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D W Kupke

Publications and source records attributed to D W Kupke.

17 recordsLinked to original sources
PubMed1992

Differential hydration of homopurine sequences relative to alternating purine/pyrimidine sequences.

The minor groove ligand distamycin A has been used to probe the relative hydration of the minor groove of eight synthetic polynucleotides of known sequence and composition. A combination of densimetric, calorimetric, and temperature-dependent spectroscopic techniques have been used to obtain complete thermodynamic profiles (delta Gzero, delta Hzero, delta Szero, and delta Vzero) for the association of distamycin A to all polymer duplexes. In 10 mM phosphate buffer, pH 7, binding of the drug to each of the polymeric duplexes resulted in characteristic negative changes in both the volume and enthalpy. Although the binding constants were found to be identical for pairs of isomer polynucleotides having identical compositions but different sequences, the values of delta Hzero, delta Szero, and delta Vzero of each such pair were remarkably different. The entropy changes were found to roughly parallel the volume changes; no such trend was seen between delta Hzero and delta Vzero. The data support the hypothesis that the volume changes observed for these systems reflect the coulombic-hydration contribution to the entropy. The heteropolymer duplexes generated much larger exothermic contributions, less favorable entropies and larger volume contractions than did the corresponding homopolymer duplexes of identical composition, and strongly suggest that polynucleotides with homopurine sequences are more hydrated than polynucleotides with alternating purine/pyrimidine sequences. In addition, it was found that duplexes containing guanine sharply reduced the affinity for the drug, also lowering the exothermicity but raising the entropy. This may be explained by the presence of an amino group in the minor groove that prevents hydrogen bonding. Substitution of the guanine with inosine reversed this trend in the thermodynamic properties. Furthermore, substitution of poly(dA) for poly(rA) in a duplex produced a similar reduction in the affinity, while raising the exothermic contribution and greatly reducing the favorable entropy effect in agreement with an apparent increase in the hydration state.

Base Sequence↗
PubMed1991

Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides.

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.

Base Sequence↗
PubMed1989

Probing the hydration of the minor groove of A.T synthetic DNA polymers by volume and heat changes.

The minor-groove ligand netropsin provides a sensitive probe of the hydration difference between poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)]. We have measured the volume change delta V accompanying binding of netropsin to these polymers, using an improved magnetic suspension densimeter. For poly(dA).poly(dT) we find delta V = +97 mL/mol of bound netropsin at pH 7.0 and 10 mM sodium phosphate buffer. For poly[d(AT)].poly[d(AT)] we find delta V = -16 mL/mol of bound netropsin. This striking differential effect suggests that the poly(dA).poly(dT) duplex compresses more water (or is more extensively hydrated). From our enthalpy and entropy results we estimate the approximately 10 water molecules, immobilized in the minor groove of this system, are displaced by each netropsin bound. The volume increase, however, is substantially larger than can be explained by a simple melting of these immobilized water molecules in the minor groove. A decompression of at least 40 water molecules must attend the complexation to the poly(dA).poly(dT) duplex. This suggests that the conformation change attending the binding of the drug to this polymer duplex causes a further dehydration, whereas no such change in dehydration and configuration for the heteropolymer system is indicated.

Binding Sites↗
PubMed1989

Volume changes in the binding of lanthanides to peptide analogues of loop II of calmodulin.

The solution expansion accompanying coordination of lanthanide ions to synthetic peptide analogues of a metal-binding loop in calmodulin was determined by a density method. This study was designed to further test the hypothesis that the nonlinear expansions observed upon sequential addition of Ca2+ to intracellular calcium-binding proteins reflect principally upon the coordination event at specific binding sequences. Three peptides of 13 residues each were synthesized as analogues of binding loop II in mammalian calmodulin: Peptide I was the native analogue; peptide II contained an aspartyl in place of an asparaginyl residue at position 5 from the N-terminus; for peptide III, the aspartyl residue in position 3 of the native analogue was interchanged with the asparaginyl residue in position 5. Thus, the number of charged-oxygen donor atoms for coordination was the same in I and in III, but the latter peptide could permit two pairs of acidic groups to converge toward the metal ion as in some loops of these proteins. The observed expansions with different lanthanide ions to the same peptide varied appreciably, suggesting dissimilar structures [Gariépy et al. (1983) Biochemistry 22, 1765-1772]; coordination to the simpler tetracarboxylate sequestrants, on the other hand, generated an expansion profile approximately as expected from the properties of the lanthanide series. The largest expansions were generated with peptide II (having the additional acidic group) for all lanthanides tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1986

Sequential volume changes on a small sample by density.

A procedure is described for generating a sequence of volume changes by density on the same sample of a solution. The entire volume-change profile over the reactant concentration range of interest can be accomplished with 0.1-0.2 mumol of a protein in 100 microliters of sample. The calcium-binding proteins, calmodulin and skeletal troponin-C, were employed to determine the volume increases attending the sequential addition of calcium ions. Both volume-change profiles exhibited nonlinear increases over the first four equivalents of calcium ion added.

Animals↗
PubMed1986

Volume changes upon addition of Ca2+ to calmodulin: Ca2+-calmodulin conformational states.

Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.

Animals↗
PubMed1977

Solid-like character of virus solutions.

The solid-like behavior of turnip yellow mosaic virus solutions following the extrusion of viral RNA in alkali was observed with a torsion-fiber balance developed for the purpose. This method provided a direct measurement of the yield stresses required to break or liquefy these solutions. The yield stresses were found to increase and to be less time dependent with increasing concentrations of the virus and they were maximal at room temperatures. If the virus had been damaged, as by freeze-thaw, little or no solid-like behavior could be demonstrated. Purified viral capsids, with or without added RNA, were also inactive. The values for the yield stresses were of the same order as the value reported previously with the use of a magnetic suspension viscometer; hence, the apparent coherency appears unrelated to the magnetic fields generated by the latter instrument. These solutions behaved as typical liquids after the required stress was applied [about 0.005 to 0.17 dyne cm-2 (0.05 to 1.7 micronN cm-2)], these forces being smaller than those usually conferred by ordinary handling.

Freezing↗
PubMed1975

Quasi-elastic behavior of solutions of viral capsid and RNA at very low shearing stresses.

By the application of shearing stresses on the order of 10(-3) dyne cm-2 (10(-2) muN cm-2), via the magnetic viscodensimeter, extremely high relative viscosities (greater than 500) were observed when turnip yellow mosaic virus was degraded in alkali into its capsid and RNA. The solutions, however, possessed a watery consistency at this stage and exhibited a quasi-elastic character by rotor-recoil experiments. The development of this curious behavior was concentration and temperature dependent; it was not seen less than 0.5% nor at 8 degrees, and appeared sooner at 30 degrees than at 20 degrees. The time of appearance was delayed as the pH was lowered; however, the effect was still observed when the pH was as low as 9. Whereas reversibility was demonstrated when the shearing stresses exceeded the elastic resistance [0.17 dyne cm-2 (1.7 muN CM-2)], thorough mixing usually resulted in a normal behavior of the solutions thereafter. Values for the modulus of rigidity at 20 degrees for about 1% virus concentration was less than 2 X 10(-2) dyne cm-2 rad-1 (0.2 muN cm-2 rad-1), which, while extremely small, was reproducible. A porous structure, possibly involving a capsid and RNA complex, is envisioned.

Mosaic Viruses↗
PubMed1974

Equilibrium sedimentation of turnip yellow mosaic virus.

Sedimentation equilibrium was achieved with turnip yellow mosaic virus at low speeds (600 rpm) in a magnetic ultracentrifuge. The experiments were carried out in the newly installed constant-speed rotor, equipped with automatic control and an electromagnetic drive. A particle mass of 5.55 x 10(6) daltons was calculated for the virus at vanishing concentrations, in essential agreement with the earlier results using the more tedious procedure with a freely coasting rotor. In order to interpret the observed departure from ideal behavior (nonassociating conditions), preferential interaction experiments were carried out by magnetic densimetry. These showed that a strong Donnan effect exists at pH 7, which contributed > 10(3) times more to the nonideality than did the excluded volume effect. A net negative charge of about 3.5 x 10(3) per particle was estimated at this pH.

Densitometry↗
PubMed1973

A magnetic suspension osmometer.

The magnetic suspension balance is used for the measurement of very small osmotic pressures. The apparatus is essentially the same as that previously used for the measurement of the density and viscosity of protein solutions except that the magnetically suspended buoy is modified to make it pressure sensitive. The method is especially useful for the measurement of osmotic pressure in small samples of dilute solutions (about 10(-6) M) or of substances with molecular weights greater than 10(5). A height-sensing device has been developed which is not dependent upon the visual precision of the operator.

Animals↗
PubMed1972

Simultaneous determination of viscosity and density of protein solutions by magnetic suspension.

The first results are reported with a magnetic suspension instrument for determination of the viscosity and density concurrently on small volumes (0.2 ml) of protein solution. Reasonable agreement was obtained with literature values for the intrinsic viscosities and specific volumes (partial or isopotential) of serum albumin and ribonuclease in native solvents, and in 6 M guanidinium chloride with or without 2-mercaptoethanol. Turnip Yellow Mosaic virus and myosin were also studied, the results with the virus being related to hydration and structure data and those with myosin to the dissociative character of the protein. The possibility of using this approach to follow the time course of viscosity and density changes during reactions is shown.

Chlorides↗
PubMed1969

Effect of pressure on the apparent specific volume of proteins.

The magnetic densimeter has been employed to measure the densities and apparent specific volumes of certain proteins in aqueous solutions as a function of pressure. The method gave values in satisfactory agreement with those found in the literature for aqueous electrolyte solutions. A change in apparent specific volume of the monomeric proteins, ribonuclease and turnip yellow mosaic virus and its capsid protein, at pressures up to 400 atmospheres at 20 degrees C was not observed within the precision of the measurements. Also, no change in the apparent specific volume of tobacco mosaic virus protein was observed as a function of these pressures whether the protein was predominantly in the polymerized or unpolymerized state. The magnetic densimeter was found to be a convenient instrument for measuring compressibilities of very small samples of solutions.

Densitometry↗