Thermal stability of proteins in intermolecular complexes.

A general phenomenological model is proposed for the estimation of the influence of the formation of complexes with ligands on thermal stability of proteins. In this model the reversible processes of unfolding-refolding and of association-dissociation of protein-ligand complexes and of the irreversible chemical degradation of the unfolded protein were analyzed jointly. By using certain approximations, the analytical expressions for both the thermodynamic and kinetic stabilization are obtained. Two thermodynamic and four kinetic regimes of stabilization and destabilization can exist in such system. Each thermodynamic regime appears to be compatible with three different kinetic regimes. The effect of the formation of complexes on thermodynamic and kinetic stability of the protein is determined by the degrees of binding of the ligand to the folded and unfolded protein species and by the rates of irreversible degradation of free protein and protein in complex.     

Thermal stability of hepatitis B surface antigen S proteins.

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.     

Thermal stability of proteins analyzed by temperature scanning pH-stat titration

Based on the fact that pH changes occur during the thermal unfolding of a protein, a pH-stat titrimetric procedure is described for the analysis of thermal stability. In all cases the agreement with other stability measurements was good, including a correlation with activity loss in enzymes. A model for the titration curves, assuming first-order denaturation kinetics, linear temperature increase, and validity of the Arrhenius equation, has been proposed and analyzed. Thus, thermodynamic constants can be calculated from tritration curves, or transition temperatures estimated if the Arrhenius constants are known. The equipment consists of a pH-stat, a programmable heating unit, and a temperature measuring/recording system. Analysis can be done quickly and on partially purified solutions, provided the buffer capacity is low, using about 20 mg protein/10 ml sample. The effects of pH, Ca²⁺ ions, substrate, chemical modification, etc., on thermal stability are conveniently analyzed up to about 90°C.     

Thermal stability of proteins does not correlate with the energy of intramolecular interactions

Small monomeric proteins from mesophilic and thermophilic organisms were studied. They have close structural and physical and chemical properties but vary in thermal stability. A thermodynamic analysis of heat unfolding was made and integral enthalpy of unfolding (DeltaH(unf)), heat capacity of hydration (DeltaC(p)(hyd)) and enthalpy of hydration (DeltaH(hyd)) and of the buried surface area (DeltaASA) of nonpolar and polar groups as well as the enthalpy of disruption of intramolecular interaction (DeltaH(int) in gas phase) at 298 K were determined. The absence of correlation between protein thermostability and energetic components suggests that regulatory mechanism of protein thermal stabilization has entropic nature.     

Thermal stability of proteins in the presence of poly(ethylene glycols)

Thermal unfolding of ribonuclease, lysozyme, chymotrypsinogen, and beta-lactoglobulin was studied in the absence or presence of poly(ethylene glycols). The unfolding curves were fitted to a two-state model by a nonlinear least-squares program to obtain values of delta H, delta S, and the melting temperature Tm. A decrease in thermal transition temperature was observed in the presence of poly(ethylene glycol) for all of the protein systems studied. The magnitude of such a decrease depends on the particular protein and the molecular size of poly(ethylene glycol) employed. A linear relation can be established between the magnitude of the decrease in transition temperature and the average hydrophobicity of these proteins; namely, the largest observable decrease is associated with the protein of the highest hydrophobicity. Further analysis of the thermal unfolding data reveals that poly(ethylene glycols) significantly effect the relation between delta H degrees of unfolding and temperature for all the proteins studied. For beta-lactoglobulin, a plot of delta H versus Tm indicates a change in slope from a negative to a positive value, thus implying a change in delta Cp in thermal unfolding caused by the presence of poly(ethylene glycols). Results from solvent-protein interaction studies indicate that at high temperature poly(ethylene glycol) 1000 preferentially interacts with the denatured state of protein but is excluded from the native state at low temperature. These observations are consistent with the fact that poly(ethylene glycols) are hydrophobic in nature and will interact favorably with the hydrophobic side chains exposed upon unfolding; thus, it leads to a lowering of thermal transition temperature.     

Thermal stability: a means to assure tertiary structure in therapeutic proteins.

To be both safe and effective, a therapeutic product must have the correct chemical structure and be free of harmful contaminants. Structure in protein therapeutic products, however, implies not only the correct sequence of amino acids (primary structure) but also the proper folding of that amino acid chain in three-dimensional space (tertiary structure). This work is part of a general strategy to develop a battery of physico-chemical methods that could give assurances of structure (and hence function) in formulated therapeutic proteins in the absence of in vivo data. It focuses on recombinant human growth hormone (rhGH), a well-characterized therapeutic protein, and examines the utility of thermodynamic parameters in assessing its tertiary structure. Resistance of solutions of formulated rhGH to thermal denaturation was followed using Fourier Transform Infrared Spectroscopy (FTIR) by observing decreases in total helicity and increases in intermolecular beta-sheet formation. Under conditions known to induce changes in the intra-molecular ionic and H-bonding patterns stabilizing the tertiary structure but not affecting the protein's secondary structure or global fold, we have observed upwards of a 12 degrees C shift in the melting temperature of the protein. Furthermore, our results indicated that the T(m) of unfolding of rhGH was sensitive to much more subtle changes in the protein structure. Thus, resistance to thermal denaturation may well be a useful means to measure structure in formulations of well-characterized therapeutic proteins.     

Thermal hysteresis proteins

Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.     

Thermal transitions of proteins

A new method for monitoring the thermal transitions of proteins is described. An unbuffered solution of native protein shows a significant and fairly abrupt change in pH as the protein becomes heat denatured. Suitable plots permit the “melting point” of the protein to be assigned. Twenty proteins have been studied with emphasis on egg albumin. The transition temperature of egg albumin is independent of protein concentration, of pH in the neutral zone, is moderately dependent on the rate of heating, increases with increasing NaCl concentration, varies inversely with the guanidine hydrochloride concentration. There is more than a 35 °C spread in the melting temperatures of the various proteins and no apparent relation exists between the melting temperature of a protein and structural features of the protein.     

Thermal stability of fish myosin

• 1.1. Thermal stability of fish myosin has been studied by using differential scanning calorimetry (DSC) and circular dichroism (CD).
• 2.2. The temperature range of the sharp decrease in α-helical content agreed very closely with that of the endothermic peaks.
• 3.3. There was a high correlation between the enthalpy of denaturation (ΔH) and the decreasing quantity in α-helicity (Δh).
• 4.4. The structure of fish myosins was much more unstable than that of rabbit.
• 5.5. The instability of fish myosins was reflected in its rod moiety.

     

Thermal stability of DNA.

Tij and Delta Hij for stacking of pair i upon j in DNA have been obtained over the range 0.034-0.114 M Na+from high-resolution melting curves of well-behaved synthetic tandemly repeating inserts in recombinant pN/MCS plasmids. Results are consistent with neighbor-pair thermodynamic additivity, where the stability constant, sij , for different domains of length N depend quantitatively on the product of stability constants for each individual pair in domains, sijN . Unit transition enthalpies with average errors less than +/-5%, were determined by analysis of two-state equilibria associated with the melting of internal domains and verified from variations of Tij with [Na+]. Enthalpies increase with Tij , in close agreement with the empirical function: Delta Hij = 52.78@ Tij - 9489, and in parallel with a smaller increase in Delta Sij . Delta Hij and Delta Sij are in good agreement with the results of an extensive compilation of published Delta Hcal and Delta Scal for synthetic and natural DNAs. Neighbor-pair additivity was also observed for (dA@dT)-tracts at melting temperatures; no evidence could be detected of the familiar and unusual structural features that characterize tracts at lower temperatures. The energetic effects of loops were determined from the melting behavior of repeating inserts installed between (G+C)-rich barrier domains in the pN/MCS plasmids. A unique set of values for the cooperativity, loop exponent and stiffness parameters were found applicable to internal domains of all sizes and sequences. Statistical mechanical curves calculated with values of Tij([Na+]) , Delta Hij and these loop parameters are in good agreement with observation.     

Thermal stabilities of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the dependence on temperature. Folding is assumed to be driven by solvophobic interactions and opposed by the conformational entropy. The temperature dependence of the solvophobic interaction is taken from the transfer experiments on amino acids by Tanford and Nozaki and on model solutes by Gill and Wads?. One long-standing puzzle has been why proteins denature upon heating, since the solvophobic force to fold strengthens with increasing temperature. This is resolved by the theory, which predicts two first-order phase transitions. "Cold denaturation" is driven principally by the weakening of the solvophobic interaction, but normal denaturation is driven principally by the gain of conformational entropy of the chain. Predictions of the thermodynamic state functions are in reasonable agreement with the calorimetric experiments of Privalov and Khechinashvili. Comparison of the theory with experiments suggests that there may be an additional enthalpic driving force toward folding which is not due to the solvophobic interactions.     

Kinetic stability of membrane proteins

Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.     

Structural stability of halophilic proteins

An examination of halobacterial amino acids exchanges as they appear in the known Spirulina platensis [2Fe-2S] ferredoxin tertiary structure indicated that most of the additional acidic residues of the halophiles occurred on the external surface of the alga structure; however, further negative changes were not placed in the ferredoxin active site region. A statistical investigation of the amino acid compositions of seven halophile and nonhalophile protein counterparts indicated that the bulkiness of amino acids used by halophiles is considerably reduced and that the overall hydrophobicity of halophilic and non halophilic molecules was essentially the same. It is suggested that the principal mode of structural stabilization for halophilic proteins is effective competition with the cytoplasmic salt for water through utilization of many external carboxyl groups of glutamic and aspartic acids. A reduction is residue bulkiness would prevent inactivation in the presence of the high molarity, antichaotropic KCl. Halophilic functionality is preserved through avoidance of additional negative charge at the active site surface.     

Conformational stability of globular proteins

The conformational stability of ribonuclease T1 has been measured as a function of the variables of most interest to biochemists: temperature, pH, salt concentration, disulfide-bond content and amino acid sequence. The results provide insight into the forces that stabilize globular proteins.     

Thermal stability and conformation of DNA and proteins under the confined condition in the matrix of hydrogels

Spatially confined environments are seen in biological systems and in the fields of biotechnology and nanotechnology. The confinement restricts the conformational space of polymeric molecules and increasing the degree of molecular crowding. Here, we developed preparation methods for agarose and polyacrylamide gels applicable to UV spectroscopy that can evaluate the confinement effects on DNA and protein structures. Measurements of UV absorbance and CD spectra showed no significant effect of the confinement in the porous media of agarose gels on the base-pair stability of DNA polynucleotides [poly(dA)/poly(dT)] and oligonucleotides (hairpin, duplex, and triplex structures). On the other hand, a highly confined environment created by polyacrylamide gels at high concentrations increased the stability of polynucleotides while leaving that of oligonucleotides unaffected. The changes in the base-pair stability of the polynucleotides were accompanied by the perturbation of the helical conformation. The polyacrylamide gels prepared in this study were also used for the studies on proteins (lysozyme, bovine serum albumin, and myoglobin). The effects on the proteins were different from the effects on DNA structures, suggesting different nature of interactions within the gel. The experimental methods and results are useful to understand the physical properties of nucleic acids and proteins under confined conditions.     

Thermal stability and phytoplankton distribution

Thermal stability is the potential of water columns to mix, and has long been known to fundamentally influence the vertical and temporal distribution of phytoplankton. Essentially this is because it indirectly controls the amount of light available to phytoplankton.Under stable conditions of strong temperature gradients algal species (or assemblages of associated species) distribute vertically because they have sufficient time to exploit the attenuated light field at their preferred depths. This encourages a species diversity which, in the Southern Hemisphere, is especially exemplified by the extremely stable conditions under the permanent ice of Antarctic lakes.In other lakes stability commonly encourages growth of blue-green algae by permitting their positive buoyancy to place them in optimal light conditions, and by inhibiting the resuspension of competing non-buoyant species. Analogous patterns occur with motile species (Dinophyceae, Cryptophyceae, etc.), and with non-motile forms whose physiological adaptations allow growth to large sub-surface peaks at preferred depths. These sub-surface maxima can be upwelled to the water surface, in a manner controlled by thermal stability and vertical shear, and horizontally transported to give large variations in horizontal distribution.At all latitudes diel stability cycles in surface waters can affect physiological properties important for growth, and in some circumstances can dominate the phytoplankton dynamics and distribution.Such short-term stability events merge with longer-term (e.g. annual) events with no conceptual distinction. A modern way to integrate this continuity is by scaling using spectral analysis of cyclicity. This allows biological variables (algal biomass, numbers, production) to be stochastically related to indices of stability (e.g. Brunt-Väisälä frequency).     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo