bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical binding of pyrene and phenanthrene to native and denatured DNA: Measurements by spectral and coupled‐column liquid chromatography methods
Authors: Nelson Henry P. , Devoe Howard
Published: December 2004

The physical (noncovalent) binding of pyrene and phenanthrene to calf‐thymus DNA in aqueous NaCl solutions was measured by a spectral method (analysis of absorption spectra by Benesi‐Hildebrand plots) and a coupled‐column liquid chromatography method (equilibration of DNA solutions with solid hydrocarbon in a generator column and analysis of dissolved hydrocarbon by liquid chromatography). The measurements yielded values of an affinity constant K′ = nK, where n is the apparent number of binding sites per nucleotide and K is the apparent binding constant. The affinity of native DNA for pyrene decreases monotonically with increasing NaCl concentration, whereas the affinity of heat‐denatured DNA exhibits a maximum at 0.10M NaCl.K′ for the binding of phenanthrene to native DNA is an order of magnitude lower than K′ for pyrene. The molar enthalpy for the binding of pyrene to native DNA in 10 mM NaCl is (−34.0 ± 1.0) kJ mol−1. The spectral method data indicate that 50 is an upper limit for the average number of base pairs between intercalation sites for pyrene along the DNA helix and that only a fraction of these sites are bound at the highest binding ratios.
Interaction of poly(I)·poly(C) with trans‐dichloro‐diammine‐platinum (II)
Authors: Hermann Dominique , Fazakerley G. Victor , Houssier Claude , Guschlbauer Wilhelm
Published: December 2004

The covalent binding of trans‐Pt (NH3)2Cl2 to the double‐stranded poly(I)·poly(C) follows three types of reactions, depending on rb and the concentration of polynucleotide in the reaction mixture. At rb ⩽ 0.1, the principal reaction is coordination to poly(I), giving rise to some destabilization of the double strand, as shown by uv and CD spectra, and a decrease in Tm values, giving rise to free loops of poly(C). At higher rb and low polynucleotide concentration, the free cytidine bases react with platinum bound on the complementary strand to form intramolecular (interstrand) crosslinks that restabilize the double‐stranded structure. At high rb and high polynucleotide concentration, while the above reaction still occurs, the predominant one is the formation of intermolecular crosslinks. Under no conditions has strand separation been observed.
Ionic and structural effects on the thermal helix‐coil transition of DNA complexed with natural and synthetic polyamines
Authors: Thomas T. J. , Bloomfield V. A.
Published: December 2004

Polyamines are ubiquitous cellular components that interacts strongly with nucleic acids. Although many of the interactions of oligocations with DNA can be rationalized with polyelectrolyte theories that treat counterions as point charges, some structural effects are evident. We have explored the effects of polyamine structure on one important aspect of DNA behavior, its thermal melting transition, by using a series of spermidine analogs NH3(CH2)3NH2(CH2)nNH, where n varies from 2 to 8 [Jorstad et al. (1980) J. Bacteriol. 141, 456–463]. For spermidine itself, n = 4. Tm for calf‐thymus DNA in the presence of each of these analogs, and the other naturally occurring polyamines putrescine2+ and spermine4+, was measured over a wide range of NaCl concentrations and polyamine:DNA phosphate ratios. There are modest, but significant structural effects. particularly with the shorter n = 2 and 3 derivatives, whose geometry my not allow full electrostatic interaction with DNA. Longer analogs, on the other hand, are not much different than spermidine in their effects on Tm, though a moderate maximum occurs at n = 5. Since polyamines are important in the cellular condensation and packaging of DNA, we have also delineated the critical polyamine and salt concentrations that are required to cause DNA aggregation. Here again, there are significant structural effects, which are not easily rationalized by any simple considerations.
pH‐jump titration of the tyrosyl groups of bovine plasma albumin
Authors: Kuwnajima Kunihiro , Matsushima Tadashi , Nitta Katsutoshi , Sugai Shintaro
Published: December 2004

Ionization properties of the tyrosyl groups of bovine plasma albumin in various conformational states—the native state (N), the two acid states (F and E), and the state (B) stable at slightly alkaline pH—were studied by means of a stopped‐flow‐pH‐jump technique. The technique allows us to obtain the tyrosyl titration curve in a conformational state that is unstable in the pH region of the titration. The pH jumps from the N and B states to various alkaline pH's, where the tyrosines ionize to bring about a time‐dependent increase in absorption at 296 nm, indicating that a number of the tyrosines buried initially become susceptible to ionization as a result of the alkaline transition occurring above pH 10.8. Extrapolation of the observed absorption change to zero time gives a spectrophotometric titration curve in the initial conformational state. Only 30–401% of the 19 tyrosines of the protein can ionize both in the N and the B state at pH 12. The pH jumps from the F and E states, on the other hand, give a decrease in absorption between pH 9 and 11.7, indicating that the tyrosyl groups initially exposed are remarked by refolding after the pH jumps, and the zero‐time titration curves show that essentially all the tyrosyl groups ionize normally in these acid states. The results are discussed in relation to the known results of the tyrosyl exposure of the protein measured by other techniques, and the consistency among them demonstrates the effectiveness of the pH‐jump titration method. Hydrogen bonding between the abnormal tyrosyl and carboxylate groups as a mechanism to stabilize native albumin is suggested from characteristics of the alkaline transition, which also involves the exposure of the tyrosyl groups, and from the tyrosyl titration curves in the native and acid states.
Solvation effects on the 31P‐nmr chemical shifts and infrared spectra of phosphate diesters
Authors: Lerner D. B. , Becktel W. J. , Everett R. , Goodman M. , Kearns D. R.
Published: December 2004

The effects of organic solvents on the 31P‐mr chemical shifts of various phosphate diesters have been investigated in water and mixed‐organic solvent systems. The addition of organic solvents to cyclic phosphates and to diethyl phosphate causes large upfield shifts of the phosphorus resonance which are attributed to solvent‐induced changes in the local hydration of the phosphodiester group. This is consistent with the fact that there is an inverse correlation between the hydrogen‐bond‐donating ability of the solvents and the magnitude of the shifts they induce. Other possible interpretations, such as solvent‐induced ion pairing and solvent‐induced conformational changes, appear to be eliminated. Fourier‐transform ir study of the cyclic nucletides reveals that there are also large solvent‐induced shifts in the frequency of the antisymmetric OPO stretching frequency, and a comparison of the two types of measurements indicates that there is a linear correlation between shifts observed in the ir and in the 31P‐nmr spectra. With UpU, the solvent‐induced 31P‐nmr shifts are ∼3 times smaller than those observed with the cyclic phosphates and the solvent‐induced shift of the OPO band is reduced (factor of ∼1.7) as compared with the cyclic phosphates. With the single‐stranded polynuclotides, poly(C) and poly(U), the solvent‐induced shifts in both the nmr and ir are quite small (∼0.1 ppm and ∼1 cm−1). The very small solvent effects observed with poly(U) and poly(C) are attributed to a combination of steric effects and a polyelectrolyte effect which maintains a high density of counterions with waters of hydration in the vicinity of the charged backbone and makes the phosphates much less susceptible to solvent‐induced changes in hydration.
Analysis of the electrophoretic properties of double‐stranded DNA and RNA in agarose gels at a finite voltage gradient
Authors: Edmondson Stephen P. , Gray Donald M.
Published: December 2004

The electrophoretic mobilities of double‐stranded (ds) DNAs and ds RNAs of various lenths, L, were measured in gels of 0.4–1.8% (w/v) agarose at a voltage gradient of 1.0 V/cm. Differences in the electrophoresis of ds DNA and ds RNA are presented and discussed. A general expression is derived that describes the electrophoretic mobility, M, of either type of ds nucleic acid as a function of the gel concentration and the nucleic acid length: M = M′1(L/L0)−x − M2, where M′1 and L0 are constants, and x and M2 depend on the agarose gel concentration. The results obtained by fitting our data with this equation are consistent with the mobilities of nucleic acids in a wide range of gel concentrations, including free electrophoresis in solution and electrophoresis in gles of high agarose concentration in which nuleic acids are expected to reptate through the gel matrix. Finally, various methods of plotting agarose gel electrophoresis data are discussed.
Molecular‐mechanical studies of hormone–protein interactions: The interaction of T4 and T3 with prealbumin
Authors: Oatley Stuart J. , Blaney Jeffrey M. , Langridge Robert , Kollman Peter A.
Published: December 2004

We present molecular‐mechanical calculations on the interactions of L‐thyroxine (T4) and 3,5,3′‐triiodo‐L‐thyronine (T3) with the binding site of human serum prealbumin. Energy refinement with restraints on the movement of the iodine atoms leads to reasonably low‐energy structures with the iodines within 0.2 Å of their x‐ray determined positions; removal of these restraints leads to larger (∼1.0 Å) deviations from these positions. However the relative energies of T3 and T4 complexation are similar in both cases, and the relative calculated energies for these complexes, when corrected by a simple empirical method for the solvation energy differences of the hormones, are in reasonable qualitative agreement with experiment.
Energetics of multihelix interactions in protein folding: Application to myoglobin
Authors: Gerritsen Mirjam , Chou Kuo‐Chen , Némethy George , Scheraga Harold A.
Published: December 2004

Conformational‐energy calculations have been carried out in order to determine favorable packing arrangements within a group of α‐helices. The influence of side chains and of the number of interacting α‐helices on the mode of packing was analyzed. In this work, our earlier methods for computing the packing energy of a pair of α‐helices [Chou, K.‐C., Némethy, G. & Scheraga, H. A. (1984) J. Am. Chem. Soc. 106, 3161–3170] have been extended to treat the interactions among several helices. Also, new algorithms allow the matching of standard peptide geometry to x‐ray coordinates of helical complexes and the analysis of interrelations between several helices. As a specific test case, the packing of three neighboring α‐helices, viz., the A, G, and H helices of sperm whale myoglobin, was considered. Minimum‐energy arrangements were computed for the separate A‐H and the G‐H α‐helix pairs as well as for the A‐G‐H three‐helix complex. For the packing of the nearly antiparallel G and H α‐helices, the same optimal structure was obtained in two‐ and three‐helix complexes, indicating that a single packing arrangement is specifically favored by interhelix interactions. For the pair of nearly perpendicular A and H α‐helices, interactions are less specific, so that there is no unique optimal structure in the two‐helix complex; in the three‐helix complex, however, a specific mode of packing is favored even for the A‐H pair. This result indicates that the presence of other nearby α‐helices can influence the packing of a given α‐helix pair. The computed arrangement of the A‐G‐H complex is very close to that of the crystallographically determined structure. These results can be used to make deductions about the likely sequence of events in protein folding, where, in this particular case, it appears that the G‐H helix pair may form first and then induce proper orientation of the A helix.
Carcinogen–base stacking and base–base stacking in dCpdG modified by (+) and (−) anti‐BPDE
Authors: Hingerty Brian E. , Broyde Suse
Published: December 2004

The low‐energy conformations accessible to dCpdG modified at guanine N2 via trans epoxide opening by (+) and (−) 7β,8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydrobenzo(a)pyrene (anti‐BPDE) have been delineated by minimized semiempirical potential‐energy calculations with all torsion angles flexible. Nearly 4000 trials were made, representing a fairly thorough investigation of the conformation space of the adducts. Carcinogen–base stacked states and base–base stacked conformers were found in the low‐energy regions of both enantiomers. Many ω′, ω, ψ domains accommodate the two types of conformations, with B‐like backbones among the most preferred states in each case. The conformational differences between the two enantiomers on the dimer level reside in subtle distinctions in orientation of the carcinogen–base linkage.
The effect of solvent viscosity and temperature on DNA viscoelastic behavior
Authors: Ostashevsky J. Y. , Lange C. S.
Published: December 2004

The effect of solvent viscosity (ηs) and temperature (T) on the shape of the concentration dependence of the principal and total recoils in creep‐recovery viscoelastometry experiments has been studied for T4 DNA solutions. The range of DNA concentration (c) was 2 – 40 μg/ml; glycerol, 70–80% v/v, sucrose, 60% v/v; NaCl, 5 mM – 1M; and T, 275 – 323 K. A linear proportionality between recoil and c was obtained at high ηs/T. At low ηs/T, the c‐dependence was nonlinear, approaching saturation at higher c. At low c, the slope of both curves was the same. Transition between “linear” and “nonlinear” values occurred over a narrow range of ηs/T (a width of 1–5 K if ηs/T was changed by varying T). (ηs/T)tr, the midpoint of the transition, was independent of solvent properties other than viscosity. Also, (ηs/T)tr increased with c. For a given c, ηs/T values above this transitional value yield linear behavior; below this, nonlinear behavior. The ratio of linear to nonlinear recoil values is a linear function of c with Kc, the slope of this dependence, independent of ηs and T. A kinetic model for the observed nonlinearity of recoil with c is presented. It explains the independence of Kc on ηs and T. An attempt has been made to explain the linear–nonlinear transitions by comparison of τ1 and TR, the lifetime of the contact points of the polymer network in the de Gennes theory. The nonlinear values are consistent with a pseudogel that exists when τ1 < TR. At τ1 > TR, the DNA behavior is similar to that in dilute solutions (linear values). Thus, the condition for transition is τ1 = TR. However, some unsolved problems remain.
The growth of large single crystals of lysozyme
Authors: Ataka Mitsuo , Tanaka Shoji
Published: December 2004

If protein single crystals larger than those suitable for x‐ray analysis are obtained, various spectroscopic, thermal, mechanical, and electrical measurements become possible. To understand the factors governing the crystal size, tetragonal lysozyme crystals were grown in batches at 15°C from solutions of different protein and salt concentrations between pH 4–7. The number and size of the crystals, and the protein concentration remaining in the supernatant, varied markedly with the initial salt amount, pH, and cation species, but large crystals always grew when the initial protein concentration (P) was in a narrow range of 2.5–3 times the crystal solubility (S). It was also shown (1) that the period before the first crystals appeared (D) varied as D ∝ (P/S)−n, where n ≃ 5, and (2) that many previous experiments used more supersaturated solutions than the optimal ones thus determined. The reason why large crystals grow only from moderately supersaturated solutions is discussed. The crystal size of the orthorhombic form grown at 40°C was less sensitive to pH and P than the tetragonal form. An effort to measure D and the solubility at 40°C revealed many differences between the two crystal forms, which we ascribe to different interactions to promote crystallization.
Effective diffusion rate through a polymer network: Influence of nonspecific binding and intersegment transfer
Authors: Berg Otto G.
Published: December 2004

The effective diffusion rate of a tracer molecule through a polymer network can be influenced by nonspecific binding. If such binding occurs, the local density fluctuations (segmental diffusion) of the network molecules will contribute to the net displacements of tracer molecules. If the network is strongly interconnected by entanglement or cross‐linking, these local motions will only carry the tracer molecules over a small region, and effective transport would require dissociation and reassociation of the tracer molecule to another part of the network. Alternatively, tracer molecules could be transferred directly (intersegment transfer) between different parts of the network whenever they are brought sufficiently close by the density fluctuations. A wormlike‐chain model for the segmental diffusion of a polymer is used to describe the network motions and to derive the effective diffusion rate for a tracer molecule as a function of network density and binding constant with or without intersegment transfer contributing. It is found that the density dependence for the effective diffusion of ethidium bromide through dense DNA solutions studied by photobleaching recovery [R. D. Icenogle and E. L. Elson (1983) Biopolymers 22, 1949–1966] agrees with an intersegment‐transfer mechanism limited by the segmental DNA motions. The calculations are also applied to a model for the intracellular diffusion of molecules loosely bound to the cytomatrix. If intersegment transfer dominates it can account for the observed size independence for the intracellular diffusion rates of various injected macromolecules.
Influence of different tripeptides on the stability of the collagen triple helix. II. An experimental approach with appropriate variations of a trimer model oligotripeptide
Authors: Thakur S. , Vadolas D. , Germann H.‐P. , Heidemann E.
Published: December 2004

A collagen model peptide comprising three covalently cross‐linked chains (Ala‐Gly‐Pro)8 with a stable triple‐helix conformation was utilized as the constant part of elongated model peptides of different composition. The tripeptides Gly‐Pro‐Hyp, Gly‐Pro‐Ala, Gly‐Pro‐Pro, Gly‐Pro‐Ser, Gly‐Ala‐Hyp, Gly‐Phe‐Hyp, Gly‐Glu‐Hyp, Gly‐Ala‐Lys, and Gly‐Pro‐Phe were coupled at the N‐terminal to the cross‐linked peptide. The transition temperatures determined by CD measurements are higher for the peptides containing the Gly‐X‐Hyp sequences followed by those with the Gly‐Pro‐Y sequences. In experiments with combinations of two different tripeptides coupled to the constant part of the cross‐linked model peptides higher transition temperatures were observed if the sequence of helix‐promoting tripeptides was not interrupted.
SANS studies of concentrated protein solutions. I. Bovine serum albumin
Authors: Nossal R. , Glinka C. J. , Chen S.‐H.
Published: December 2004

Small‐angle neutron scattering (SANS) was used to examine concentrated bovine serum albumin solutions of up to 20% protein w/v. At higher protein concentrations, scattering data show distinct features that can be ascribed to strong intermolecular interactions. Differential scattering cross‐sections are fitted to a theoretical model of interparticle potential consisting of a hard core plus an exponentially decaying “tail.” For moderate ionic strength (0.03M K Acetate, pH 5.9), the intermolecular interaction agrees with the double‐layer repulsive part of the well‐known DLVO (Derjaguin, Landau, Verwey, Overbeek) theory for interacting colloidal particles. We thus demonstrate that it is possible to determine size parameters and the surface charge of protein molecules in dense solutions. At high salt concentrations (≥0.2M NaCl) data can be fitted by the same potential model, although interpretation in terms of DLVO theory is not possible. Even in this case, however, “effective” molecular size and potential parameters can be determined.
Collagen fibrillogenesis in vitro: An investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering
Authors: Payne K. J. , King T. A. , Holmes D. F.
Published: December 2004

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large‐scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.
The interaction of Ca2+‐binding proteins with the carbocyanine dye stains‐all
Authors: Caday Cornelio G. , Lambooy Peter K. , Steiner Robert F.
Published: December 2004

The interaction of the carbocyanine dye Stains‐all with the Ca2+‐binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca2+, complexes with Stains‐all of all three proteins exhibit at high dye: protein mole ratios an intense J absorption band at 600–650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J‐band is progressively lost as the dye: protein ratio decreases and is replaced by bands of the γ and β types at 450–550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J‐band is observed; its intensity is undiminished at the lowest dye: protein ratios examined. In the presence of excess Ca2+ the J‐band is lost for all three proteins. For calmodulin and troponin C it is replaced by σ‐ and β‐bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.
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

Bovine 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

Changes 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

The 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

The 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

The 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

A 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

Holographic 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

An 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

The 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

The 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

The 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

Small 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

The 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

The 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

In 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

We 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

Poly(trimethyl‐L‐lysine), [Lys(Me3)]n, is converted from random coil to α‐helix at about 1/30 of the NaClO4 concentration required by poly(L‐lysine), (Lys)n. NaClO4 generates turbidity in [Lys(Me)3]n at concentrations above that required for helix formation, and decreases turbidity above lM NaClO4. The turbidity runs parallel to enhanced, and then decreased, fluorescence of a dansyl label. Helix formation per se does not induce enhanced fluorescence. Increasing NaClO4 concentration increases 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.
Differential polarization microscopy (cd and linear dichroism) of polytene chromosomes and nucleoli from the dipteran sarcophaga footpad
Authors: Livolant Françloise , Mickols William , Maestre Marcos F.
Published: December 2004

Circular Dichroism (CD) and Linear Dichroism (LD) Microscopy measurements of single live polytene nuclei of the fleshfly Sarcophaga bullata suggest the existence of two groups of data. This was observed in both the linear and circular dichroism spectra indicating a difference in both the linear and chiral arrangements of the chromatin. The two groups of data were averaged separately and called nucleolus type 1 and type 2. The CD of nucleoli type 1 all show positive CD in the 260 nm wavelength. For the nucleoli type 2 the CD are very different, always negative, with maximum amplitude at 250 nm. The CD of the band, interband and transcriptionally active puffs show very similar CD spectra at 260 nm but differ at lower wavelength (between 245 and 260 nm). Differences are also observed in the LD measurements for nucleoli type 1 (LD maximum at 265 nm) vs. nucleoli type 2 (LD maximum centered at 250 nm). These differences are interpreted as a manifestation of large transcriptional activity for nucleolus type 1, and a more condensed structure and low transcriptional activity for nucleolus type 2.