Interactions of urea with native and unfolded proteins: a volumetric study.

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

Folding thermodynamics of the hybrid-1 type intramolecular human telomeric G-quadruplex.

Guanine-rich DNA sequences that may form G-quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G-quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G-quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G-rich sequences in the presence or the absence of their complementary C-rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26-meric human telomeric sequence d[A3G3(T2AG3)3A2]. In the presence of K+ ions, the latter adopts the hybrid-1 G-quadruplex conformation, a tightly packed structure with an unusually small number of solvent-exposed atomic groups. The K+-induced folding of the G-quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G-quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G-quadruplex formation. Based on our volume data, 432±19 water molecules become released to the bulk upon the G-quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute-solvent interactions all over the surface of the folded structure.

Interactions of glycine betaine with proteins: insights from volume and compressibility measurements.

We report the first application of volume and compressibility measurements to characterization of interactions between cosolvents (osmolytes) and globular proteins. Specifically, we measure the partial molar volumes and adiabatic compressibilities of cytochrome c, ribonuclease A, lysozyme, and ovalbumin in aqueous solutions of the stabilizing osmolyte glycine betaine (GB) at concentrations between 0 and 4 M. The fact that globular proteins do not undergo any conformational transitions in the presence of GB provides an opportunity to study the interactions of GB with proteins in their native states within the entire range of experimentally accessible GB concentrations. We analyze our resulting volumetric data within the framework of a statistical thermodynamic model in which each instance of GB interaction with a protein is viewed as a binding reaction that is accompanied by release of four water molecules. From this analysis, we calculate the association constants, k, as well as changes in volume, ΔV(0), and adiabatic compressibility, ΔK(S0), accompanying each GB-protein association event in an ideal solution. By comparing these parameters with similar characteristics determined for low-molecular weight analogues of proteins, we conclude that there are no significant cooperative effects involved in interactions of GB with any of the proteins studied in this work. We also evaluate the free energies of direct GB-protein interactions. The energetic properties of GB-protein association appear to scale with the size of the protein. For all proteins, the highly favorable change in free energy associated with direct protein-cosolvent interactions is nearly compensated by an unfavorable free energy of cavity formation (excluded volume effect), yielding a modestly unfavorable free energy for the transfer of a protein from water to a GB/water mixture.

A short DNA aptamer that recognizes TNFα and blocks its activity in vitro.

Tumor necrosis factor-alpha (TNFα) is a pivotal component of the cytokine network linked to inflammatory diseases. Protein-based, TNFα inhibitors have proven to be clinically valuable. Here, we report the identification of short, single-stranded DNA aptamers that bind specifically to human TNFα. One such 25-base long aptamer, termed VR11, was shown to inhibit TNFα signaling as measured using NF-κB luciferase reporter assays. This aptamer bound specifically to TNFα with a dissociation constant of 7.0 ± 2.1 nM as measured by surface plasmon resonance (SPR) and showed no binding to TNFß. Aptamer VR11 was also able to prevent TNFα-induced apoptosis as well as reduce nitric oxide (NO) production in cultured cells for up to 24 h. As well, VR11, which contains a GC rich region, did not raise an immune response when injected intraperitoneally into C57BL/6 mice when compared to a CpG oligodeoxynucleotide (ODN) control, a known TLR9 ligand. These studies suggest that VR11 may represent a simpler, synthetic scaffold than antibodies or protein domains upon which to derive nonimmunogenic oligonucleotide-based inhibitors of TNFα.

Volumetric characterization of interactions of glycine betaine with protein groups.

We report the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides and oligoglycines at glycine betaine (GB) concentrations ranging from 0 to 4 M. We use these results to evaluate the volumetric contributions of amino acid side chains and the glycyl unit (-CH(2)CONH-) as a function of GB concentration. We analyze the resulting GB dependences within the framework of a statistical thermodynamic model and evaluate the equilibrium constant for the reaction in which a GB molecule binds each of the functionalities under study replacing four water molecules. We calculate the free energy of the transfer of functional groups from water to concentrated GB solutions, ΔG(tr), as the sum of a change in the free energy of cavity formation, ΔΔG(C), and the differential free energy of solute-solvent interactions, ΔΔG(I), in a concentrated GB solution and water. Our results suggest that the transfer free energy, ΔG(tr), results from a fine balance between the large ΔΔG(C) and ΔΔG(I) contributions. The range of the magnitudes and the shape of the GB dependence of ΔG(tr) depend on the identity of a specific solute group. The interplay between ΔΔG(C) and ΔΔG(I) results in pronounced maxima in the GB dependences of ΔG(tr) for the Val, Leu, Ile, Trp, Tyr, and Gln side chains as well as the glycyl unit. This observation is in qualitative agreement with the experimental maxima in the T(M)-versus-GB concentration plots reported for ribonuclease A and lysozyme.