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The negative enthalpy change (deltaH(sub obs)) is examined over a much wider temperature range and as a function of monovalent salt concentration and type for SSB binding to (dT)70 under solution conditions that favor the fully wrapped (SSB)65 complex using isothermal titration calorimetry. Over this wider temperature range, a strongly temperature-dependent negative heat capacity change (deltaC(sub p,obs) is observed.

Results show that Escherichia coli homotetrameric SSB protein is transferred from one single-stranded DNA molecule to another with the need for a protein intermediate. Data indicate that the rate of transfer is dependent on the amount of free DNA binding sites within the initital SSB tetramer-single-stranded DNA donor complex.

Research has been conducted on the homotetrameric Escherichia coli SSB protein's binding to the single-stranded oligodeoxynucleotides. The kinetic mechanism of this binding has been investigated under conditions allowing the fully wrapped ssDNA complex formation and the results are reported.

Salt concentration was shown to be significant in the stability and specificity of protein-nucleic acid interactions, as evidenced by the results of binding the tetrameric Escherichia coli single-stranded binding (SSB) protein to the poly(U) nucleic acid. Possible explanation for the event is the role of cations as they are bound to nucleic acids. Equilibrium association constant decreased with increased salt concentrations at all temperatures used when binding SSB proteins with the linear poly(U) nucleic acid.

The equilibrium binding Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) to ssDNA is examined for comparison with that of Escherichia coli SSB (EcoSSB). The binding enthalpy has shown a large sensitivity to NaCl concentration, similar to that seen for EcoSSB and these common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.

Isothermal titration calorimetry was used to probe the specificity and examine the thermodynamics of single-stranded DNA binding protein (SSB) binding to two Escherichia coli proteins important for DNA replication, the x subunit of DNA polymerase III holoenzyme and the PriA helicase. The results suggested that DNA binding proteins could use the interaction of SSB with both x and PriA to gain access to ssDNA by first interacting with the SSB C-termini.

A study shows that Deinococcus radiodurans SSB (DrSSB) facilitates a local denaturation of the DNA helix at the ss-dsDNA junction of partially duplex DNAs with overhangs of both 15 and 30 nucleotides. Results reveal that DrSSb protein can destabilize duplex DNA immediately adjacent to both 3' and 5' ssDNA extensions and may have relevance for a variety of bacterial DNA metabolic processes.

The single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium is examined to determine whether scRPA could bind ssDNA in multiple binding modes. Results reveal that ssRPA could bind to long ssDNA in at least two binding modes in vitro and that the relative stability of these two modes is influenced by the salt concentration.

Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) originates from a radiation-resistant bacterium and participates in DNA recombination, replication, and repair. Although it functions as a homodimer, it contains four DNA binding domains (OB-folds) and thus is structurally similar to the Escherichia coli SSB (EcoSSB) homotetramer. We examined the equilibrium binding of DrSSB to ssDNA for comparison with that of EcoSSB. We find that the occluded site size of DrSSB on poly(dT) is ∼45 nucleotides under low-salt conditions (<0.02 M NaCl) but increases to 50-55 nucleotides at ⩾0.2 M NaCl. This suggests that DrSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB, although the site size difference between modes is not as large as for EcoSSB. suggesting that the pathways of ssDNA wrapping differ for these two proteins. The occluded site size corresponds well to the contact site size (52 nucleotides) determined by isothermal titration calorimetry (ITC). Electrophoretic studies of complexes of DrSSB with phage MI3 ssDNA indicate the formation of stable, highly cooperative complexes under low-salt conditions. Using ITC. we find that DrSSB binding to oligo(dT)s with lengths close to the determined site size (50-55 nucleotidesl is stoichiometric with Δ Hobs of approximately -94 ± 4 kcal/mol. somewhat smaller than that for froSSB (approximately - 130 kcal/mol) under the same conditions. The observed binding enthalpy shows a large sensitivity to NaCl concentration, similar to that observed for EcoSSB. With the exception of the less dramatic change in occluded site size, the behavior of DrSSB is similar to that of EcoSSB protein (although clear quantitative differences exist). These common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo. [ABSTRACT FROM AUTHOR]

The Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the Χ subunit of DNA polymerase III holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last ~9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over Χ due primarily to a more favorable enthalpic component. PriA and Χ also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both Χ and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini. [ABSTRACT FROM AUTHOR]

Reveals that adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates. Use of isothermal titration calorimetry; Negatives values of enthalpy change for dT(pT)34, dC(pC)34 and dA(pA)34; Thermodynamic properties for SSB-dA(pA) binding.

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families (a) to bind to CP, (b) to allosterically inhibit barbed-end capping by CP, and (c) to allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs across all protein families [Hernandez-Valladares, M., et al. (2010) Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. Nat. Struct. Mol. Biol. 17 , 497-503; Bruck, S., et al. (2006) Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein. J. Biol. Chem. 281 , 19196-19203]. These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.