Folding Thermodynamics of the Hybrid-1 Type Intramolecular Human Telomeric GQuadruplex

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 K1 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 K1-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, 432619 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. VC 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014. Keywords: G-quadruplexes; conformational transitions; volume; compressibility; expansibilit

Solvation and Conformational Stability of Proteins and DNA

The work performed in this dissertation is devoted to understanding and quantifying solute-solvent interactions in the folded and unfolded states of proteins and DNA G-quadruplexes using volumetric techniques. We characterized the interactions of the protein stabilizer, glycine betaine (GB), with proteins and their functional groups through a combination of partial molar volume and adiabatic compressibility measurements of N-acetyl amino acid amides, oligoglycines, cytochrome c, ribonuclease A, lysozyme, and ovalbumin at GB concentrations ranging from 0 to 4 M. We evaluated the equilibrium (binding) constant, k, for the reaction in which a GB molecule binds each of the functionalities under study replacing four water molecules. We found that GB forms direct interactions with all protein groups studied here. In addition, the differential free energy of solute-solvent interactions in a concentrated GB solution and water, ΔΔGI, is negative for all the proteins studied and ΔΔGI becomes more favourable as the concentration of GB increases. We also employed volumetric measurements of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A in the solutions at 0 to 8 M urea to quantify the urea-induced solvation changes in the native and unfolded states of proteins. An increase in the concentration of urea to 8 M leads to a ~20% increase in the solvent accessible surface area of apocytochrome c. The urea-induced unfolding of ribonuclease A and α-chymotrypsinogen A is accompanied by increases in solvent accessible surface area of 1.9 ± 0.4 and 2.0 ± 0.6 times that of the native states, respectively. Furthermore, we characterized the volumetric properties of the folded and unfolded states of the Na+-stabilized antiparallel G-quadruplex conformation of Tel22, d[A(GGGTTA)3GGG], and the K+-stabilized hybrid-1 conformation of Tel26, d[AAAGGG(TTAGGG)3AA]. The coil-to-G-quadruplex transition of Tel26 accompanies a release of 434 ± 19 water molecules from its hydration shell to the bulk, which is more than four times the number of waters released compared to Tel22 (103 ± 44). We proposed that this extensive DNA dehydration originates from both the waters in direct contact with the domains that become buried in G-quadruplex formation and a general decrease in solute-solvent interactions all over the surface of the folded structure.Ph.D

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 <i>c</i>, 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, <i>k</i>, as well as changes in volume, Δ<i>V</i>₀, and adiabatic compressibility, Δ<i>K</i>_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