Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway

The C2 domain is a targeting domain that responds to intracellular Ca²⁺ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P₂ specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca²⁺ specifically binds to the Ca²⁺-binding region, facilitating PtdIns(4,5)P₂ access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P₂ binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P₂ binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P₂ and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P₂ is hydrolyzed to generate diacylglycerol and IP₃, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.     

Structural and thermodynamic studies of simple aldose reductase-inhibitor complexes

The competitive inhibition constants of series of inhibitors related to phenylacetic acid against both wild-type and the doubly mutanted C298A/W219Y aldose reductase have been measured. Van't Hoff analysis shows that these acids bind with an enthalpy near -6.8 kcal/mol derived from the electrostatic interactions, while the 100-fold differences in binding affinity appear to be largely due to entropic factors that result from differences in conformational freedom in the unbound state. These temperature studies also point out the difference between substrate and inhibitor binding. X-ray crystallographic analysis of a few of these inhibitor complexes both confirms the importance of a previously described anion binding site and reveals the hydrophobic nature of the primary binding site and its general plasticity. Based on these results, N-glycylthiosuccinimides were synthesized to demonstrate their potential in studies that probe distal binding sites. Reduced alpha-lipoic acid, an anti-oxidant and therapeutic for diabetic complications, was shown to bind aldose reductase with a binding constant of 1 microM.     

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.     

A simple hydrogen bond potential function for conformational studies

Since hydrogen bonding plays an important role in determining energetically favourable conformations of biological molecules, a computationally simple and convenient semiempirical function has been evaluated by constraining a function to conform to the experimentally observed data for isolated simple systems of hydrogen bonded dimers. The function is found to be satisfactory for use in conformational energy calculations.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. IV. Enthalpies of hydration of 5-alkyluracils

Enthalpies of sublimation, DeltaH degrees (subl) and of solution in water, DeltaH degrees (sol) were determined for a series of crystalline 1,3-dimethyl-uracil derivatives substituted at the C5-ring carbon atom with alkyl groups (-C(n)H(2n+1), n = 2-4) and some of their C(5.6)-cyclooligomethylene analogues (-(CH2)(n)-, n = 3-5). From these data. enthalpies of hydration DeltaH degrees (hydr)= DeltaH degrees (sol) - DeltaH degrees (subl) were calculated and corrected for energies of cavity formation in pure liquid water in order to obtain enthalpies of interaction, DeltaH degrees (int) of the solutes with their hydration shells. The latter are discussed together with the recalculated DeltaH degrees (int) for variously methylated uracils, obtained previously according to a simplified correction procedure, in terms of perturbations in the energy and scheme of hydration of the diketopyrimidine ring brought about by alkyl substitution. It was found that each -CH2-group added with an alkyl substitution contributes favorably about -20 kJ mol(-1) toDeltaH degrees (int).This contribution is partially cancelled by the unfavorable contribution to DeltaH degrees (int) connected with removal of some water molecules bound in the first and subsequent hydration layers by an alkyl substituent. This is particularly evident on substitution at the polar side of the diketopyrimidine ring on which water molecules are expected to be bound specifically.     

Calculation of thermodynamic properties of protein in Escherichia coli K-12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K-12 protein. These resulted in values fro DeltaG(f) of -38.09 kJ (-9.10 kcal)/ UCFW, for DeltaH(f) of -68.18 kJ (-16.29 kcal)/UCFW, and for DeltaS(f) of -94.2 J (-22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K-12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for DeltaG, 0.75 kJ (0.18 kcal)/UCFW for DeltaH, and -4.09 J (-0.98 cal)/UCFW deg for DeltaS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.     

Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state

Two endothermic peaks could be observed for five commercial samples of bovine serum albumin (BSA). The smaller peak observed by differential scanning calorimetry (DSC) corresponded to enthalpy relaxation. This peak was followed on storage of BSA, in its glassy state, after it had been heated above its denaturation temperature. Enthalpy and peak temperature increased with duration of storage. On storage for one week at 60 degrees C, a sample at 8.3% moisture showed a peak at 100 degrees C with an energy value of approximately 2 J per g protein. BSA samples were heated within the DSC sufficiently to eliminate the lower enthalpy peak but without altering the denaturation enthotherm. The amount of physical aging shown by these BSA samples was similar to that of the heat-denatured samples. It was concluded that the heating endotherms of dry BSA reflect both the storage and thermal history of the sample. Possible implications of the enthalpy relaxation of BSA on the behavior of this important protein are considered.     

Thermodynamic consequences of disrupting a water-mediated hydrogen bond network in a protein:pheromone complex

The mouse pheromones (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT) and 6-hydroxy-6-methyl-3-heptanone (HMH) bind into an occluded hydrophobic cavity in the mouse major urinary protein (MUP-1). Although the ligands are structurally unrelated, in both cases binding is accompanied by formation of a similar buried, water-mediated hydrogen bond network between the ligand and several backbone and side chain groups on the protein. To investigate the energetic contribution of this hydrogen bond network to ligand binding, we have applied isothermal titration calorimetry to measure the binding thermodynamics using several MUP mutants and ligand analogs. Mutation of Tyr-120 to Phe, which disrupts a hydrogen bond from the phenolic hydroxyl group of Tyr-120 to one of the bound water molecules, results in a substantial loss of favorable binding enthalpy, which is partially compensated by a favorable change in binding entropy. A similar thermodynamic effect was observed when the hydrogen bonded nitrogen atom of the heterocyclic ligand was replaced by a methyne group. Several other modifications of the protein or ligand had smaller effects on the binding thermodynamics. The data provide supporting evidence for the role of the hydrogen bond network in stabilizing the complex.     

Enantioseparation of racecadotril using polysaccharide‐type chiral stationary phases in polar organic mode

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide‐type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile) at 5 different temperatures (5–40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector—and mobile‐phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R‐enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.