thermodynamics of azurin folding

14864 J. Phys. Chem. 1995, 99, 14864-14870

Thermodynamics of the Thermal Unfolding of Azurin

Carmelo La Rosa,* Danilo Milardi, and Domenico Grasso Dipartimento di Scienze Chimiche, Universitb di Catania, V.le A. Doria, 6-95125 Catania, Italy

Rita Guzzi and Luigi Sportelli Dipartimento di Fisica, Universitb della Calabria, 87036 Arcavacata di Rende, Cosenza, Italy

Received: January 4, 1995; In Final Form: July 19, 1995@

The thermal denaturation of azurin from Pseudomonas aeruginosa was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR), and optical density (OD) experiments, with the aim of determining its thermodynamic stability and the thermally induced conformational changes of its active site. DSC experiments have shown an irreversible and complex unfolding path. In order to characterize the kinetically controlled step, DSC measurements were carried out at different scan rates. An extrapolation of the experimental heat capacity data to infinite scan rate allowed all the kinetic and thermodynamic parameters related to the process to be obtained. All these parameters extracted from the calorimetric data were verified by means of a curve-fitting program using an equation containing all information necessary to fully describe the unfolding process in details. Thermal denaturation, followed up to 82 "C by ESR and OD measurements, allowed us to study the structural variations of the copper environment at different temperatures. The AHLI thermodynamic, together with the value of AC, calculated according to an approach taking into account the common features of protein unfolding and dissolution of hydrophobic compounds, was used to evaluate the thermodynamic stability (AG) for the reversible process over the entire temperature range of denaturation. The high value of the maximum stability thus calculated was explained by the stabilizing effect of copper.

Introduction Azurin is a small blue copper protein' that acts as an electron

transfer agent in the redox systems of certain bacteria. Together with plastocyanin, azurin is the best characterized protein of this class. Recently, studies have been concentrated on the distinct spectroscopic properties of azurins and plastocyanins.2 These proteins exhibit a very intense absorption band in the visible region of the electromagnetic spectrum with 595 < A,,,= < 630 nm and E FZ 500 M-' cm-I, unusually high redox potentials (240-400 mV), and a characteristic narrow hyperfine splitting in the ESR ~ p e c t r a . ~ . ~ X-ray diffraction studies on crystals of small blue copper proteins have led to the determi- nation of the high-resolution three-dimensional structures of various a z u r i n ~ . ~ . ~ The structure of azurin from Pseudomonas aeruginosa (P. aeruginosa) has also been p~b l i shed .~ .~

The thermodynamic stability of the tertiary structure of this enzyme has been the focal point of intense research over the past few years. In particular, previous works7 showed a structure which is highly resistant to thermal unfolding: temperatures exceeding 70 "C are necessary for irreversible unfolding. This unusual thermal resistance has been generally ascribed to a number of factors, including disulfide bridges, intramolecular hydrogen bonds, hydrophobic effects, and sta- bilization by Cu2+ bindi~~g.~-'O

The whole of these effects can be detected by micro- differential scanning calorimetry (micro-DSC) measurements, when samples of enzyme in a suitable environment are temperature scanned.'

The thermodynamic analysis of the calorimetric profiles is not directly possible because the thermal denaturation of azurin is irreversible. This irreversibility makes both the application of statistical-mechanical deconvolution methods' and classical thermodynamic analysis impossible.

* To whom correspondence should be addressed. @Abstract published in Advance ACS Abstracts, September 15, 1995.

0022-365419512099- 14864$09.0010

The remarkable asymmetry of the DSC curves at the end of the transition is a further complication in the analysis of the unfolding process. It is ascribable both to exothermic phenomena and to the occurrence of kinetic factors.I3

So far, the thermodynamic analysis of the DSC curves of proteins showing calorimetric irreversibility has in general been considered impossible. However, in some cases, under specific conditions, even in the presence of calorimetric irreversibility, a thermodynamic analysis has been carried outsi4

In this paper we separate the effects associated with the reversible step of the denaturation process from those associated to the irreversible step, calculating the characteristic parameters in each case. These parameters were subsequently used to simulate the complete C,,,, profiles by means of a best fit program using the SIMPLEX minimization algorithm based on an equation previously developed on the basis of the hypoth- esized pathway of unfolding. The model used describes the denaturation path as the sum of two effects: an endothermic effect comprising the energy involved in the destruction of the protein's three-dimensional structure and an exothermic effect ascribable to the aggregation of the polypeptidic chain network. The first of these effects is, as will be explained, reversible, and in this case a thermodynamic analysis is possible. In contrast the second effect, which is assumed to be slow with respect to the reversible unfolding equilibrium, is irreversible and kinetically controlled.

Optical density (OD) measurements, carried out in the same experimental conditions as the calorimetric ones, allowed us to calculate all kinetic variables associated with the transition involving the copper environment. The geometry of copper environment in the different states was investigated by means of electron spin resonance (ESR) experiments that made the correct interpretation of all the conformational changes reliable.

0 1995 American Chemical Society


40

I

I

-vv

50 55 60 65 70 75 80 85 90 95 100

Temperature ('C)

Figure 1. DSC thermogram of azurin after subtraction of the buffer- buffer base line: Protein concentration, 1.25 mg/mL, pH = 7.03; ionic strength, 0.1 M in NaC1; scan rate, 0.5 "Clmin. The base line (dashed curve) was obtained as described in the text.

Experimental Section

Azurin from P. aemginosa (MW 14 600) was obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. The protein concentration was determined by the procedure of Lowry et al.I5 Potassium phosphate (analytical grade) was obtained from FLUKA Chemie AG (Buchs, Swit- zerland).

DSC scans were carried out with a SETARAM (Lyon, France) micro-differential scanning calorimeter (micro-DSC) with stainless steel 1-mL sample cells, interfaced with a BULL 200 Micra1 computer. The sampling rate was 1 pointh in all measuring ranges. The enzyme (1.25 mg/mL) was dissolved in 10 mM phosphate buffer at pH = 7.03. The ionic strength was adjusted at 0.1 M by sodium chloride. The same solution without the protein was used in the reference cell. Both the sample and reference were scanned from 30 to 100 "C with a precision of f0.08 "C at the scanning rates of 0.3,0.5,0.7, and 1 "Chin. The calorimetric scans were carried out under an extra nitrogen pressure of 1.5 bar.

In order to obtain the C, curves, buffer-buffer base lines were obtained at the same scanning rate and then subtracted from the sample c ~ r v e s . ~ ~ , ' ~ All the C, exc curves were obtained using a fourth-order polynomial fit as the base line, as reported in Figure 1. The average level of noise was about f0.4 pW, and the reproducibility at refilling was about 0.1 d/(K/mL).

The calibration in energy was obtained by giving a definite power supply, electrically generated by an ET2 SETARAM Joule calibrator within the sample cell.

OD measurements were carried out with a JASCO 7850 spectrophotometer equipped with a Haake thermostated bath Model D8 G (f0.02 "C). Quartz cuvettes with a 1-cm optical path were used throughout. The temperature of the samples was measured directly by a YSI precision thermistor dip in the reference cuvette. The experiments were started 3 min after samples were positioned in the thermostated sample holder at the initial temperature of 50 "C. The heating rates were 0.3, 0.7, and 1 "Chin.

The ESR measurements were carried out with a Bruker ER 200D-SRC X-band spectrometer equipped with the ESP 1600 data system. All the ESR spectra were recorded at 120 K. A cold nitrogen flow was used to reach this temperature which was controlled with an accuracy of f 0 . 3 K by means of the ER 41 11 VT temperature control system.

J. Phys. Chem., Vol. 99, No. 40, 1995 14865

TABLE 1: Scanning Rate Effect on Azurin Aqueous Solution Obtained at Constant Concentrationa

scanhate ("Clmin) Tmb ("C) AHc (kJ/mol) AHCalIAPH 0.3 81.0 276.7 5 20 0.20 0.5 83.3 409.6 f 22 0.25 0.7 84.4 478.6 f 29 0.39 1 .o 84.7 512.3 f 21 0.55 cad 86.3 624.6 5 73 1.00

a pH and ionic strength was 7.03 and 0.1 M in NaCI, respectively. The estimated uncertainty of these values is f0.5 "C. Enthalpies

expressed as mean f standard deviation. dValues obtained for extrapolation as reported in the text.

-- 72 76 80 84 88 92 96

Temperature ('C)

Figure 2. Effect of scanning rate on the excess heat capacity function of azurin: (a) 0.3, (b) 0.5, (c) 0.7, and (d) 1 "C/min. Protein concentration was the same (1.25 mg/mL) in all experiments.

Results and Discussion

In Figure 1 the calorimetric profile of azurin at the scan rate of 0.5 OC/min is reported. No calorimetric reversibility was observed; Le., a second run of a previously scanned sample did not show any endothermic peak. This means that the thermal unfolding of Azurin is, on the whole, irreversible and it cannot be analyzed in the light of classical thermodynamics. From the Van't Hoff ratiosi8 calculated at different scan rates, it can be easily deduced that the thermal transition is not of the type "all or none" (see Table 1). Since the transition is irreversible, it is necessary to establish if the reaction is under kinetic control and if there is a change of molecularity during the heating of the ~olution. '~ DSC scans at different scan rates allow us to determine if the process is under kinetic control, while a change of molecularity during heating is impossible in this case because azurin is a monomeric protein.

Scanning Rate Effect on DSC Thermograms. In Figure 2, DSC scans of azurin obtained at different scan rates (between 0.3 and 1 "Chin) in the 72-96 "C range are reported. Protein concentration was maintained at 1.25 mg/mL (7.81 x M), so that intermolecular interactions are not probable. From the scanning rate effect, it is possible to calculate the apparent activation energy (Eapp) of the unfolding process using the following e q u a t i ~ n : ' ~ . ~ ~

where v is the scan rate ("C/min) and T, the temperature of the maximum heat absorption. From the slope of the linear plot of ln(vlTm2) vs UT,,,, reported in Figure 3, we can calculate the Eapp of the process, which, in our case, is 356 kJ/mol. It can be noted that the exothermic peak, localized at the end of the

14866 J. Phys. Chem., Vol. 99, No. 40, 1995

600

La Rosa et al.

v = C D .....'. - .- .. . v-1 'c/mln -11 I- -----I

-11.5 t Y

-12.5-

-1 3

-11.5 -

-1 3.5 -I I I I 1

2.793 2.798 2.803 2.808 2.813 2.818 2.823

irr, * 103(Ko

Figure 3. Linear plot of ln(VlT,,,*) vs UT,,,. The slope of this line represents the apparent activation energy of the denaturation process.

transition, diminishes when the scan rate increases, while calorimetric enthalpy (AH) and the temperature T m increase with increasing scan rates (Table 1). In a previous work,21 we have shown that the scan rate influences differently the exothermic from the endothermic phenomena; even if both peaks become sharper and shift toward the low temperature side when v decreases, this effect is much greater for the exothermic peak than for the endothermic one.

Since the exothermic peak decreases with increasing scan rates, we can conclude that this exothermic contribution is time dependent. It is therefore reasonable to consider the reversible step separable from the irreversible one by means of the extrapolation of the calorimetric curves to infinite scanning rate^.'^**^

The Cpexc curve at infinite scanning rate was obtained by means of the following procedure: the cumulative enthalpy functions (AH) were calculated from the experimental calorimetric profiles obtained at different scan rates by using the equation

where TO is the temperature at which all molecules are in the initial state, and Cpexc is the specific excess heat calculated according to Privalov and Potekhin'* (see Figure 2).

In the denaturation range (AH) profiles depend on the scan rate (Figure 4), Le., at T = Ti, (AH)T=T~ is a function of the scan rate.

For a first order process, the relationship between (AH), (AH)rev, T, and v is given by the following equation:13

where AH is the calorimetric enthalpy calculated at the chosen scan rate, (AH),, represents the cumulative enthalpy function containing the information pertinent only to the species that are in thermodynamic equilibrium, and kapp is the apparent kinetic constant. Obviously, when v + 00, (AH) + (AH)rev exponen- tially.

In order to obtain the (AH)rev function over the entire denaturation range, the numerical values of (AH)v=O.~,T=T,,

temperature Ti, vs llv. In the exponential graphs shown in Figure 5, the intercept values with the y axis give the desired value of (AH)rev at a given temperature. For the sake of clarity we have reported the points obtained at T = 82 and 83 "C only.

(Wv=O.S,T=T,, (mu=0.7,T=T,, ( ~ F I ,T=T, are plotted, for a given

70 72 74 76 78 80 82 84 86 88 90 92 94 Temperature ('C)

Figure 4. Cumulative enthalpies obtained at different scan rates. The square points (m) represents the thermodynamic cumulative enthalpy calculated as described in the text over all the temperatures within the denaturation range.

250 I 200 c A

h - 150

4 loo

O r I I I I I I I 0 0.5 1 1.5 2 2.5 3

l /v (K1 min)

Figure 5. Examples of exponential plots of the cumulative enthalpies of denaturation (AH) vs l l v obtained at T = 82 and 83 "C.

However, the curve represented by square points (M) shown in Figure 4 is made by all the intercepts obtained in the above explained way, over the entire denaturation range. In other words, each single point (M) of the curve represents the (AH),, value obtained by the intercept with the y axis of the exponential fits of Figure 5 at any given temperature. Deriving the (AH)rev

profile with respect to temperature, we obtain the Cp exc profile at infinite scanning rate with a AH = 624.6 kJ/mol and a T m = 86.3 "C.

Mechanical-Statistical Analysis of the Time-Independent Cpexc Profile. In order to establish the path of the folding- unfolding transition, a mechanical-statistical analysis of the thermodynamic Cp exc profile, obtained from the first derivative of (AH),,, respect to temperature, was carried out using the classic deconvolution algorithm of Freire and B i l t ~ n e n . ' ~ ? ~ ~ From this analysis we obtained an unfolding path of the all or none type. This result was also conf i ied by the Van't Hoff analysis (see Table 1 and Figure 6).

From the above discussion we deduce that the unfolding path of azurin occurs in a first approximation in two steps: one reversible time independent, all or none type and one irreversible and under kinetic control.

Optical Density and Electron Spin Resonance Measure- ments. The thermally-induced modifications of the active site of azurin were studied by means of optical density measurements (1 = 625 nm), in the same conditions as those adopted for calorimetric measurements at scan rates of 0.3, 0.7, and 1 "C/


, , 8 - 1 I I

100, 1700

80 I d g 401

0

J. Phys. Chem., Vol. 99, No. 40, 1995 14867

2600 3100 3600 HI Gauss

1.0 n 0

0.8

"." 56 60 64 68 I2 16 80 84

TIT

Figure 7. Normalized ODs25 variation as a function of temperature, recorded at different scan rates.

min. The copper region is optically characterized by an intense absorbance in the visible region deriving from a "charge transfer" transition pnS(Cys) - d~z-~z (Cu2+). The energy of this transition depends both on the relative charge on atoms S and Cu and on the geometry of the c o m p l e ~ . ~ , ~ ~ , ~ ~

The OD denaturation curves can be separated into three different zones (Figure 7): in the low temperature region, up to about 67 "C, optical density does not change; this means that, up to 67 "C, the geometry of the copper-sulfur bond (responsible for the absorbance at 625 nm) does not change. From 67 to 76 "C, the OD curves show a linear reduction, with a slope that is independent from the adopted scan rate. Moreover, with the rescanning of the samples previously scanned up to 76 "C, an OD curve identical to the first cycle is obtained. This means that the transformation occurring in this temperature range is reversible. The zone beyond 76 "C is characterized by a differentiation of the OD curves, depending on the scan rate adopted. In this zone irreversible processes occur, caused by conformational deep changes in the copper environment. This behavior is common to other copper proteins.26

In order to characterize the azurin active site in the "linear- reduction region" of the OD denaturation curve, ESR measurements were carried out to show the modifications in the symmetry of the copper site. In Figure 8 the magnetic spectra for the states of azurin obtained after heating to 78 "C (curve c) and to 84 "C (curve d), registered at 120 K, are reported. Curve Sa represents the ESR spectrum of native azurin. From the comparison between the ESR signal relative to the native state and the state represented by curve c, we can note that in the latter a new peak at 2700 G is present together with a

Figure 8. ESR spectra of azurin recorded at 120 K in the (a) native state, (cj the intermediate state, and the (d) final state. Curve b represents the simulated ESR spectrum of azurin in the intermediate state.

TABLE 2: Magnetic Parameters Used To Simulate the ESR Spectra of Azurin (Az) in the Native (N) and Final (F) States

RII R.t Ail (GI A i (GI Az (Nj 2.271 2.052 55 15 A z P ) 2.284 2.045 160 15

sensitive increase in the value of 41 (from 55 to 60 G). No appreciable variations can be noted in the perpendicular region of the spectrum. It is our opinion that these variations are ascribable to two effects: (a) a small percentage of molecules that are not in the native state N; (b) an enfeeblement of the three-dimensional structure of the protein induced by temperature, causing the progressive transformation from state N to an intermediate state (enfeeblement of the coordination bonds between copper and its neighbors). In fact, the optical density curve maintains its profile unaltered compared to the native protein, while its intensity decreases by about 20% at 78 "C. Moreover, the ESR spectrum of this intermediate state was obtained by means of computer simulation (curve 8b) which considered the sum between curves 8a and 8d. The magnetic parameters used in the simulation are reported in Table 2.

To conclude, the ESR spectrum relative to the state represented by curve d is very similar to that usually obtained for a planar complex of the type C U ~ + - ~ N - ~ O . ~ ' S ~ ~ This means that heating the protein to 84 "C causes the substitution of the two sulfur atoms with oxygen without ligands.

The meaning of the optical analysis has to be considered with great attention because its wrong interpretation can induce to overestimate of the contribution of copper to the global stability of the structure. We note that optical analysis is based on a n charge transfer transition related to the overlap of the atomic orbitals of copper and sulfur. So the reduction of the optical absorbance does not necessarily mean a loss of copper from its environment. The only sure conclusion we can make is that the geometry of the copper environment undergoes a tethrae- dral-planar transition up to about 76 "C whose transition temperature depends on scan rate. This effect is not ac- companied by any significant thermal effect, as the absence of evident signals in the corresponding C,,,, curves shows.

The trend of OD curves at different scan rates can be summarized in the following scheme:

14868 J. Phys. Chem., Vol. 99, No. 40, 1995

N-N,-N,

La Rosa et al.

TABLE 3: Thermodynamic and Kinetic Parameters Obtained from the Curve Fitting Operations Carried Out at Different Scan Rates As Described in the Texl?

u AHu Tli2 AHag E P mb 8 where N represents the native protein, which maintains its structure unchanged up to 67 "C. In state NI, obtained after heating the solution up to 76 "C, the copper ion is bound with its ligands, but an enfeeblement of the copper-sulfur bond occurs as evidenced by the slow decrease of optical absorbance. As we have already shown, the transition N Q Nl is perfectly reversible. The subsequent step (that occurs beyond 76"C), represents, as we have yet explained, the change of coordination of copper and is an irreversible process.

By supposing that the transformation process of the active site is a first-order p r o c e ~ s ~ ~ , ~ ~ and using eq 1, we obtained a value of 451 kJ/mol for the apparent activation energy. The Eapp value was also obtained using the variation of optical density as a function of time at a fixed temperature. The fit of the experimental points expressed as follows

OD, = OD,,, + [OD,=, - OD,,,]e-kr (4)

made it possible to calculate the values of k relative to the reaction at the temperatures Ti of 80.0,79.0,78.0, and 77.0 "C. The values for k are as follows: kgo = 0.197, k79 = 0.121, k7g = 0.062, and k77 = 0.042 min-I. By means of k data the activation energy, Eapp, was calculated using the Arrhenius equation:

EaPP I n k = c - - RT,

From the corresponding plot of In k vs UTi, a value of Eapp = 441 kJ/mol was obtained. This value corresponds with that previously found studying the OD scan rate dependence, but it is different from the value found by calorimetry (Eapp = 356 kJ/mol).

The real cause of this difference has to be found in the fact that spectroscopic investigation is, by its very nature, limited to a well defined spatial region of the protein: the active site Cu2+-N2SS*, while calorimetric measurements involve the whole structure of the protein.

Simulation of the Calorimetric Profiles. All data collected therefore suggest a denaturation path of the following type for azurin:

N - U - F (4 where N is the native state, U is the unfolded state, and F is the final state. Of course, chemical equilibrium between N and U is assumed to be always established.

In order to determine the kinetic and thermodynamic parameters of the steps of this process, the equations developed by Sanchez-RuizI9 have been applied. The approach of Sanchez- Ruiz starts from the classical Lumry and Eyring models,30 considering the heat exchange associated with the final step U - F to be negligible. Milardi et a1.2' have recently developed the mathematical background for the analysis of the process of type a when the enthalpy of the process U - F (AHag) is not negligible. The experimental curves, obtained at different scan rates (Figure 2), were fitted with the following mathematical equation simulating this process:

0.3 633 85.9 -337 45 79.1 3.2 15.4 0.5 648 85.8 -369 85 86.8 2.4 11.2 0.7 648 85.8 -372 80 86.7 3.1 18.0 1 653 86.0 -327 30 80.9 2.9 15.8

a Enthalpy and activations energies values are given in W/mol, temperature values in "C, scan rates in "Clmin. The minimum increments in the minimization procedure are 1 W/mol for the enthalpies and 0.1 "C for the temperatures. The starting parameters are AH" = 624 kJ/mol (range &73W/mol) and T ~ D = 85.8 "C (range f 0 . 2 "C). The kinetic parameters are freely floating. b m (W K-' mol-]) is a measure of the accuracy of the fitting operation. It is defined as: m = iX,((C,')lheor - (C,')expl)/nl, where (Cpl)exp is the ith value of the experimental C, thermogram, (Cpi)lheor is the corresponding calculated value, and n is the total number of points of the scan. 6 is the standard deviation of the (C,i)theor - (Cpl)exp function calculated in the denaturation range.

where K is the thermodynamic equilibrium constant associated with the reversible step

and k is the kinetic constant of the irreversible step

(7)

AHu is the thermodynamic enthalpy variation associated with the N - U unfolding process, E is the activation'energy of the irreversible step, AHag is the enthalpy associated with the irreversible process, and T112 and r* are the temperatures at which the thermodynamic equilibrium constant K and the kinetic constant k approach unity, respectively. The other symbols have the usual significance. In eq 6 the unknown parameters are AHag, E, and r". We must emphasize that the activation energy of the irreversible step is different from the apparent activation energy (Eapp) obtained by means of eq 1. In fact, E and Eapp are bounded by the following relati~nship:'~

Eapp = E + AHu + AHag (9)

In this case AHu is a known parameter, but AHag, and E must be considered independent variables. These two quantities can be obtained only by fitting eq 6 with the experimental curve by means of the SIMPLEX algorithm. The fitting operation gives the results reported in Table 3.

These results give different kinetic values at different scan rates. This is ascribable to the fact that the final state, as it is obtained in an irreversible way, depends on the scan rate.

In Figure 9, the complete result of fitting the curve calculated by means of eq 6 with that obtained experimentally is reported. The scan rate is 0.5 "C/min.

The only way to justify the validity of the parameters extracted from the extrapolation procedure consists of calculating the relative populations of the unfolded state U over the entire denaturation range by using the equationI9

The relative populations of species N and U can be calculated by means of the following equations:

(1 1) X J X , = K, X, + Xu + X , = 1


200, 1

-50 75 80 05 90 95

Temperature ("2)

Figure 9. Curve fitting of the experimental C, exc profile: scan rate, 0.5 "C/min. Equation 6 reported in the text was used for the fitting program. The optimized parameters are AHTJ = 648 Id/mol, TI/Z = 359 K, r$ = 360 K, AHHag = -369 kJ/mol, and E = 85 Idlmol.

0.8 L o,6. v-0.3 W m l n V

N 0.8 -

0 8 -

v= 10 Wmin 0.4 -

0.2 -

'50 55 80 65 70 75 80 85 80 95 100 Temperature ( 'C )

Figure 10. Relative populations of the species N, U, and F, calculated at 0.3, 1, and 10 "Clmin.

Only in the case of a significant amount of species U existing during the thermally induced transition the extraction of thermodynamic informations from calorimetric data is reliable. In Figure 10 we have calculated the relative populations of the species N, U, and F, by using eqs 10 and 11 and the parameters reported in Table 3. We can note from Figure 10 that at 1 "C/ min, (our highest experimental scan rate) the maximum amount of species U is about 20%, a value considered sufficient to

J. Phys. Chem., Vol. 99, No. 40, 1995 14869

justifyI3 the validity of the operation. The effect of scan rate on the relative population of the species involved is, on the other hand, also confirmed by the lower panel of Figure 10 obtained at the hypothetical scan rate of 10 "C/min: the relative population of U increases at increasing scan rate.

Thermodynamic Analysis of the Unfolding Process. The calculation of the Gibbs free energy relative to the unfolding process in all the temperature ranges considered requires three parameters: MU, TII~, and AC,. Unfortunately, the value of AC, (Cp u - C, N) cannot be calculated experimentally, because the value of C, at the offset temperature is ascribable to the final (F) state and not to the unfolded (U) state. For this reason AC, was calculated by means of the Murphy and Gill3' model. In accordance with the considerations reported in that paper, the value of AC, can be evaluated on the basis of correlations with model compounds, and by consideration of the apolar and polar surface exposed to the solvent after the opening of the polypeptidic chain. It has also been shown that the apolar surface is proportional to the number of apolar hydrogen (AC,-CH-),~* while the polar surface is proportional to the number of peptidic bonds (Nres).33934 On the basis of all these considerations, Murphy and Gill proposed a set of equations for the calculation of AC,:

AC, = AC, ap + AC, pol (12)

Acp ap =faflCH(Acp)oCH

fa, = 0.574 + 0.000702Nre, (14)

(ACp)o-coNH- = -60 f 6 (JK mol-') (16)

(ACp)"+H- = 28 f 1 (JK mol-') (17)

As there are 757 apolar hydrogen atoms and 128 aminoacidic residues in azurin, AC, is 8.5 kJ/(Wmol). By collecting the data obtained and by means of the following formulas

MT = - Acp(Tl/2 - r ) (18)

(20)

Tu2 - T AGT = AHu- - ACp(TL,2 - r ) + TAC, In T

it is possible to evaluate AG, AH, and AS, as a function of temperature." The AHu and Tl/2 parameters used are the ones obtained by the curve fitting operation carried out at 0.5 "C/ min, reported in Table 3. The results are reported in Figure 11. It is interesting to note that the maximum value of AG is 63 kJ/mol. This value is very close to the maximum stability predicted by P r i ~ a l o v ~ ~ for a protein with 128 residues (64 kJ/ mol). This stability, extremely high if compared with many other globular proteins, is ascribed to the stabilizing effect of copper.

Conclusions Taken as a whole, the DSC, ESR, and OD data made it

possible to highlight many fundamental aspects of the thermal denaturation of azurin, calculating (when possible) the principal thermodynamic and kinetic parameters. In particular, it was possible to elucidate the following aspects:

14870 J. Phys. Chem., Vol. 99, No. 40, 1995 La Rosa et al.

G. Arena for helpful discussions during the preparation of the manuscript.

ur -400 $

-20

-40 ' " " " ' '-1,000 -20 -10 0 10 20 30 40 50 60 70 80 90 100

Temperature ('C)

Figure 11. Temperature dependence of the Gibbs energy (AG) (solid line), enthalpy (AH), and entropy changes (-TAS) (dashed lines): pH = 7.03, ionic strength, 0.1 M; protein concentration, 1.25 mg/mL.

(a) From a calorimetric point of view, the thermal denaturation of azurin can be described in terms of the sum of two effects of opposite sign, the exothermic step being responsible for the calorimetric irreversibility. The possibility of calculating the AC, by means of the method of Murphy and Gill, together with the extrapolation procedure of the calorimetric traces discussed in the text, made the thermodynamic analysis of the time independent process possible. Moreover, all thermodynamic and kinetic parameters were tested by means of a curve- fitting program running with a previously developed equation, which describes the C, exc function in all the temperature ranges investigated.

(b) Optical and ESR analyses made it possible to study the response of copper and its chemical environment to temperature variations. From the scan rate dependence of the OD curves, it was possible to calculate the kinetic parameters associated with this process. ESR investigations made it possible to study the geometry of the Cu ligands in all temperature ranges of the scans. The OD linear reduction represents a reversible distortion of the copper-sulfur bond, while the scan rate dependent zone consists of the irreversible substitution of the S(Cys) and S(Met) by oxygen atoms. As a consequence, a change in the coordination environment of copper occurs.

Acknowledgment. This work was partially supported by MURST (Minister0 della Universith e della Ricerca Scientifica e Tecnologica) and CNR Rome, Progetto Finalizzato Biotec- nologie e Biostrumentazione, and GNCB-CNR. We thank Prof.

References and Notes (1) Adman, E. T. In Topics in Molecular and Structural Biology:

Metalloproteins; Momson, P. M., Ed.; Chemie Verlag: Weinheim, Germany, 1969; Vol. 6, Part 1, pp 1-42.

(2) Gray, H. B.; Solomon, I. In Copper Proteins; Spiro T., Ed.; John Wiley and Sons Inc.: New York, 1981; pp 1-39.

(3) Fee, J. A. Struct. Bonding (Berlin) 1975, 23, 1-60. (4) Gus, J. M.; Freeman, H. C. J. Mol. Biol. 1983, 169, 521-563. ( 5 ) Nar, H.; Messerschmidt, A.; Huber, B.; Van de Kamp, M.; Canters,

(6) Adman, E. T.; Stenkamp, R. E.; Sieker, L. C.; Jensen, L. H. J.

(7) Engeseth, H. R.; McMillin, D. R. Biochemistry 1986, 25, 2448-

(8) Pnvalov, P. L. Adv. Protein Chem. 1979, 33, 167-239. (9) Muller, N. Biopolymers 1993, 33, 1185- 1193.

G. W. J. Mol. Biol. 1991, 218, 427-447.

Mol. Biol. 1978, 123, 35-47.

2455.

(10) Coleman, J. E.; Chlebowski, J. F. Adv. Znorg. Biochem. 1979, 1,

(1 1) Privalov, P. L.; Khechinashvili, N. N. J. Mol. Biol. 1974,86,665-

(12) Freire, E.; Biltonen, R. L. Biopolymers 1978, 17, 463-479. (13) Freire, E.; Van Odsol, W. W.; Mayorga, 0. L.; Sanchez-Ruiz, J.

(14) Edge, V.; Allewell, N. M.; Sturtevant, J. M. Biochemistry 1985,

(15) Lowry, 0. H.; Rosenbrough, N. J.; Fan, A. L.; Rendall, R. J. J.

(16) Sturtevant, J. M. Annu. Rev. Phys Chem. 1987,38, 463-512. (17) Connelly, P.; Ghosaini, L.; Hu, C. Q.; Kitamura, S.; Tanaka, A,;

(18) Privalov, P. L.; Potekhin, S. A. Methods Enzymol. 1986,4,4-51. (19) Sanchez-Ruiz, J. M. Biophys. J. 1992, 61, 921-935. (20) Sanchez-Ruiz, J. M.; Lbpez-Lacomba, J.; Cortijo, M.; Mateo, P.

L. Biochemistry 1988, 27, 1648-1652. (21) Milardi, D.; La Rosa, C.; Grasso, D. Biophys. Chem. 1994, 52,

183- 189. (22) Hernandez-Arana, A.; Rojo-Dominguez, A.; Altamirano, M. M.;

Calcagno, M. L. Biochemistry 1993, 32, 3644-3648. (23) Biltonen, R.; Freire, R. L. CRC Crit. Rev. Biochem. 1978,5, 85-

124. (24) Romero, A.; Hoitnik, C. W. G.; Nar, H.; Huber, R. A.; Messer-

Schmidt, A.; Canters, G.W. J . Mol. Biol. 1993, 229, 1007-1021. (25) Solomom, E. I.; Baldwin, M. J.; Lowery, M. D. Chem. Rev. 1992,

92, 521-542. (26) Gross, E. L.; Draheim, J. E.; Curtiss, A. S.; Crombie, B.; Scheffer,

A.; Pan, B.; Chiang, C.; Lopez, A. Arch. Biochem. Biophys. 1992, 298, 413-419.

(27) Peisach, J.; Blumberg, W. E. Arch. Biochem. Biophys. 1974, 691- 708.

(28) Den Blaauwen, T.; Canters, G. W. J . Am. Chem. SOC. 1993, 115, 1 12 1 - 1 129.

(29) Galisteo, M. L.; Sanchez-Ruiz, J. M. Eur. Biophys. J. 1993,5-30. (30) Lumry, R.; Eyring, H. J. Phys. Chem. 1954,58, 110-120. (31) Murphy, K. P.; Gill, S. J. J. Mol. Biol. 1991, 222, 699-709. (32) Jorgensen, W. L.; Gao, J.; Ravimohan, C. J. Phys. Chem. 1985,

(33) Chothia, C. J. Mol. Biol. 1976, 105, 1-14. (34) Pnvalov, P. L.; Gill, S . J. Adv. Protein Chem. 1988,39, 191-234. (35) Pnvalov, P. L. Thermochim. Acta 1990, 163, 33-46.

1-67.

684.

M. Annu. Rev. Biophys. Chem. 1990, 19, 159-188.

24, 5899-5906.

Biol. Chem. 1951, 193, 265-275.

Sturtevant, J. M. Biochemistry 1991, 30, 1887-1895.

89, 3470-3473.

Jp950365J

thermodynamics of azurin folding

Documents