Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Heat denaturation of human high density lipoproteins and chylomicrons

1. The objective of this investigation was to determine whether structural differences between apolipoproteins could be detected by heat denaturation. 2. The apoproteins of human serum high density lipoprotein (HDL2, d = 1.070-1.125 and HDL3, d = 1.125-1.21 g/ml), their major polypeptide constituents (R-Thr and R-Gln), and apochylomicrons were investigated. 3. Heat denaturation was found to be reversible in the temperature range from 20 to 80 degrees. 4. The thermodynamic parameters of heat denaturation delta F, delta H, delta S and delta Cp were calculated on the basis of a single transition from the "native" to "denatured" state for apo-HDL2, apochylomicrons, R-Thr and R-Gln; for apo-HDL3 these parameters were calculated on the basis of two transitions. 5. The thermodynamic parameters, with the exception of delta F, which describe heat denaturation of high density apolipoprotein, of high density apolipoprotein polypeptides and of apochylomicrons were found to be similar on a molar basis and to have approximately the same values as the thermodynamic parameters which describe heat denaturation of non-lipid binding proteins; on a weight basis differences were apparent between the apolipoproteins and the polypeptides or non-lipid binding proteins.     

The thermodynamics of protein stability. Cold destabilization as a general phenomenon

A theoretical analysis of the temperature/stability profiles of proteins shows that, where a two-state model represents the denaturation, and where the free energy of denaturation delta G(T) shows a strong temperature dependence, then the protein becomes subject to both high- and low-temperature destabilization. In the simplest case delta G(T) is parabolic, therefore the high temperature TH, where delta (G(TH) = 0, is complemented by a low temperature TL, where delta G(TL) = 0. It is generally stated that the partial molal heat capacity change delta C accompanying the heat denaturation is positive and independent of the temperature. This implies that heating the protein through TL results in a negative delta C which seems physically unsatisfactory. The constant delta C model is explored and a physically more realistic model is advanced which allows for a temperature-dependent delta C which changes sign at some temperature within the range of stability of the native protein; delta G(T) then has the form of a skewed parabola. Experimental heat capacity data for native lysozyme and for a flexible polymer lend support to this model. The molecular basis of cold inactivation of proteins is discussed in the light of the thermodynamic analysis.     

Stability of recombinant Lys25-ribonuclease T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

Direct Correlation between Molecular Dynamics and Enzymatic Stability: A Comparative Neutron Scattering Study of Native Human Butyrylcholinesterase and its “Aged” Soman Conjugate

An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its “aged” soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.     

Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state.

We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.     

A Monte Carlo simulation study of protein-induced heat capacity changes and lipid-induced protein clustering.

Monte Carlo simulations were used to describe the interaction of peripheral and integral proteins with lipids in terms of heat capacity profiles and protein distribution. The simulations were based on a two-state model for the lipid, representing the lipid state as being either gel or fluid. The interaction between neighboring lipids has been taken into account through an unlike nearest neighbor free energy term delta omega, which is a measure of the cooperativity of the lipid transition. Lipid/protein interaction was considered using the experimental observation that the transition midpoints of lipid membranes are shifted upon protein binding, a thermodynamic consequence of different binding constants of protein with fluid or gel lipids. The difference of the binding free energies was used as an additional parameter to describe lipid-protein interaction. The heat capacity profiles of lipid/protein complexes could be well described for both peripheral and integral proteins. Binding of proteins results in a shift and an asymmetric broadening of the melting profile. The model results in a coexistence of gel and fluid lipid domains in the proximity of the thermotropic transition. As a consequence, bound peripheral proteins aggregate in the temperature range of the lipid transition. Integral proteins induce calorimetric melting curves that are qualitatively different from that of peripheral proteins and aggregate in either gel or liquid crystalline lipid phase. The results presented here are in good agreement with calorimetric experiments on lipid-protein complexes and have implementations for the functional control of proteins.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.     

Heat of denaturation of lysozyme

The enthalpy of denaturation of lysozyme was determined by measuring the heat, capacity of an aqueous solution of this protein in the vicinity of the transition temperature, 46 °C at pH 1. Within experimental error the calorimetric, heat (56 ± 8 kcal/mole) was found to agree with the van't Hoff transition enthalpy (63 ± 6 kcal/mole) determined from optical rotation measurements as a function of temperature. This indicates that denaturation of this protein can be interpreted in terms of a two-state model. Successive measurements of the same sample showed, from several lines of evidence, that the transition was about 80% reversible for the particular environmental conditions and thermal history involved in the study.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly(ethylene glycol)-induced fusion and destabilization of human plasma high-density lipoproteins

High-density lipoproteins (HDL) are macromolecular complexes of specific proteins and lipids that mediate the removal of cholesterol from peripheral tissues. Chemical unfolding revealed that HDL fusion and rupture are the two main kinetic steps in HDL denaturation. Here we test the hypothesis that lipid fusogens such as poly(ethylene glycol) (PEG) may promote lipoprotein fusion and rupture and thereby destabilize HDL. We analyze thermal disruption of spherical HDL in 0-15% PEG-8000 by calorimetric, spectroscopic, electron microscopic, and light scattering techniques. We demonstrate that the two irreversible high-temperature endothermic HDL transitions involve particle enlargement and show a heating rate dependence characteristic of kinetically controlled reactions with high activation energy. The first calorimetric transition reflects HDL fusion and dissociation of lipid-poor apolipoprotein A-1 (apoA-1), and the second transition reflects HDL rupture and release of the apolar lipid core. Neither transition involves substantial protein unfolding; thus, the transition heat originates from lipid and/or protein dissociation and repacking. At room temperature, PEG-8000 induces HDL fusion that is distinct from the heat-, denaturant-, or enzyme-induced fusion since it leads to formation of larger particles and does not involve apoA-1 dissociation. Increasing the PEG concentration in solution from 0 to 15% leads to low-temperature shifts by approximately -18 degrees C in the two calorimetric HDL transitions without altering their nature. Thus, consistent with our hypothesis, PEG-8000 induces fusion and reduces the thermal stability of HDL. Our results suggest that PEG is useful for the analysis of the molecular events involved in metabolic HDL remodeling and fusion.     

A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin

Bovine alpha-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, alpha-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine alpha-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca2+ for alpha-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for alpha-lactalbumin has been determined to be 2.9 x 10(8) M-1.     

Thermodynamics of phospholipid-sucrose interactions.

The effect of 0-1.0 M sucrose on the phase-transition properties of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (1,2-DPPC) was examined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. Increasing the concentration of sucrose caused a small, but experimentally significant, increase in the temperature (Tm) of maximal excess apparent specific heat (Cmax) and in delta T 1/2 (the transition width at 1/2 Cmax), a reduction in Cmax, and a small decrease (approximately 8-10% at 1.0 M sucrose compared with 0 M sucrose) in the calorimetric enthalpy (delta Hcal) of the gel-to-liquid crystalline transition. The calorimetric parameters of the pretransition of 1,2-DPPC were not significantly affected by sucrose in the concentration range examined, except there was a 1.0 degree C increase in the temperature (Tp) of maximal excess apparent specific heat in the presence of 1.0 M sucrose. The results are discussed in terms of the possible molecular mechanisms that could have caused the observed changes and are contrasted with the results obtained by C. -H. Chen et al. (1981, Biophys. J., 36:359-367).     

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry

Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.     

Stability parameters for one-step mechanism of irreversible protein denaturation: a method based on nonlinear regression of calorimetric peaks with nonzero deltaCp

Thermal transitions of many proteins have been found to be calorimetrically irreversible and scan-rate dependent. Calorimetric determinations of stability parameters of proteins which unfold irreversibly according to a first-order kinetic scheme have been reported. These methods require the approximation that the increase in heat capacity upon denaturation deltaCp is zero. A method to obtain thermodynamic parameters and activation energy for the two-state irreversible process N --> D from nonlinear fitting to calorimetric traces is proposed here. It is based on a molar excess heat capacity function which considers irreversibility and a nonzero constant deltaCp. This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (Tm), (2) calorimetric enthalpy at Tm (deltaHm), (3) deltaCp, and (4) activation energy (E). The thermal irreversible denaturation of subtilisin BPN' from Bacillus amyloliquefaciens was studied by differential scanning calorimetry at pH 7.5 to test our model. Transitions were found to be strongly scanning-rate dependent with a mean deltaCp value of 5.7 kcal K(-1)mol(-1), in agreement with values estimated by accessible surface area and significantly higher than a previously reported value.     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.