Biomedical subjects

D P Remeta

Publications and source records attributed to D P Remeta.

10 recordsLinked to original sources

PubMed1999

A quantitative method for evaluating the stabilities of nucleic acids.

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed "reference" duplex (usually a Watson-Crick duplex) and a related "test" duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Base Sequence↗

PubMed1999

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin.

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.

Animals↗

PubMed1998

Characterization of the structure, function, and conformational stability of PorB class 3 protein from Neisseria meningitidis. A porin with unusual physicochemical properties.

PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710-10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic beta-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.

Amino Acid Sequence↗

PubMed1998

Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein.

A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared from CPS serotypes 6B, 14, 19F, and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild type counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 micrograms CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30,000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular, both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysin-induced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines.

Animals↗

PubMed1997

Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity.

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of approximately 1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (Tm = 88 degrees C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60 degrees C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.

Amino Acid Sequence↗

PubMed1993

Thermodynamic characterization of daunomycin-DNA interactions: comparison of complete binding profiles for a series of DNA host duplexes.

Using a combination of spectroscopic and calorimetric techniques, we have determined complete thermodynamic binding profiles (delta G degree, delta H degree, and delta S degree) for the complexation of daunomycin to a series of 10 polymeric DNA duplexes. We find the resulting drug binding data to be sensitive to the base composition and sequence of the host duplex, with the binding free energies ranging from -7.5 to -10.8 kcal/mol of bound drug and the binding enthalpies ranging from +4.11 to -10.76 kcal/mol of bound drug at 25 degrees C. The smaller range in the free energy term reflects the impact of large enthalpy-entropy compensations. We observe that the three synthetic duplexes which exhibit the highest daunomycin binding affinities all contain GC (or IC) base pairs as part of alternating purine/pyrimidine sequence motifs, with these high binding affinities being strongly enthalpy driven at 25 degrees C. Specific comparisons between the binding profiles for daunomycin complexation with select pairs of host duplexes lead to the following observations: (1) The presence or absence of a major-groove methyl group does not alter daunomycin binding thermodynamics. (2) The presence or absence of a minor-groove amino group does alter daunomycin binding thermodynamics. (3) Duplexes with different base compositions but identical minor-groove functionality exhibit similar daunomycin binding thermodynamics. (4) Homopolymeric duplexes composed of either AT or AU base pairs, but not GC base pairs, exhibit large enthalpy-entropy compensations in their daunomycin binding profiles. We propose interpretations of these and other features of our thermodynamic data in terms of specific daunomycin-DNA interactions deduced from available structural data.

Base Composition↗

PubMed1991

Thermodynamic characterization of daunomycin-DNA interactions: microcalorimetric measurements of daunomycin-DNA binding enthalpies.

We report the first direct determination of binding enthalpies for the complexation of monomeric daunomycin with a series of 10 polymeric DNA duplexes. These measurements were accomplished by using a recently developed stopped-flow microcalorimeter capable of detecting reaction heats on the microjoule level. This enhanced sensitivity allowed us to measure daunomycin-DNA binding enthalpies at monomeric drug concentrations (e.g., 10-20 microM), thereby precluding the need to correct for daunomycin self-association, as has been required in previous batch calorimetric studies [Remeta, D. P., Marky, L. A., & Breslauer, K. J. (1984) Abstracts of Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, 838a; Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., Snyder, J. G., & Marky, L. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926]. We correct the published daunomycin-DNA binding enthalpies measured by batch calorimetry at higher drug concentrations (e.g., 0.5-1.0 M) for the enthalpy contribution associated with the binding-induced disruption of drug aggregates. The requisite correction term was obtained from a van't Hoff analysis of temperature-dependent NMR measurements on daunomycin solutions. We find remarkable agreement between the net binding enthalpies derived from these corrected batch calorimetric data and the corresponding binding enthalpies measured directly by stopped-flow microcalorimetry. The enhanced sensitivity of the stopped-flow instrument also allowed us to evaluate the influence of drug binding density on the daunomycin-DNA binding enthalpies. This assessment was accomplished by conducting stopped-flow calorimetric measurements over a range of seven different drug-to-phosphate ratios (r). For most of the 10 DNA host duplexes studied, we find that the daunomycin binding enthalpies exhibit small but significant r dependencies. The sensitivity of the stopped-flow instrument also enabled us to detect significant dilution enthalpies for several of the drug-free DNA duplexes, a quantity generally assumed to be negligible in previous studies. We discuss the binding enthalpies, their dependence on binding density, and the duplex dilution enthalpies in terms of the influence of base composition, sequence, conformation/hydration, and binding cooperativity on the sign and the magnitudes of the daunomycin-DNA binding enthalpy data reported here.

Binding Sites↗

PubMed1989

Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites.

We have employed a combination of temperature-dependent UV absorption spectroscopy, circular dichroism, and batch calorimetry to characterize the binding of actinomycin D to a series of oligomeric DNA duplexes. We find the duplex [d(CGTCGACG)]2 to be unique in its ability to bind actinomycin D strongly despite the absence of a classic GpC site. We present evidence that this non-GpC-containing duplex binds two actinomycin D molecules in an apparently cooperative manner to form a complex that exhibits aberrant spectroscopic and calorimetric behavior. We propose that these observations are consistent with actinomycin D exhibiting a high-affinity, sequence-dependent DNA-binding mode distinct from its classic binding to isolated GpC sites.

Base Sequence↗

PubMed1987

Enthalpy-entropy compensations in drug-DNA binding studies.

We present a comparative study of calorimetrically derived thermodynamic profiles for the binding of a series of drugs with selected DNA host duplexes. We use these data to demonstrate that comparisons between complete thermodynamic profiles (delta G zero, delta H zero, delta S zero, delta Cp) are required before drug binding can be used as a probe of DNA conformation, since enthalpy-entropy compensations can cause two drug-DNA binding events to exhibit similar binding free energies (delta G zero) despite being driven by entirely different thermodynamic forces (delta H zero, delta S zero). In this work, we employ a combination of spectroscopic and calorimetric techniques to characterize thermodynamically the DNA binding of netropsin and distamycin (two minor groove-directed ligands), ethidium (an intercalator), and daunomycin (a combined intercalator/groove binder). Our free energy data (delta G zero) show that each drug exhibits similar binding affinities at 25 degrees C for the alternating copolymer duplex poly[d(A-T)].poly[d(A-T)] and for the homopolymer duplex poly(dA).poly(dT). However, our calorimetric measurements reveal that the nature of the thermodynamic forces (delta H zero, delta S zero) that drive drug binding to these two host duplexes at 25 degrees C are entirely different, despite similar binding free energies (delta G zero) and similar salt dependencies (lnK/ln[Na+]). Specifically, the 25 degrees C binding of all four drugs to the alternating copolymer poly[d(A-T)].poly[d(A-T)] is overwhelmingly enthalpy driven, whereas the corresponding binding of each drug to the homopolymer duplex poly(dA).poly(dT) is overwhelmingly entropy driven. Thus, the similar binding free energies (delta G zero) we measure for complexation of each drug with poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) result from compensating changes in the enthalpy and entropy terms. Comparison with the thermodynamic profiles for the complexation of these drug molecules to other DNA host duplexes at 25 degrees C reveals that the binding of each is strongly enthalpy driven, except when the poly(dA).poly(dT) homopolymer serves as the host duplex. This comparison allows us to conclude that poly[d(A-T)].poly[d(A-T)] behaves thermodynamically as the more "normal" host duplex toward drug binding, whereas the entropy-driven binding to the poly(dA).poly(dT) duplex represents "aberrant" behavior. Furthermore, since each of the four drugs exhibits different modes of DNA binding, we conclude that the observed entropy-driven behavior for binding to poly(dA).poly(dT) reflects an intrinsic property of the homopolymer duplex that is perturbed in a common manner upon ligation rather than a common property of all four binding ligands. To rationalize the large positive entropy changes that drive drug complexation with poly(dA).poly(dT) duplex, we propose a model that emphasizes binding-induced perturbations of the more highly hydrated, altered B conformation of the homopolymer. Our results suggest that an aberrant thermodynamic binding profile may reflect an unusual DNA conformation in the host duplex. However, before such a conclusion can be reached, complete thermodynamic binding profiles must be examined, since enthalpy-entropy compensations can cause two binding events to exhibit similar binding constants even when they are driven by very different thermodynamic forces.

Calorimetry↗

PubMed1983

Thermodynamics of drug-DNA interactions.

Batch calorimetry, differential scanning calorimetry (DSC), uv/vis absorption spectroscopy, fluorescence spectroscopy, and circular dichroism (CD), have been used to detect, monitor, and thermodynamically characterize the binding of daunomycin, dipyrandenium, dipyrandium, and netropsin to poly d(AT) and actinomycin D to salmon testes (ST) DNA. The following thermodynamic binding profiles have been obtained. (table; see text) All the poly d(AT) binding studies were done at 25 degrees C while actinomycin binding to ST DNA was performed at 1 degree C to enhance drug solubility. These thermodynamic parameters are interpreted in terms of specific interactions that have been proposed as part of models for the binding of each drug.

Animals↗