bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective fluorescent labeling of amino groups of bovine pancreatic tryspin inhibitor by reductive alkylation
Authors: Amir Dan , Levy Daniel P. , Levin Yehuda , Haas Elisha 
Published: December 2004

AbstractBovine pancreatic trypsin inhibitor (BPTI) was reductively alkylated with 2‐methoxy‐1‐naphthyl aldehyde and sodium cyanoborohydride (NaCNBH3). All five possible derivatives, each labeled at one of the primary amino groups of BPTI, were obtained. The distribution of yields of the various derivatives can be controlled by changing the reaction conditions. Products were identified by high‐performance liquid chromatography (HPLC) tryptic peptide mapping. This procedure was used for the preparation of three pure 2‐methoxy‐1‐naphthyl‐methylenyl‐BPTI (MNA‐BPTI) derivatives. Purification was achieved by means of affinity chromatography and HPLC. The spectral characteristics of the probe, notably monoexponential decay with a lifetime of 6.8 ± 0.1 ns and moderate limiting fluorescence polarization, P = 0.3 ± 0.015, make it a very useful donor in energy‐transfer measurements.
Phosphorus nmr spectra of natural DNA fragments in the course of the B‐to‐A conformational transition
Authors: Kypr Jaroslav , Sklenář Vladimír , Vorlíč‐ková Michaela 
Published: December 2004

AbstractChanges in the 31P‐nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B‐to‐A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the 31P‐nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B‐to‐A transition was reflected in a cooperative downfield shift of the DNA 31P‐nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double‐and single‐stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A‐form resonances were 30–40% narrower than the B‐form resonances, which is attributed to marked sequence‐dependent variations in the latter conformation and to their reduction in the former. (5) The B‐form DNA aggregated in the concentrated 31P‐nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its 31P‐nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A‐form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B‐to‐A interconversion.
Specificity in DNA interactions: An nmr investigation of the interaction of propidium with oligodeoxyribonucleotides containing normal and G · T base pairs
Authors: Wilson W. David , Jones Robert L. , Zon Gerald , Banville Debra L. , Marzilli Luigi G. 
Published: December 2004

AbstractThe interaction of propidium with three self‐complementary oligodeoxyribonucleotides has been investigated by 1H‐ (base‐pair imino proton assigned by 1D NOE and saturation transfer methods) and 31P‐nmr as a function of ratio of propidium to oligomer (from zero to saturation) and temperature. The three oligomers are dTATATGCGCATATA (1), dTATATGTGCATATA (2), which has the same sequence as 1 except for the mismatched base pair at position 7, and dTATGTGCATA (3), which is a shortened version of 2. The imino proton chemical‐shift changes of 1 on titration with propidium can be explained by the effects of the ring‐current anisotropy of propidium at intercalation (3.4 Å) and next‐neighbor sites (6.8 Å). The results indicate that propidium binds with neighbor exclusion but with no significant specificity for any intercalation site in the sequence of 1. The addition of propidium to 1 results in general downfield shifts of all 31P signals, as expected for a nonspecific intercalator. Imino and 31P‐nmr spectra for 2 indicate that this oligomer forms a hydrogen‐bonded G · T base pair at position 7 with little change in base pairing and stacking of base pairs 1–6 compared to 1. The results for addition of propidium to 2 and 3 are quite different than with 1. At low ratio only secondary shifts (6.8 Å) are seen for the G and T imino protons of base‐pair 7 on addition of propidium. At higher ratios of propidium, the signals for these G and T protons are lost in 2 and severely broadened in 3, even at low temperature. The other potential intercalation sites in 2 and 3 appear to bind propidium strongly and without significant specificity as with 1. 31P spectra of 2 in the presence of propidium show the expected downfield shifts and broadening. Thus, the minor differences in local helix geometry in 1, and in 2 and 3, away from the G · T base pair do not significantly affect propidium intercalation specificity. Having one or two G · T base pairs at a site, however, makes intercalation in the standard manner significantly less favorable.
Gramicidin S analogs with a D‐Ala, Gly, or L‐Ala residue in place of the D‐Phe residue: Molecular conformations and interactions with phospholipid membrane
Authors: Higashijima Tsutomu , Miyazawa Tatsuo , Kawai Masao , Nagai Ukon 
Published: December 2004

AbstractThe proton nmr and CD spectra of gramicidin S (GS) cyclic‐(Val1,1′‐Orn2,2′‐Leu3,3′‐D‐Phe4,4′‐Pro5,5′)2 and of GS analogs—namely, [D‐Ala4,4′]‐GS, [Gly4,4′]‐GS, and [L‐Ala4,4′]‐GS—were analyzed. The molecular conformation of [D‐Ala4,4′]‐GS is similar to that of GS, with the trans form about the D‐Ala‐Pro peptide bond. The molecular conformation of [Gly4,4′]‐GS depends on the solvent composition of dimethylsulfoxide‐d6/trifluoroethanol (DMSO)‐d6/TFE and DMSO‐d6/H2O as well as the solute concentration. In DMSO‐d6 solution, [Gly4,4′]‐GS forms the GS‐type conformation of the monomer at lower concentration. At higher concentration, the GS‐type conformer is converted to the other one that forms molecular aggregates. The cis form about the X‐Pro peptide bonds is found for [Gly4,4′]‐GS and [L‐Ala4,4′]‐GS in DMSO‐d6 and for [L‐Ala4,4′]‐GS in TFE solution. The large temperature dependences of α‐proton chemical shifts of [L‐Ala4,4′]‐GS in DMSO‐d6 solution indicate that the conformer equilibrium changes with temperature. The GS‐type conformation is not formed in [L‐Ala4,4′]‐GS. The two active peptide analogs, [D‐Ala4,4′]‐GS and [Gly4,4′]‐GS, interact with the phospholipid membrane, taking the GS‐type conformation. By contrast, an inactive analog, [L‐Ala4,4′]‐GS, does not interact with phospholipid membrane. The activities of GS analogs are found to correlate to the formation of the GS‐type conformation upon binding with phospholipid membrane.
Binding of porcine pancreatic secretory trypsin inhibitor to bovine β‐trypsin: A kinetic study
Authors: Ascenzi Paolo , Amiconi Gino , Bolognesi Martino , Menegatti Enea , Guarneri Mario 
Published: December 2004

AbstractThe kinetics of the formation of the complex between bovine β‐trypsin and the porcine pancreatic secretory trypsin inhibitor (PSTI; Kazal‐type inhibitor) was investigated following the spectral changes associated with the displacement of proflavine from the enzyme, upon inhibitor binding, between pH 3.5 and 8.0 (I = 0.1M) at 21 ± 0.5°C. With inhibitor in excess over the enzyme ([PSTI] ≥ 5 × [bovine β‐trypsin]), the time course of the reaction corresponds to a pseudo‐first‐order process. Over the whole pH range explored, the concentration dependence of the rate is second order at low PSTI concentrations but tends to first order at high inhibitor concentrations. This behavior may be explained by a relatively fast pre‐equilibrium followed by a limiting first‐order process. Values of kinetic parameters for PSTI binding to bovine β‐trypsin depend, between pH 3.5 and 8.0, on the acid–base equilibrium of a single ionizing group (probably His‐57 of bovine β‐trypsin) that undergoes an acidic pKa shift from 7.0 in the free bovine β‐trypsin to 5.5 in the enzyme:PSTI complex. Kinetics of the bovine β‐trypsin:PSTI adduct formation has been analyzed and compared with that of other (pro)enzyme:inhibitor reactions. Considering the known molecular structures of free serine (pro)enzymes, of Kazal‐ and Kunitz‐type inhibitors, as well as of their complexes, the binding behavior of PSTI to bovine β‐trypsin has been related to the inferred stereochemistry of the proteinase:inhibitor contact region.
A simplified analysis of scatchard plots for systems with two interacting binding sites
Authors: Galley W. C. , Bouvier M. , Clas S.‐D. , Brown G. R. , St‐Pierre L. E. 
Published: December 2004

AbstractA simple graphical method for calculating stoichiometric and site binding constants for systems with two initially equivalent interacting sites is derived from a modified Scatchard equation. The binding constants can be calculated from Scatchard plots (r/[A] as a function of r) using the values of r/[A] (r is the molar ratio of bound ligands to total protein and [A] is the equilibrium concentration of free ligand) when r = 0 and r = 1 (half‐saturation). The applicability of the method to the adsorption of bilirubin by peptide pendants immobilized on a polyacrylamide support is demonstrated.
Transport of probe molecules through fibrin gels as observed by means of holographic relaxation methods
Authors: Stewart Ubonwan A. , Bradley Michael S. , Johnson Charles S. , Gabriel Don A. 
Published: December 2004

AbstractHolographic relaxation spectroscopy (HRS) has been used to study transport of benzospiropyran (SP), BSA labeled with azobenzene (BSA‐ABITC), and IgG‐κ labeled with fluorescein (IgG‐FITC) through fibrin gels formed under various conditions. The structures of the gels were controlled by means of the concentrations of fibrinogen, thrombin, and Ca2+ present during assembly of the fibrin. The diffusion coefficient of free dye (SP) was found to be independent of the fibrinogen concentration. The diffusion rate of labeled BSA reflected the assembly conditions of the gel for fibrinogen concentrations above approximately 6 g/L. In particular, the diffusion coefficient was higher in gels formed in the presence of 5 mM Ca2+. The labeled IgG showed photoinduced aggregation, as previously reported, as well as photoinduced attachment to the gel network to produce a permanent diffraction grating. Thus IgG is not a probe in the classical sense, but provides a model for protein diffusion and interactions in gels. These studies indicate that HRS is well suited to the study of molecular transport in fibrin gels.
Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels
Authors: Slater Gary W. , Rousseau Jean , Noolandi Jaan , Turmel Chantal , Lalande Marc 
Published: December 2004

AbstractAn extensive series of experiments has been performed to study the mobility of DNA fragments ranging in size from 2.0 to 48.5 kilobose pairs. By varying the agarose concentration in the gels and the electric field strength, three DNA electrophoresis regimes were clearly identified: the Ogston regime (small DNA fragments in large pores of agarose), the reptation regime without DNA chain stretching (small pores of agarose and weak electric fields), and the reptation regime with DNA chain stretching (small pores of agarose, strong electric fields, and large DNA fragments). Here we report on the experimental identification of these regimes and on the conditions governing the transition between each of them. The onset of reptation and of stretching of DNA chains in gel electrophoresis are described quantitatively for the first time, and a phase diagram for the dynamics of DNA during electrophoresis is presented.
Base sequence effects in double‐helical DNA. III. Average properties of curved dna
Authors: Maroun Rachid C. , Olson Wilma K. 
Published: December 2004

AbstractThe matrix‐generator methods set forth in the preceding paper for treating rodlike DNA are adapted here to the calculation of average chain extension, macroscopic flexibility, and terminal residue orientation in curved duplexes. The different characteristics of curved vs rodlike chains are illustrated with the hypothetical poly[d(A5G5)] · poly[d(T5C5)] duplex. The curved helix is both more compact and macroscopically stiffer than either the poly(dA) · poly(dT) or the poly(dG) · poly(dC) chain. The calculations have also been extended to simple repetitive DNA sequences generated by synthetic ligation studies and the computed average chain properties compared with observed gel mobilities. The predicted chain extension is also checked against the measured persistence lengths of the rodlike poly[d(GC)] and poly[d(AT)] alternating copolymers, and the known cyclization tendencies of selected repeating sequences. Chains are generated from local potential energy maps describing the morphology and flexibility of adjacent base pairs. The energy maps, while approximate, are more accurate descriptors of local structure than many of the intuitive models of DNA curvature offered to date. According to the energy surfaces, the intrinsic bending of curved DNA can be traced to asymmetry in the bending of the Gs and Cs that join half‐helical turn stretches of adenines in these chains. The oligo A stretches are analogous to residues of a perfectly elastic DNA that bend with equal likelihood in opposing directions. In other models of DNA curvature, the (G · C) base pairs are presumed to adopt the classical B‐DNA structure, while the (A · T) base pairs are thought to be in some perturbed conformation.
Sedimentation coefficients of DNA topoisomers: The helical wormlike chain
Authors: Shimada Jiro , Yamakawa Hiromi 
Published: December 2004

AbstractThe sedimentation coefficient sN of the DNA topoisomer with the linking number N is evaluated as a function of N and chain length on the basis of a (circular) twisted wormlike chain, i.e., a special case of the helical wormlike chain. Evaluation is carried out by an application of the Oseen–Burgers procedure of hydrodynamics to the cylinder model with the preaveraged Oseen tensor. The necessary mean reciprocal distance between two contour points is obtained by a Monte Carlo method. It is shown that sN increases as |ΔN| is increased from 0 in the range of small |ΔN|, where ΔN = N − N, with N the number of helix turns in the linear DNA chain in the undeformed state. It is found that there is semiquantitative agreement between the Monte Carlo values and the experimental data obtained by Wang for sN.
Conformation of the second disulfide loop in human transforming growth factor‐α studied by two‐dimensional nmr spectroscopy
Authors: Han Kyou‐Hoon , Niu Chien‐Hua , Roller Peter P. , Ferretti James A. 
Published: December 2004

AbstractThe determination of the conformation of a cyclic heptadecapeptide derived from the second loop of human transforming growth factor‐α, [Ala21]‐hTGF‐α‐(16–32), is described. Two‐dimensional homonuclear Hartmann–Hahn and rotating‐frame cross‐relaxation spectroscopy in H2O were used to obtain the complete proton resonance assignments and the necessary distance constraints between nonbonded hydrogen atoms to derive a conformation without involving any energy minimization. The result is an ellipsoidal‐shaped structure with a turn at Gly19 and a bend formed by residues 26–29, Gln‐Glu‐Asp‐Lys. Comparison is made with the second loop of human epidermal growth factor and the results are discussed in terms of receptor binding and biological activity.
Small angle neutron and x‐ray scattering studies of concentrated protein solutions II. Cytochrome C
Authors: Wu Chuan‐Fu , Chen Sow‐Hsin 
Published: December 2004

AbstractSmall angle neutron scattering (SANS) and small angle x‐ray scattering (SAXS) techniques were used to study two series of concentrated protein solutions containing horse heart cytochrome C (a spherical protein molecule with hydrated diameter σH = 32.6 Å and pI = 10.2) at pD = 2.9 and 11.9, respectively. The concentration of the protein ranges from 1.2 to 24 g/dL. A titration experiment was made to determine the protein charge (Zt) for each sample. Since the form factor of the protein was already determined from a previous experiment [C. F. Wu and S. H. Chen (1987) J. Chem. Phys. 87, 6199–6205] at pD = 6.8, the present series of experiments was used to test the effect of protein charge on the interparticle structure factor. It is demonstrated that the absolute values of both SANS and SAXS cross sections can be calculated with a theory (called the generalized one‐component macroion theory) of the interparticle structure factor, which contains a single unknown parameter, the effective protein charge Zp. Comparison of Zt and Zp thus obtained shows that when the bare surface charge density of the protein calculated by using Zt exceeds about 3 μC/cm2, Zp becomes much smaller than Zt. This is interpreted as a charge renormalization phenomenon similar to that known to occur for highly charged polystyrene latex particles in aqueous solution [S. Alexander, P. M. Chaikin, P. Grant, G. J. Morales, and P. Pincus (1984) J. Chem. Phys. 80, 5776–5781].
Electric birefringence studies of rabbit skeletal tropomyosin
Authors: Swenson Charles A. , Stellwagen Nancy C. 
Published: December 2004

AbstractThe electric birefringence of rabbit skeletal tropomyosin and its nonpolymerizable derivative was studied as a function of protein concentration, pulse length, and electric field strength. Analyses of the zero field birefringence decay curves show that nonpolymerizable tropomyosin, which has had on average six C‐terminal residues removed, and tropomyosin are both well approximated by rigid cylinders in solution at low salt concentrations at 20°C. The measured relaxation times for the monomers of polymerizable and nonpolymerizable tropomyosin are 1.5 ± 0.4 μs and 1.30 ± 0.2 μs, respectively, in good agreement with the values calculated from the known dimensions. For tropomyosin the electrical pulse induces the formation of linear dimers. Orientation occurs primarily by a permanent dipole mechanism. Permanent and induced dipole moments were calculated from reversing field experiments and from the saturation of the birefringence. Removal of the six C‐terminal residues decreases the measured permanent dipole moment by 9.5%, from 6300 to 5700 Debyes, which is in good agreement with the 7% decrease calculated for permanent dipole contributions arising from the peptide dipoles and from the asymmetric distribution of the formal charges. This change is due primarily to the removal of Asp 280.
Stability of polypeptide conformational states. II. Folding of a polypeptide chain by the scanning simulation method, and calculation of the free energy of the statistical coil
Authors: Meirovitch Hagai , Vásquez M. , Scheraga Harold A. 
Published: December 2004

AbstractThe scanning simulation method suggested by Meirovitch is extended to a study of the stability of decaglycine at 100 and 300 K. The model is based on the potential energy function ECEPP (Empirical Conformational Energy Program for Peptides) with rigid geometry and without solvent. The free energy of the statistical coil, which is defined over the whole phase space excluding the region of the right‐handed α‐helix, is calculated. At 100 K, the molecule is found to be unstable in the statistical coil region, and the method generates (i.e., “folds”) conformations that are left‐handed or right‐handed α‐helices with very high preference. Their free energy is found to be comparable with that obtained by another method developed in our previous paper (paper I) [H. Meirovitch, M. Vásquez, and H. A. Scheraga, (1987) Biopolymers 26, 651–671]. At 300 K the statistical coil becomes the most stable state; sample conformations of the coil are generated efficiently with the scanning method and the free energy is calculated. It appears that both the scanning method and the method of paper I can be used to carry out a complete analysis of the stability of a polypeptide based on free energy considerations.
Highly cooperative binding to DNA by a histone‐like, sperm‐specific protein from Spisula solidissima
Authors: Libertini Louis J. , Ausió Juan , Van Holde K. E. , Small Enoch W. 
Published: December 2004

AbstractIn sperm of the bivalve mollusc, Spisula solidissima, the predominant protein from the highly condensed chromatin shares characteristics with both protamines and somatic histones. We have examined, in vitro, the binding of this unusual protamine‐like protein (PLP) to DNA. The binding stoichiometry was estimated by DNA thermal melting and by fluorescence titration to be about 70 base pairs of DNA per PLP molecule; thus, since the overall charge on PLP is about 140, the saturated complex should be essentially charge neutral. In order to decrease problems of aggregation, PLP was labeled with fluorescein isothiocyanate (PLP‐F) to allow binding studies at very low concentrations of the complex. However, even at the lowest protein concentrations studied (2.5 nM), aggregation was easily detected for stoichiometries near 1. The binding was therefore studied at low ratios of PLP‐F to DNA by examining the effect of increasing salt concentration on the complexes. The resulting salt‐dissociation curves were analyzed by an approach based on the exact binding isotherm of McGhee and von Hippel [(1974) J. Biol. Chem. 86, 469; (1976) 103, 679]. This approach allows direct simulation of the salt‐dissociation curves and can thus be used for least squares fitting of the experimental data to determine the binding parameters. The results indicate that the binding is extremely cooperative (ω ∼ 105) and involves a surprisingly small number of monovalent ions (∼ 3–5).
Three persistence lengths for a stiff polymer with an application to DNA B‐Z junctions
Authors: Manning Gerald S. 
Published: December 2004

AbstractWe discuss three persistence lengths for any locally stiff polymer: the familiar persistence length for bending fluctuations, a persistence length for twisting fluctuations [introduced first by J. Wilcoxon and J. M. Schurr (1983) Biopolymers 22, 2273–2321], and a persistence length for contraction/extension fluctuations. They correspond to the three classical modes of elastically resilient responses to forces and torques on a thin elastic rod—bending, twisting, and contraction/extension. Although numerically distinct (the persistence length for twist is especially large), the order of magnitude of all three is 100 base pairs (bp) for DNA. With some confidence, therefore, we conclude that DNA exhibits elastic resilience on a scale of 100 bp. Perturbations at both ends of a 100‐bp segment are thus expected to propagate as structural effects throughout the length of the segment, as has been observed experimentally when a 99‐bp segment of B‐DNA is flanked at both ends by sequences with B‐Z transition capability [R. M. Wartell, J. Klysik, W. Hillen, and R. D. Wells (1982) Proc. Natl. Acad. Sci. USA 79, 2549–2553]. We predict that the observed effect will disappear if Z‐susceptible flanking occurs at only one end of the elastically resilient segment. We suggest, moreover, that similar experiments can determine the maximum length of a DNA segment capable of elastic response.
Formation and stability of helical poly (Nϵ, Nϵ, Nϵ‐trimethyl‐L‐lysine) in sodium perchlorate solution
Authors: Bello Jake 
Published: December 2004

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