Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

Water structures of differing order and mobility in aqueous solutions of schizophyllan, a triple-helical polysaccharide as revealed by dielectric dispersion measurements

Dielectric dispersion measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan over a wide concentration range 10-50 wt % at -45 to +30 degrees C. In the solution state, three different water structures with the different relaxation times tau were found, namely, bound water (taul), structured water (taus), and loosely structured water (tauls) in addition to free water (tauP). Structured water is less mobile and loosely structured water is nearly as mobile as free water, but bound water with taul is much less mobile, thus taul > taus > tauls greater, similar tauP. The order-disorder transition accompanies the conversion between structured water and loosely structured water. However, the species with taus remains even in the disordered state and constitutes part of bound water in the entire temperature range. In the frozen state, in addition to bulk water formed by partial melting, two mobile species existed, which were assigned to liquidlike bound water and found to be a continuation of bound water in the solution state. These relaxation time data are discussed in connection with the entropy levels of the four structures deduced from heat capacity data (cf. Yoshiba, K.; et al. Biomacromolecules 2003, 4, 1348-1356).     

Contribution of secondary structure to the heat capacity and enthalpy distribution of the unfolded state in proteins.

We have recently shown that one can construct the enthalpy distribution for protein molecules from experimental knowledge of the temperature dependence of the heat capacity. For many proteins the enthalpy distribution evaluated at the midpoint of the denaturation transition (corresponding to the maximum in the heat capacity vs temperature curve) is broad and biphasic, indicating two different populations of molecules (native and unfolded) with distinctly different enthalpies. At temperatures above the denaturation point, the heat capacity for the unfolded state in many proteins is quite large and using the analysis just mentioned, we obtain a gaussian-like enthalpy distribution that is very broad. A large value of the heat capacity indicates that there are structural changes going on in the unfolded state above the transition temperature. In the present paper we investigate the origin of this large heat capacity by considering the presence of changing amounts of secondary structure (specifically, alpha-helix) in the unfolded state. For this purpose we use the empirical estimates of the Zimm-Bragg sigma and s factors for all of the native amino acids in water as determined by Scheraga and co-workers. Using myoglobin as an example, we calculate probability profiles and distribution functions for the total number of helix states in the specific-sequence molecule. Given the partition function for the specific-sequence molecule, we can then calculate a set of enthalpy moments for the molecule from which we obtain a good estimate of the enthalpy distribution in the unfolded state. This distribution turns out to be quite narrow when compared with the distribution obtained from the raw heat capacity data. We conclude that there must be other major structural changes (backbone and solvent) that are not accounted for by the inclusion of alpha-helix in the unfolded state.     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.     

Thermodynamics of conformational transition and chain association of ι-carrageenan in aqueous solution: Calorimetric and chirooptical data

Isothermal microcalorimetry, differential scanning calorimetry (DSC), and chirooptical data obtained for ι-carrageenan in NaCl, LiCl, and NaI aqueous solutions are presented. The experiments have been performed as a function of concentration both for the polymer and for the simple salt as a cosolute. The experimental findings consistently show the occurrence of a salt-induced disorder-to-order transition. From microcalorimetric experiments the exothermic enthalpy of transition ΔH_tr is obtained as the difference between the theoretical, purely electrostatic ΔH_el enthalpy change and the actual mixing enthalpy ΔH_mix, measured when a ι-carrageenan salt-free solution at constant polymer concentration is mixed with a 1:1 electrolyte solution of variable concentration. In the case of added NaCl, the absolute values of enthalpy changes |ΔH_tr| are in good agreement with those obtained for the opposite process, at comparable polymer and salt concentrations, from DSC melting curves. The microcalorimetric results show that the negative maximum value of ΔH_tr corresponding to the interaction of Li⁺ counterion with ι-carrageenan polyion results to be significantly lower than the corresponding values obtained for Na⁺ counterion. At variance with the microcalorimetric data, chirooptical results show that the salt-induced disorder-to-order transition, occurring in the 0.02–0.2M salt concentration range, appears to be complete at a concentration of about 0.08–0.1M of the simple ion, irrespective of the polymer concentration and of the nature of added electrolyte. © 1998 John Wiley & Sons, Inc. Biopoly 45: 105–117, 1998     

Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding.

Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (delta Cp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and delta Cp.     

Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.