Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.     

The conformational transition of horse heart porphyrin c

The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having time constants of 20 and 200 ms as detected by stopped flow absorbance or fluorescence measurement, with about 80% of the observed change in the faster phase. The kinetics of porphyrin c refolding are not significantly altered by increasing the viscosity of the refolding solvent 15-fold by addition of sucrose. We suggest that the folding of guanidine denatured cytochrome c is not a diffusion-limited process and that the requirement for protein axial ligation elicits the slow (s) kinetic phase observed in the refolding of cytochrome c.     

The self-association of chymotrypsinogen

The self-association of chymotrypsinogen A is reduced by decreasing the pH below pH 8 or by an increase in electrolyte level. The latter effect is correlated with ionic strength for several electrolytes with different properties. The self-association in H₂O is almost independent of temperature. Substitution of D₂O for H₂O increases the extent of association. Conversion of the two reactive tyrosines to nitrotyrosine extends the zone of stability of the complex species to more acid pH. From these results it is concluded that the dominant stabilizing force is electrostatic in origin, but that hydrophobic forces also make a significant contribution.     

The reversible heat denaturation of chymotrypsinogen

Within a restricted range of pH and protein concentration crystalline chymotrypsinogen undergoes thermal denaturation which is wholly reversed upon cooling. At a given temperature an equilibrium exists between native and reversibly denatured protein. Within the pH range 2 to 3 the amount of denatured protein is a function of the third power of the hydrogen ion activity. The presence of small amounts of electrolyte causes aggregation of the reversibly denatured protein. A specific anion effect has been observed at pH 2 but not at pH 3. Both the reversible denaturation reaction and the reversal reaction have been found to be first order reactions with respect to protein and the kinetic and thermodynamic constants for both reactions have been approximated at pH 2 and at pH 3. Renatured chymotrypsinogen has been found to be identical with native chymotrypsinogen with respect to crystallizability, solubility, activation to δ-chymotrypsin, sedimentation rate, and behavior upon being heated. Irreversible denaturation of chymotrypsinogen has been found to depend on pH, temperature, protein concentration, and time of heating. Irreversible denaturation results in an aggregation of the denatured protein.     

The role of conformational energetic disorder in the catalytic activity of immobilized enzymes

We analyze cooperative behavior in a system of immobilized enzymes which incorporates the notion of heterogeneity or disorder in the interactions. In addition to equilibrium phase changes, this system exhibits vitrification or glass-like transitions in which the overall catalytic activity freezes into one of many possible states. It is shown that these long-lived metastable phases can be produced by a combination of disorder, systematic surface and intermolecular interactions, and chemical association effects such as ligand binding. Biophysical consequences of this frozen state include greatly diminished sensitivity of enzymatic activity to thermal and chemical perturbations. This effect coincides with the appearance of a multitude of possible macroscopic catalytic states rather than a single equilibrium state. Our analysis also suggests that high surface coverages will tend to be catalytically inactive if they are fully equilibrated; rather, high activity with high surface coverage is more likely to be associated with vitrified states of surface immobilization and deep or abrupt chemical quenches.     

Electrophoretic separation of molecular species associated with the thermal transition of chymotrypsinogen A

Solutions of thermally unfolded chymotrypsinogen A were rapidly cooled to 0 °C and subjected to gel electrophoresis. In addition to the native protein, two other electrophoretic species were observed. Both appear to result from reversible interaction with the transition process. Of the three electrophoretic species revealed, that which represents the reversibly unfolded state is the intermediate as gauged by its mobility.     

ATP-induced conformational transition of denatured proteins.

Although the conformational change occurring in proteins upon ATP binding is important in many biological reactions, the mechanism by which ATP binding induces the conformational change is unknown. We found that ATP induces acid-unfolded (pH 2) ferricytochrome c or apomyoglobin to adopt a compact structure with a significant amount of alpha-helix and increased hydrophobicity. A very similar conformational transition was observed at neutral pH for an amphiphilic model polypeptide. The effectiveness of various adenine nucleotides in inducing the conformational transition was found to be proportional to their phosphate group contents, i.e., adenosine tetraphosphate greater than ATP greater than ADP greater than AMP. These results should be important when considering the mechanism of the ATP-induced conformational change in proteins during various biological reactions.     

Acid- and temperature-induced conformational changes in human chorionic gonadotropin and the mechanism of subunit dissociation

Human chorionic gonadotropin undergoes a conformational transition in acid which at 4 °C is characterized by: (i) a reversible increase in the polarization of tyrosyl fluorescence, P, with a midpoint at pH 5, (ii) a slight decrease in the elution volume on Sephadex G-100 at pH 3 relative to pH 7, (iii) a slight decrease in s_20,w. (iv) a small positive near uv difference spectrum (Δ? ~2%), and (v) the appearance of a positive CD feature at 235 nm. These observations are compatible with an acid-expanded form of the hormone in which the rotational freedom of one or more tyrosine residues is restricted and/or their proximity to potential quenching groups is altered. The increased value of P following acidification is stable at temperatures below 10 °C, but at higher temperatures it decreases with time to an extent which is dependent on the temperature. A substantial portion of this decrease occurs before subunit dissociation can be detected, reflecting the occurrence of a thermal transition with a midpoint near 26 °C. A similar transition was observed at neutral pH with a midpoint near 22 °C. These results suggest the occurrence of at least two conformationally distinct forms of hCG which may be sequentially encountered prior to subunit dissociation in acid. The kinetics will be either biphasic or strictly first order, depending on the temperature at which the hormone is acidified.     

The conformational transition of κ-carrageenan using microcalorimetry

The interactions between inorganic cations and κ-carrageenan in aqueous solutions have been studied using microcalorimetry. The enthalpy of dilution and the enthalpy of mixing of κ-carrageenan in the coil conformation with an alkaline metal chloride have been investigated and are in agreement with theoretical predictions. The formation of helical dimer in a K⁺ salt form in the absence of a gel formation is confirmed.     

On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions

Poly-N-isopropylacrylamide (PNIPAM) is a chemical isomer of poly-leucine, having the polar peptide group in the side-chain rather than in the backbone. It has been demonstrated experimentally that PNIPAM dissolved in aqueous solution undergoes a collapse transition from coil to globule on increasing temperature above the θ-point. By a careful reviewing of existing experimental data, we emphasize that such coil to globule collapse has to be considered an intramolecular first-order transition, analogous to the cold renaturation of small globular proteins. The main theoretical approaches to the coil to globule collapse in homopolymers are discussed briefly, and a critical comparison between the existing models is performed. We point out that, as a general result, the coil to globule collapse is expected to be a first-order transition for rigid and semi-rigid macromolecules. Finally, taking advantage of the analogy between the coil to globule collapse of PNIPAM and the cold renaturation of small globular proteins, we try to clarify some important and intriguing aspects of protein thermodynamics. This leads to the conclusion that the amphiphilic nature of polypeptide chain plays the fundamental role for the existence of two temperature-induced conformational transitions.