐L‐Ala sequence, on the one hand, and in the Gly‐L‐Pro and L‐Ala‐L‐Pro derivatives, on the other hand. In L‐Pro‐Sar and X‐L‐Pro models, the cis–trans isomerism around the middle tertiary amide function is observed. Thus cis L‐Pro‐Sar and L‐Ala‐L‐Pro conformers are folded by an intramolecular i + 3 → i hydrogen bond, whereas cis D‐Ala‐L‐Pro and Gly‐L‐Pro molecules accommodate an open conformation. In dimethylsulfoxide the βII‐ and βII′‐folding modes are not essentially destabilized, as contrasted with the βI conformation, which is less populated. In water solution all the above‐mentioned conformations, with the possible exception of the βII′‐folding mode for D‐Ala‐L‐Pro molecules, seem to vanish. Solute conformations are also compared with the crystal structures of four proline‐containing tripeptides.
Dynamic light scattering from collagen solutions. II. Photon correlation study of the depolarized light
Authors: Thomas John C. , Fletcher Guy C.
Published: December 2004

The depolarized forward‐scattered light from solutions of rat tail collagen has been studied by photon correlation spectroscopy. The measured autocorrelation function is seen to decay on two widely different time scales. The decay time for the fast component is consistent with the rotational diffusion of rodlike collagen monomers. The slowly decaying autocorrelation component is attributed to large nonspecific aggregates of collagen. A substantial fraction of the collagen is in this aggregated form. Extrapolation of the faster decay times to zero concentration yields a value of θ = 1082 ± 30 sec−1 for the rotational diffusion coefficient of the collagen monomer.
Mechanism for unwinding of double‐helical polynucleotides by formaldehyde
Authors: Chay Teresa Ree
Published: December 2004

We have formulated a mathematical model for the unwinding process of polynucleotides induced by formaldehyde by making use of the master equation approach. Our model assumes that the formaldehyde kinetics follows the helix–coil theory of Zimm and Bragg. The model incorporates experimental features such as the interplay between pH‐dependent and ‐independent reactions and the dependence of the initial and final helix stability on the unwinding. That is, our model incorporates the existence of the critical point such that if the pH of the system is high enough (i.e., pH ≳ 7), the unwinding of polynucleotides occurs by means of two separate chemical reactions, either by the HCHO–imino reaction alone or by the HCHO–amino reaction alone. The critical point depends on the ionic strength, temperature, and the formaldehyde concentration satisfying the relations= 1 +KUλ, wheresis the helix stability parameter,KUis the binding constant for the HCHO–imino reaction, and λ is the formaldehyde concentration. Thus above the critical point, i.e.,s< (1 +KUλ), the unwinding is due to the reaction of imino groups with formaldehyde, and below the critical point, i.e.,s> (1 +KUλ), the unwinding is due to the reaction of amino groups with formaldehyde. Although, below the critical point, the imino group does not participate directly in the rate‐determining step, it participates indirectly in such a manner that it reduces the initial helix stability parameterstos/(1 +KUλ) by forming a complex. The kinetic constants in the master equation have been determined by making use of the principle of detailed balance and the breathing mechanism proposed in the past. With this model we have quantitatively explained the anomalous temperature dependence observed in the kinetics of formaldehyde‐induced poly[d(A‐T)] and the salt dependence observed in poly(A·U).
Difference in substrate binding processes between intact and iodine‐inactivated lysozyme
Authors: Segawa Shin‐Ichi , Nakayama Masaaki
Published: December 2004

The binding of glycol chitin to intact and iodine‐inactivated lysozyme was studied by measuring the absorbance of the complex with N‐methylnicotinamide chloride, which binds to the subsite C in lysozyme as a competitive inhibitor. The association constant of glycol chitin to inactivated lysozyme was determined from static experiments to be 1.7 × 104M−1. The kinetics of the substrate binding to intact and iodine‐inactivated lysozyme were measured by the stopped‐flow method at 23°C and pH 5.6. The binding to inactivated lysozyme was clearly monophasic, whereas in intact lysozyme it consisted of multiple phases. In the substrate binding to intact lysozyme, a fast bimolecular process and two subsequent slow unimolecular processes were observed besides the hydrolysis process of polymer substrate. These slow phases were missing completely in inactivated lysozyme. It results from the alteration in the local structure occurring at the subsite D in inactivated lysozyme. These results mean that the slow phases are important for catalytic action of lysozyme. The rate constants of association and dissociation in the fast bimolecular process were determined in this paper. Furthermore, the association constant of the substrate to intact lysozyme was also determined kinetically to be 6.5 × 103M−1.
7‐Amino‐actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA
Authors: Chiao Yu‐Chih Chen , Rao K. Gurudath , Hook John W. , Krugh Thomas R. , Sengupta Sisir K.
Published: December 2004

Fluorescence, CD, absorption, and 1H‐nmr studies are reported for complexes of 7‐amino‐actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG‐dC)· poly(dG‐dC). The optical spectra for the 7‐amino‐actinomycin D complex with pdG‐dC, pdG‐dC‐dG‐dC and pdC‐dG‐dC‐dG are similar in shape to the 7‐amino‐actinomycin D complex with either DNA or poly(dG‐dC). The changes in the 1H chemical shifts of the 7‐amino‐actinomycin D and the pdG‐dC resonances that accompany complex formation show that 7‐amino‐actinomycin D forms a minature intercalated complex with two pdG‐dC molecules. The magnitudes of the induced chemical shifts for the 7‐amino‐actinomycin D complex formation with pdG‐dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG‐dC molecules. The pdN‐dG dinucleotides (N = C, A, or T) form stacked complexes with 7‐amino‐actinomycin D. The presence of the 7‐amino‐group results in a larger dimerization constant (in aqueous solution) for 7‐amino‐actinomycin D [KD(6°C) = 4.4 × 103M−1], as compared to actinomycin D [KD(6°C) = 1.7 × 103M−1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7‐amino‐actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG‐dC)·poly(dG‐dC), and poly(dA‐dC)·poly(dG‐dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.
Bromophenol red as a probe of lysozyme‐active site environment: A temperature‐jump study
Authors: Krishnamoorthy G. , Prabhananda B. S. , Gurnani S.
Published: December 2004

Spectrophotometric techniques have been employed to study the binding of bromophenol red (BPR) to hen egg white lysozyme and the consequent inhibition of enzyme activity. Experimental evidence is given from the dye binding studies in the presence of hexasaccharide and from the studies on activity that BPR binds at a site outside the proposed cleft region (A–F) in such a way that it inhibits the lytic activity towards cell walls but does not inhibit the activity towards hexasaccharide. These observations are consistent with the kinetics of binding [studied using temperature‐jump (T‐jump)] in the presence of Co++ or chitotriose in large concentrations and the experiments with acetylated lysozyme which suggest that the binding site of BPR is closer to a lysine residue near the cleft. It is suggested that the binding site of BPR could be important in positioning the peptide segment of the cell walls, which are cleaved in the cleft.

Evidence for the statement that this binding takes place at least by a two‐step process, in which the bimolecular step is followed by a slower monomolecular step, is given from the observations of two types of 1:1 complexes at 24°C in equilibrium studies and from the concentration dependence of the relaxation observed at 605 nm in the T‐jump experiments. The binding process is examined by analyzing the T‐jump data obtained between 18 and 33°C in the pH range 5.2–9.2 and ionic strength 0.01–01. The ionic strength and pH dependences of the equilibrium constant associated with the bimolecular step k2/k1 and the forward rate constant associated with monomolecular step k3 have been given as evidence for the suggestion that a Coulombic interaction is involved in the first step of binding. However, the final state of binding is hydrophobic in nature. The enthalpy of activation ΔH and the entropy of activation ΔS associated with kf[= k3(k1/k2)] showed compensation behavior with pH variation, with maxima around pH ∼ 7.5 in H2O. This has been interpreted as a maximal disordering of water structure in a region of the enzyme at this pH during the monomolecular step. However, the binding of chitotriose or Co++ in the cleft reduces the ΔH and ΔS associated with the monomolecular step of BPR binding, probably by disordering the structured water during their binding in the cleft. The differences in the kinetic parameters obtained in H2O and in D2O probably arise due to subtle differences in the conformation of the enzyme in the two solvents and apart from isotope effects.

The correlation between the pH (or pD) dependence of the “intrinsic activity” towards cell walls and ΔH or ΔS indicates that ordered water structure could be playing a role in controlling the catalytic activity. It is also suggested that this factor is associated with the rate constant k3s of the monomolecular step leading to the formation of the final bound state of the substrate in cell lysis, which is also a factor controlling kcat.
Circular dichroism study on polyelectrolytic properties of carboxymethyldextran
Authors: Gekko Kunihiko
Published: December 2004

The effects of neutralization, charge density, concentration, counterion, and added salts on CD spectra of carboxymethyldextran (Cm‐dextran) were systematically investigated in order to understand the polyelectrolytic effects of ionic polysaccharides on CD spectra. CD curves of sodium Cm‐dextran showed a negative band near 214 nm due to the carboxyl n – π* transition, but the acid spectrum indicated an additional positive band near 225–228 nm, depending on the charge density. The increase in charge density caused the increase in the magnitude of ellipticity and the red shift of the band. The magnitude of the CD band showed a remarkable concentration dependence in water without any change of position and shape but was independent of concentration in the presence of salt. CD spectra of Cm‐dextran were drastically influenced by the kind of counterion and added salts. These variations of CD spectra under different conditions are discussed from the viewpoint of ion binding and the intermolecular and intramolecular electrostatic interactions of this polysaccharide.
Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt‐free tRNA in solution
Authors: Patkowski A. , Chu B.
Published: December 2004

The influence of sodium ion concentration in solution on the initially salt‐free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (DT) and rotational (DR) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H2O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl2 solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low‐salt or salt‐free tRNA solutions, we detected in the linewidth distribution function an extra‐fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light‐scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long‐range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt‐free or low‐salt solutions.