Possible mechanism of specific action of pulsed UHF-fields

Experimental data were obtained for conditions of stimulation of mechanical vibrations in the liquid models of microwave fields. Possible role of different elastic waves modes in the formation of specific effects in pulsed HF-fields is discussed. Mathematical estimation suggests that excited shearing waves may have biological action. The results obtained suggest an acoustic nature of the action of pulsed HF-fields on biological objects.     

Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses.

In this work, we evaluate the stability, dynamics and protein-nucleic acid interaction in Flock House virus (FHV). FHV is an RNA insect virus, non-enveloped, member of the family Nodaviridae. It is composed of a bipartite single-stranded RNA genome packaged in an icosahedral capsid of 180 copies of an identical protein (alpha protein). A fundamental property of many animal viruses is the post-assembly maturation required for infectivity. FHV is constructed as a provirion, which matures to an infectious virion by cleavage of alpha protein into beta and gamma subunits. We used high pressure, temperature and chemical denaturing agents to promote perturbation of the viral capsid. These effects were monitored by spectroscopy measurements (fluorescence, light scattering and CD) and size-exclusion chromatography. The data showed that FHV was stable to pressures up to 310 MPa at room temperature. The fluorescence emission and light scattering values showed small changes that were reversible after decompression. When we combined pressure and sub-denaturing urea concentrations (1 M), the changes were more drastic, suggesting dissociation of the capsid. However, these changes were reversible after pressure release. The complete dissociation of FHV could be observed only under high urea concentrations (10 M). There were no significant changes in emission spectra up to 5 M urea. FHV also was stable when we used temperature treatments (high and low). We also compared the effects of urea and pressure on FHV wild type and cleavage-defective mutant VLPs (virus-like particles). The VLPs and authentic particles are distinguishable by protein-RNA interactions, since VLPs pack cellular RNA and native particles contain viral RNA. Our results demonstrated that native particles are more stable than VLPs to physical and chemical treatments. Our data point to the specificity of the interaction between the capsid protein and the viral RNA. This specificity is crucial to the stability of the particle, which makes this interaction an excellent target for drug development.     

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.     

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a³E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.     

Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.     

Possible mechanism of CO2 interaction with NAD-dependent malate dehydrogenase

IR spectra of NAD-dependent malate dehydrogenase in the deuterium oxide solutions were studied in the absence of CO2 and at solution saturation with it. The presence of CO2 in the system results in weakening the absorption band intensity at 1650 cm-1 and in the appearance of the band at 1543 cm-1, which is explained by the formation of carbamates under conditions of the protein molecules free amino groups interaction with CO2.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Roles of intrinsic disorder in protein-nucleic acid interactions

Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.     

Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes

A phenomenological theory of gel electrophoresis is elaborated for protein-DNA complexes involving one, two, or three binding sites on the DNA molecule. The computed electrophoretic patterns simulate experimental patterns shown by both prokaryotic and eukaryotic systems. The mechanism whereby the electrophoretic protein-DNA ladder is generated upon titration of the operator with repressor is embodied in theory of mass transport coupled to reversible interactions under chemical kinetic control. In contrast to strong interactions (association constant greater than 10(12) M-1), patterns observed with weak complexes (K less than 10(10) M-1) could be simulated only by applying the cage effect, a model of which is formulated. Theoretical underpinning is provided for the electrophoretic estimation of equilibrium association constants, and requisite chemical kinetic conditions are elucidated for direct estimation of the rate constant for dissociation of the protein-DNA complex from gel patterns. The theory thus affords an experimenter with a means for determining the conditions required to render the gel retardation method a valid procedure for evaluating equilibrium constants and/or kinetic parameters for the particular protein-nucleic acid system under investigation. These several considerations apply not only to interactions of proteins with nucleic acids (DNA or RNA) but also to a wide range of macromolecular interactions involving peptides, drugs, and other ligands as well as large assemblies such as multienzyme complexes.