Yuen Lai Shek

Urea interactions with protein groups: A volumetric study

We determined the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides, N-acetyl amino acid methylamides, N-acetyl amino acids, and short oligoglycines as a function of urea concentration. We analyze these data within the framework of a statistical thermodynamic formalism to determine the association constants for the reaction in which urea binds to the glycyl unit and each of the naturally occurring amino acid side chains replacing two waters of hydration. Our determined association constants, k, range from 0.04 to 0.39 M. We derive a general equation that links k with changes in free energy, DeltaGtr, accompanying the transfer of functional groups from water to urea. In this equation, DeltaGtr is the sum of a change in the free energy of cavity formation, DeltaDeltaGC, and the differential free energy of solute-solvent interactions, DeltaDeltaGI, in urea and water. The observed range of affinity coefficients, k, corresponds to the values of DeltaDeltaGI ranging from highly favorable to slightly unfavorable. Taken together, our data support a direct interaction model in which urea denatures a protein by concerted action via favorable interactions with a wide range of protein groups. Our derived equation linking k to DeltaGtr suggests that DeltaDeltaGI and, hence, the net transfer free energy, DeltaGtr, are both strongly influenced by the concentration of a solute used in the experiment. We emphasize the need to exercise caution when two solutes differing in solubility are compared to determine the DeltaGtr contribution of a particular functional group.

Hydration changes accompanying helix-to-coil DNA transitions.

We applied ultrasonic velocimetric and high-precision densimetric measurements to characterizing the helix-to-coil transition of the GGCATTACGG/CCGTAATGCC decameric DNA duplex. The transition was induced either by temperature or by mixing the two complementary single strands at isothermal conditions. The duplex dissociation causes increases in volume and expansibility while resulting in a decrease in compressibility. Our volumetric data in conjunction with computer-generated structural information are consistent with the picture in which the duplex dissociation is accompanied by an uptake of ∼180 water molecules from the bulk phase into the hydration shell of the DNA. Analysis of our compressibility and expansibility data reveals that the single-stranded conformation is likely to exist as a heterogeneous mixture of nearly isoenergetic subspecies differing in volume and enthalpy. We use our estimate of the change in hydration to evaluate the hydration and configurational contributions to the helix-to-coil transition entropy. The duplex dissociation is accompanied by an increase in configurational entropy, ΔSconf, of ∼23 cal mol(-1) K(-1) per nucleotide, which signifies liberation of manifold frozen degrees of freedom involved in maintaining the conformational stability of the duplex and the related stiffening of the heterocyclic bases and the sugar-phosphate backbone. To the best of our knowledge, this is the first experimental estimate of the change in configurational entropy associated with the helix-to-coil transition of a DNA.

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.

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.

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.

Polyelectrolyte effects in G-quadruplexes

The role of counterion condensation as a dominant force governing the stability of DNA duplexes and triplexes is well established. In contrast, the effect of counterion condensation on the stability of G-quadrupex conformations is poorly understood. Unlike other ordered nucleic acid structures, G-quadruplexes exhibit a specific binding of counterions (typically, Na(+) or K(+)) which are buried inside the central cavity and coordinated to the O6 carbonyls of the guanines forming the G-quartets. While it has been known that the G-quadruplex-to-coil transition temperature, TM, increases with an increase in the concentration of the stabilizing ion, the contributions of the specific (coordination in the central cavity) and nonspecific (condensation) ion binding have not been resolved. In this work, we separate the two contributions by studying the change in TM of preformed G-quadruplexes following the addition of nonstabilizing ions Li(+), Cs(+), and TMA(+) (tetramethylammonium). In our studies, we used two G-quadruplexes formed by the human telomeric sequences which are distinct with respect to the folding topology and the identity and the number of sequestered stabilizing ions. Our data suggest that the predominant ionic contribution to G-quadruplex stability comes from the specifically bound Na(+) or K(+) ions and not from counterion condensation. We offer molecular rationalizations to the observed insensitivity of G-quadruplex stability to counterion condensation and emphasize the need to expand such studies to assess the generality of our findings.

Interactions of Urea with the Folded and Unfolded States of Proteins

Urea is a strong protein denaturant, yet, the mechanism of action of urea on the protein unfolding process is still largely unknown. To tackle this problem, we determined the partial molar volumes, Vo, and adiabatic compressibilities, KoS, of a set of model proteins to examine their interactions with urea. Specifically, we measured the partial molar volume and adiabatic compressibility of ribonuclease A, α-chymotrypsinogen A, lysozyme, and apocytochrome c in aqueous solutions of urea at concentrations between 0 and 8M. At pH 2 and pH3, ribonuclease A and α-chymotrypsinogen A, respectively, exhibit a two-state transition of unfolding over the range of urea concentrations studied. Even in 8M urea, lysozyme retains its native conformation and apocytochrome c remains unfolded at pH 7. The fact that lysozyme and apocytochrome c do not undergo any conformational transitions in the presence of urea provides an opportunity to study the interactions of urea with proteins in the native and unfolded states within the entire range of experimentally accessible urea concentrations. We analyze our resulting volumetric data within the framework of a statistical thermodynamic model in which each instance of urea interaction with a protein is viewed as a binding reaction that is accompanied by release of two water molecules. From this analysis, we calculate the association constants, k, as well as changes in volume, ΔVO, and adiabatic compressibility, ΔKSO, accompanying each urea-protein association event in an ideal solution. By comparing these parameters with similar characteristics determined for low-molecular weight analogues of proteins, we quantify the extent of cooperative effects involved in interactions of urea with any of the proteins studied in this work.

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 (–CH2CONH–) 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, ΔGtr, as the sum of a change in the free energy of cavity formation, ΔΔGC, and the differential free energy of solute-solvent interactions, ΔΔGI, in a concentrated GB solution and water. Our results suggest that the transfer free energy, ΔGtr, results from a fine balance between the large ΔΔGC and ΔΔGI contributions. The range of the magnitudes and the shape of the GB dependence of ΔGtr depend on the identity of a specific solute group. The interplay between ΔΔGC and ΔΔGI results in pronounced maxima in the GB dependences of ΔGtr 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 TM-versus-GB concentration plots reported for ribonuclease A and lysozyme.

Molecular Mechanism of Urea-Induced Protein Denaturation

For more than half a century, urea has been used as a strong denaturant in protein folding/unfolding studies. However, the molecular mechanisms of urea-induced protein unfolding still remain unclear. This lack of understanding is to some extent reflects the scarcity of direct thermodynamic information that can be used to characterize interactions of urea with amino acid side chains and the peptide group. We recently demonstrated that volumetric measurements combined with statistical thermodynamic approach may represent a novel and effective way to tackle this problem [Lee, S. & Chalikian, T. V. (2009) J. Phys. Chem. B. 113, 2443-2450]. In this work, we employ high precision acoustic and densimetric techniques to quantify the solvation properties of solutes in the presence of urea. Specifically, we report the partial molar volumes, V°, and adiabatic compressibilities, KS°, of N-acetyl amino acid amides containing all 20 naturally existing amino acid side chains and oligoglycines, (Gly)1-5, at urea concentrations ranging from 0 to 8 M. Using our developed statistical thermodynamic approach, that links volumetric observables of a solute with solute-solvent and solute-cosolvent interactions in binary solvents, we evaluate the binding constants, k, and elementary changes in volume, ΔV, and compressibility, ΔKS, accompanying the replacement of water in the vicinity of the solutes with a urea molecule. While the binding constants are essentially similar for all protein groups, the magnitude and the sign of the determined values of ΔV and ΔKS vary markedly. The latter values reflect the nature of urea interactions with specific functional groups and the concomitant changes in hydration. In general, our results are consistent with a picture in which urea interacts with polar, non-polar and charged groups with comparable affinities, although the underlying forces stabilizing each type of interaction depend on the chemical nature of the interacting group.