Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.

In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance.     

Conformational stability of spectrin and fodrin

The conformational stability of erythrocyte spectrin and brain spectrin-like protein (fodrin) has been studied by circular dichroism. In agreement with previous reports the circular dichroism spectra of both proteins in the peptide region were almost identical. The essential differences, on the other hand, were found in the near u.v. range, most probably due to differences in the conformation of intrachain disulphide bonds. Heat denaturation curves, relating to the level of secondary structure (ellipticity at 221 nm) showed that fodrin is more stable than spectrin: curves of reversible as well as irreversible denaturation are shifted to higher temperatures and also the amount of alpha-helices in the denatured state is higher. Spectrin conformation was found to be very sensitive to the presence of water-soluble organic solvents; the denaturation curves exhibit maxima and minima not typical of protein isothermic denaturation. The observed low conformational stability of spectrin is discussed in the context of its molecular environment and function in the red cell membrane.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Thermal properties of young red blood cells are indicative of an age-dependent regulation of membrane-skeleton interaction

The effects of red blood cell (RBC) age on membrane thermal properties have been investigated by using a 16-nitroxide stearic acid spin probe. We detected in unfractionated and most dense cells (2% fraction of circulating cells) a thermal transition at 40 degrees C that in young cells (1% fraction) was lowered at 33-35 degrees C. Spectrin seems to be directly involved in the transition detected in both young and unfractionated cells, as showed by the disappearance of the breaks after low salt extraction of spectrin. A further indication for a role of spectrin in this transition comes from its characteristic thermal unfolding above 40 degrees C. However, young cells did not show changes either in the thermal unfolding of spectrin or in the distribution of spectrin dimer, tetramer, and high oligomeric forms. These data rule out that spectrin of young RBC is modified in its thermal properties and indicate that young cells may have a different spectrin-membrane interaction. Treatment of unfractionated ghosts with an antibody specific for a fragment of the 10K domain of protein 4.1, which is fully competent for the spectrin-actin binding, produced an evident lowering of the transition temperature. The same antibody did not affect the thermal transition of young ghosts. Our results suggest that spectrin-membrane interactions may be regulated during RBC lifespan.     

Extremely high thermal stability of streptavidin and avidin upon biotin binding

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.     

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

The nuclear matrix is a thermolabile cellular structure

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.     

Differential scanning calorimetric investigation of pea chloroplast thylakoids and thylakoid fractions.

High sensitivity differential scanning calorimetry (DSC) was employed to study the thermal denaturation of components of pea chloroplast thylakoid membranes. In contrast to previous reports utilizing spinach thylakoids, several transitions are reversible, and deconvolution of the calorimetric curves indicates nine transitions in both first and second heating scans, but overlapping transitions obscure at least three transitions in the first heating scans of control thylakoids. Glutaraldehyde fixation increases the denaturation temperature of several transitions which is consistent with a reported increase in thermal stability of thylakoid function due to fixation. Acidic pH treatment has little effect on the DSC curves, although it has been reported to have a significant effect on membrane structure. Separation of grana from stroma thylakoids indicates that components responsible for transitions centered at approximately 56, 73, 77, and 91 degrees C are predominantly or exclusively associated with grana thylakoids, whereas components responsible for transitions centered at approximately 63 and 81 degrees C are predominantly associated with stroma thylakoids. A broad transition centered at 66 degrees C is associated with grana thylakoids, whereas a sharp transition at the same temperature is due to a component associated with stroma thylakoids. Evidence obtained by washing treatments suggests the latter transition originates from the denaturation of the thylakoid ATPase (CF1). Analysis of the calorimetric enthalpy values indicates most components of the grana thylakoids denature irreversibly at high temperature, whereas components associated with the stroma thylakoids have a considerable degree of thermal reversibility.     

Temperature- and denaturant-induced unfolding of two thermophilic esterases.

We studied the temperature- and denaturant-induced denaturation of two thermophilic esterases, AFEST from Archeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, by means of circular dichroism measurements. Both enzymes showed a very high denaturation temperature: 99 degrees C for AFEST and 91 degrees C for EST2. They also showed a remarkable resistance against urea; at half-completion of the transition the urea concentration was 7.1 M for AFEST and 5.9 M for EST2. On the contrary, both enzymes showed a weak resistance against GuHCl; at half-completion of the transition the GuHCl concentration was 2.0 M for AFEST and 1.9 M for EST2. The thermodynamic parameters characterizing urea- and GuHCl-induced denaturation of the studied enzymes have been obtained by both the linear extrapolation model and the denaturant binding model. The dependence of the thermal stability on NaCl concentration for both esterases has also been determined. A careful analysis of the data, coupled with available structural information, has allowed the proposal of a reliable interpretation.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.     

Changes in passive electric parameters of human erythrocyte membrane during hyperthermia: role of spectrin phosphorylation.

In prefixed by 1 mmol/l OsO4 human erythrocytes, the discocyte shape was preserved upon heating to temperatures which include the denaturation temperature of the main peripheral protein spectrin. Nevertheless, the suspension of fixed cells displayed threshold decrease in its capacitance and resistance at the temperature range where spectrin denaturates. The same changes were established using intact cells and their resealed ghosts. For packed cells (ghosts), the capacitance and resistance decreased about 17% (31%) and 30% (19%). These data indicate a decrease in the beta dispersion of erythrocyte membrane associated, according to a previous study (Ivanov 1997), with the heat denaturation of spectrin at 49.5 degrees C. The amplitude of the 49.5 degrees C decrease in beta dispersion was reversibly reduced in intact erythrocytes and white ghosts following reversible decrease in the phosphorylation of their membrane proteins. It was fully eliminated in ghosts following their resealing with alkaline phosphatase (0.1 mg/ml) which dephosphorylated membrane proteins. These findings are discussed in relation to similar changes found in normal and tumour tissues and cells during hyperthermia.     

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712). The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.     

A differential scanning calorimetric study of brain clathrin

The thermal denaturation of clathrin-coated vesicles isolated from bovine brain tissue has been studied by differential scanning calorimetry and has been compared to basket structures reformed from isolated triskelion trimers of clathrin and to isolated triskelions. The coated vesicles and reformed baskets displayed similar, yet distinct, thermal behavior. Calorimetric data of the coated vesicles exhibited a single denaturation transition peak at 55.9 +/- 0.1 degrees C, skewed to low temperatures whereas the thermograms for the reformed baskets exhibited a broad transition peak at 53.1 +/- 0.1 degrees C and a peak at 56.3 +/- 0.1 degrees C. Neither transition was reversible. The specific transition enthalpy was 11.5 +/- 1.0 J g-1 for the coated vesicles and the total transition enthalpy was 9.1 +/- 0.3 J g-1 for the reformed baskets. In contrast, isolated triskelions showed no thermal transition between 15 and 90 degrees C. Although the coated vesicles and the reformed baskets have similar stability reflecting their similar structures, the coated vesicles appear to be marginally more stable than the reformed baskets. The complexity of the transition profiles and their lack of symmetry suggest the existence of several, somewhat independent, domains unique to the cage-like structure of the coated vesicles and reformed baskets.     

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.