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Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii
Biochimica et Biophysica Acta 1597 (2002) 51 – 59 www.bba-direct.com
Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii II. Holo form Roberto Favilla a,b,*, Matteo Goldoni a,c, Fabio Del Signore a,c, Paolo Di Muro d, Benedetto Salvato d,e, Mariano Beltramini d,e,* a INFM Unit, University of Parma, Parma, Italy Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy c Department of Physics, University of Parma, Parma, Italy d CNR Center for Biomedical Technologies, Section of Padua, University of Padua, Padua, Italy e Department of Biology, University of Padua, Padua, Italy b
Received 23 April 2001; received in revised form 8 January 2002; accepted 4 February 2002
Abstract The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl < 1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C1/2 = 1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C1/2 = 1.4 M). Cooperative transitions with C1/2 values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C1/2 = 1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hemocyanin; Unfolding; Carcinus; Kinetics; Simulation; Guanidinium hydrochloride
Abbreviations: Hc(s), hemocyanin(s); holo-CaeSS2, holo (copper containing) hemocyanin subunit; oxy-CaeSS2, oxygenated holo subunit; deoxy-CaeSS2, deoxygenated subunit; apo-CaeSS2, apo (copper free) subunit; ANS, 8-anilino-1-naphtalene sulphonate; TRIS, 50 mM Tris/HCl buffer at pH 7.0; GuHCl, guanidinium hydrochloride; MG, molten globule; LS, light scattering; CD, circular dichroism; ABS, absorbance; IF, intrinsic (protein tryptophan) fluorescence; EF, extrinsic (protein bound ANS) fluorescence; SF, stopped flow; PDB, protein data bank * Corresponding authors. R. Favilla is to be contacted at Dept. of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy. Tel.: +39-0521-905488; fax: +39-0521-905223. M. Beltramini is to be contacted at Dept. of Biology, University of Padua, Via Ugo Bassi 58, 35121 Padua, Italy. Tel.: +39-049-8276337; fax: +39-049-8276300. E-mail addresses: [email protected] (R. Favilla), [email protected] (M. Beltramini). 0167-4838/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 2 ) 0 0 2 7 9 - 0
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1. Introduction The biological role of hemocyanins (Hcs)1 is based on their capability to bind molecular oxygen reversibly [1,2]. Thus, the holo protein exists in two forms, oxy and deoxyHc, the equilibrium between them depending on the oxygen partial pressure. The functionally active forms of mollusc and arthropod Hcs both contain a pair of copper ions in their active site. In all cases, each copper ion (CuA and CuB) is bound to three histidine residues. The deoxygenated form of Hcs contains the metal ions in the Cu(I) state, is colourless and diamagnetic. Binding of oxygen occurs via a reversible one-electron transfer from each copper ion to the ligand, thus the resulting complex is depicted as a [Cu(II)O22 Cu(II)] complex, where the peroxide dianion is bound in a A:D2 –D2 bridging mode. This coordination is responsible for the main peculiar optical absorption properties of oxy-Hcs: their absorption spectrum in the near UV region exhibits an intense band with maximum near 340 nm (e c 20,000 M 1 cm 1), attributed to a peroxide-to-Cu(II) charge transfer transition [3,4]. X-ray crystallography studies have shown that, although the tertiary and quaternary folds of mollusc and arthropod Hcs are markedly dissimilar, the structures of the active sites of oxy-Hcs are almost identical. The metal – metal distance is 0.35 –0.36 nm in the oxy holo-Hc from the octopod Octopus vulgaris [5] and the horseshoe crab Limulus polyphemus [6], as well as in the deoxy-Hc of the lobster Panulirus interruptus [7]. Furthermore, by superimposing the X-ray crystallographic map of the active site region of Octopus and Limulus oxy-Hcs, all residues within a radius of 0.5 nm from the Cu –Cu midpoint are not only conserved in the sequence but also occupy similar positions [8]. In addition to the functional role, the active site bound copper ions were found to play a stabilising role concerning the conformational properties of Hcs [9 –11]. On the basis of the information derived by X-ray crystallography, the two sets of histidines, each including three residues, that coordinate to the two copper ions belong to two different antiparallel a-helices [6,7]; thus, metal coordination is expected to increase the conformation rigidity. Moreover, the molecular oxygen binding mode, in the form of an equatorial bridge between the two metal ions [6,7], is possibly expected to contribute to the stabilisation of the protein conformation. Along lines similar to those exploited in our accompanying paper [12], we describe here the unfolding process of the holo form, which offered us the possibility to follow two additional parameters: the residual copper and peroxide-bound to the protein as guanidinium hydrochloride (GuHCl) concentration was increased. The affinity of Hcs for molecular oxygen is strongly modulated by protein conformation [13] and the X-ray crystallographic studies are aimed to disclose how the conformational changes occurring within one subunit are propagated through the oligomer [14]. Thus, it could be possible to observe changes in the charge transfer band
intensity at such a low denaturant concentration where the active site copper ions are still bound to the protein matrix.
2. Materials and methods 2.1. Preparation of protein samples Samples of the 5S non-reassociating Hc subunit from C. aestuarii (CaeSS2) in its functional form, i.e. containing the two active site copper ions (holo-CaeSS2), were prepared according to a procedure already described [15]. Concentrated stock samples (several mg/ml), stored in 20% sucrose at 30 jC, were dialysed at 4 jC against TRIS before measurements. Protein concentration was calculated using an extinction coefficient of 1.24 mg 1 ml cm 1 at 278 nm, at pH 7.0 and 20 jC, identical to that of apo-CaeSS2. The content of the oxy form in holo-CaeSS2 samples was determined spectrophotometrically by measuring the absorbance ratio A337/A280 and using the value of 0.21 for a 100% oxy holo-CaeSS2 solution in oxygen saturated buffer [15]. The deoxy form of holo-CaeSS2 was prepared by treatment of the oxy form with 1 mM sodium dithionite in tris(hydroxymethyl)aminomethane hydrochloride and the loss of oxygen was monitored by the disappearance of the absorbance band at 337 nm. Due to the low oxygen affinity of the isolated subunit, holo-CaeSS2 in air-equilibrated TRIS exists as a 65– 35% mixture of oxy and deoxy forms, respectively, as expected from its oxygen saturation curve [15] and as shown by the A337/A280 ratio of 0.14. 2.2. Reagents and buffer 8-Anilino-1-naphtalenesulphonate (ANS; ammonium salt), GuHCl, tris(hydroxymethyl)aminomethane hydrochloride and sodium chloride were all from Sigma Chem. Co. Solutions buffered with TRIS were used throughout. 2.3. Spectroscopic measurements All spectroscopic measurements were performed on 1 AM holo-CaeSS2, unless otherwise stated. Protein solutions were previously incubated for several hours in TRIS, containing the appropriate GuHCl concentration, in order to allow pseudo-equilibrium conditions to be reached, as manifested by the constancy of the spectroscopic signal under investigation. Actually, our spectroscopic data could not be treated as true equilibrium values, due to the irreversible loss of copper ions upon denaturation. However, refolding of the apo protein was reversible and, in fact, quite similar to that observed with apo-CaeSS2 [12]. This notwithstanding, fitting of the experimental data was performed using sigmoidal functions, in order to derive the corresponding C1/2 values (Table 1). In the case of ANS, a double sigmoidal function was used, since two consecutive transitions were observed. The protein concentration used here was 1 AM, i.e. the same
R. Favilla et al. / Biochimica et Biophysica Acta 1597 (2002) 51–59 Table 1 C1/2 values of the unfolding transitions of holo-CaeSS2, induced by GuHCl Parameter IF IF ABS337 AA EFa EFb CD detected (intensity) (kmax) oxygen (copper) (ANS) (ANS) 225 nm C1/2 (M)
1.0
1.4
1.4
1.4
1.0
1.6
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(IF) changes were detected by excitation at 280 nm (5-nm slitwidth) and emission above 300 nm, using an appropriate cutoff filter. Other experimental and instrumental details were as previously described [12].
1.8
They are best-fit values, obtained using a two-state transition fitting function to each experimental data set. IF: intrinsic tryptophan fluorescence, ABS: absorbance, AA: atomic absorbance (for copper release), EF: (ANS) extrinsic fluorescence, CD: far UV circular dichroism. The error in all cases is within F 10%. a First transition. b Second transition.
as that used with apo-CaeSS2 [12]. At this concentration, holo-CaeSS2 exhibited only a little aggregation in the low denaturant concentration range, which however did not affect the spectroscopic measurements, as described below. The experimental error was estimated to be below 10% in all measurements. Instrumentation used for light scattering fluorescence, absorbance and circular dichroism measurements were the same as previously described [12]. The protein secondary structure percent content, derived from far UV CD spectra, was evaluated according to the methods described for apo-CaeSS2 [12]. For LS measurements a wavelength of 450 nm was used, instead of 350 nm chosen for apo-Hc [12], to avoid any absorbance effect due to the oxygen to copper charge transfer band. 2.4. Protein copper content Copper concentration in holo-CaeSS2 samples was determined by atomic absorption spectroscopy using a Perkin-Elmer Analyst 100 flame spectrophotometer. Protein samples (4 ml each), previously equilibrated in TRIS containing different concentrations of GuHCl, were ultrafiltered using Ultrafree Millipore filters (5 kDa cutoff) to separate released copper ions from the protein and washed twice with the cognate solution. Copper concentration in the ultrafiltrate and protein samples was then measured by atomic absorption, after calibration of the instrumental response with standard copper solutions (0.2, 0.4, 0.6 Ag/ml) in the same medium used for holo-CaeSS2. The concentration of holo-CaeSS2 was so low as to give negligible matrix effects on the atomic absorption measurements. The total copper concentration in the ultrafiltrate and holo-CaeSS2 solutions was found to be constant in all samples, indicating quantitative recovery of the metal. Copper concentration in each holo-CaeSS2 sample was then correlated to the protein concentration determined as described above and converted to copper-to-protein stoichiometry assuming the presence of two g-atoms of Cu per 75 kDa protein in the native sample.
3. Results and discussion 3.1. Unfolding under pseudo-equilibrium conditions 3.1.1. Light scattering The 450-nm LS intensity of 1 AM oxy holo-CaeSS2, detected at 90j with respect to the incident beam, was found to increase above 0.05 M GuHCl, reaching a maximum value, about 4-fold higher than in TRIS, between 0.2 and 0.4 M. Above 0.4 M GuHCl, the LS intensity decreased steadily and at 1.2 M GuHCl had the same value as in TRIS (Fig. 1). This effect is qualitatively similar to that observed with apo-CaeSS2, though an order of magnitude lower than with apo-CaeSS2 [12], confirming the ability of GuHCl to induce protein aggregation in the low denaturant concentration range. The smaller degree of aggregation may be attributed to the presence of copper ions at the active site of holo-CaeSS2, which probably remains in a more native-like state, in contrast to apo-CaeSS2, already present in a more labile molten globule-like conformation in TRIS [12]. It should finally be mentioned that holo-CaeSS2 is also very prone to aggregate, depending on temperature and protein concentration, as already documented in a previous SAXS study by us [16]. For instance, a 1-AM protein solution becomes slightly opalescent at 25 jC, but not at 20 jC, as well as at 20 jC a 4 AM, but not a 2 AM, solution containing 1 M GuHCl. 3.1.2. Intrinsic fluorescence IF measurements were negligibly affected by the very limited increase of LS intensity observed at low denaturant
2.5. Kinetic measurements A three-syringe stopped flow apparatus (model SFM3, Bio-Logic) was used to follow the unfolding of holoCaeSS2 induced by GuHCl. Protein intrinsic fluorescence
Fig. 1. LS intensity of holo-CaeSS2 as a function of GuHCl. Holo-CaeSS21 AM in TRIS, LS at 90j from excitation (kexc = 450 nm), 20 jC.
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Fig. 2. Fluorescence spectra of holo-CaeSS2 (—), apo-CaeSS2 (. . .) and holo-CaeSS2 denatured in 4 M GuHCl (- - - -). Inset: fluorescence spectra of holo-CaeSS2 a (60:40 mixture of oxy and deoxy forms) (- - -) and the fully deoxy form (—).
concentration. The fluorescence spectra of holo-CaeSS2 and apo-CaeSS2 in TRIS are shown in Fig. 2, together with that of holo-CaeSS2 denaturated in 4 M GuHCl. In the absence of denaturant, the IF spectrum of holo-CaeSS2 was quenched (about one third) as compared to apo-CaeSS2, while the maximum emission wavelengths were almost the same (338 and 340 nm, respectively). The much lower fluorescence quantum yield of holo-CaeSS2, with respect to that of apo-CaeSS2, already observed in several other Hcs [1], can be attributed to quenching effects by both copper ions and bound oxygen. More precisely, whereas quenching in oxy holo-CaeSS2 can be mainly attributed to energy transfer from excited trp residues to the copper –oxygen complex, quenching in deoxy holo-CaeSS2 can only be due to the presence of copper ions. Actually, this quenching of IF is already abolished in apo-CaeSS2 [12]. Furthermore, the small difference in the emission maximum (1– 2 nm) of the two forms may suggest either a little increase of solvent exposure of at least some tryptophan residues or the onset of fluorescence from a different class of fluorophores that is quenched in the native protein. The quenching effect exerted by bound oxygen can be appreciated by comparing the spectra of the holo-protein in air and of its deoxy form: the emission intensity of this latter derivative is about 40% more intense than the oxy form, but the spectral shape is similar, confirming a quantitatively relevant quenching effect by oxygen, without any appreciable increase of solvent exposure of the trp residues. It is worth noting that the fluorescence spectrum of holo-CaeSS2, shown in Fig. 2 inset (dashed line), is relative to a mixture of 65% oxy-Hc and 35% deoxy-Hc. Thus, on account of this relative abundance of the two species, the fluorescence spectrum of deoxy-Hc turned out to be 1.8-fold more intense than that of oxyCaeSS2. The fluorescence spectrum of holo-CaeSS2, denatured in 4 M GuHCl, is largely perturbed and identical to that of apo-CaeSS2 denatured under similar conditions [12], thus demonstrating that both holo- and apo-CaeSS2 con-
verge to an unfolded form exhibiting the same emissive properties. The interpretation of the emissive properties of denatured apo-CaeSS2 also applies to holo-CaeSS2, since the two copper ions are released from the active site upon denaturation. In particular, the shift of emission maximum from 338 to 355 nm is compatible with a full solvent exposure of the trp residues. Two different curves were observed when the IF intensity or maximum emission wavelength values was plotted as a function of GuHCl concentration (Fig. 3A). The corresponding best-fit values, obtained by using a two-state model for each curve, are shown in Table 1. Two transitions (midpoints near 1.0 and 1.4 M, respectively) were detected from IF intensity data when excitation was at 280 nm, whereas only the first one was detected when excitation occurred at 295 nm (Fig. 3B). This difference suggests that the transition at higher GuHCl is due to the loss of tyrosine-to-tryptophan energy transfer, resulting in a decrease of tryptophan emission intensity. Such energy transfer does not occur upon excitation at 295 nm—where only trp residues absorbed, and therefore a single transition is expected. The initial increase of IF intensity, observed at low GuHCl, can be attributed to oxygen
Fig. 3. Unfolding curves of holo-CaeSS2, as obtained by plotting the protein IF as a function of GuHCl. (A) Integrated spectrum intensity (.) and maximum emission wavelength (o) upon excitation at 280 nm; (B) Integrated spectrum intensity (n) upon excitation at 295 nm. Solid lines: best-fit curves, from a simple two-state function. Dashed line: monoexponential fit to the second half of the data points.
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and copper release that occur before denaturation. The observed decrease of IF intensity above 1 M GuHCl points to a more substantial unfolding, including loss of tyrosine to tryptophan energy transfer and also involves an increase of exposure of the trp residues to water. This conclusion is confirmed by similar transition obtained following the red shift of the maximum emission peak (Fig. 3A). According to these results, the two transitions are rather well separated, and therefore occur sequentially in an almost uncoupled way.
ence of the population fraction of each species as a function of GuHCl concentration was thus calculated and is shown in Fig. 4 (inset), whereas the corresponding unfolding parameter values are reported in Table 1. The intermediate Hc form detected here can be considered similar to apoCaeSS2. This interpretation is supported by the competence of the intermediate to bind ANS and by the almost coincident C1/2 value (1.6) of the two transitions (APO ! U here, apo-CaeSS2 ! U in the previous case [12]).
3.1.3. Extrinsic fluorescence (EF) In our previous paper on the unfolding of apo-CaeSS2 [12], we have shown that ANS was able to bind to apoCaeSS2 before denaturation, as demonstrated by the increase of the fluorescence intensity of the dye (EF) in the presence of protein without denaturant. Thus, based on this result, ANS was also exploited here to know as to up to what extent it is able to bind to holo-CaeSS2 as a function of denaturant concentration. In the absence of GuHCl, the EF properties of ANS in buffer did not change in the presence of holoCaeSS2, demonstrating that the dye does not bind. Hence, the presence of a properly folded active site prevents ANS binding. At intermediate GuHCl concentrations, an appreciable increase of the EF intensity was observed, with a maximum value near 1.3 M. By plotting the integrated EF intensity in the presence of holo-CaeSS2 as a function of GuHCl concentration, a bell-shaped curve was obtained (Fig. 4). The simplest interpretation of this result is that the native-like structure of oxy holo-CaeSS2 is changed into a molten globule-like intermediate (C1/2 = 1 M), before being extensively denatured at higher denaturant concentrations (at about 1.6 M) with concomitant loss of the ANS binding ability. Based on this, the overall process could be fitted by a three-state cooperative unfolding model involving native (N), intermediate (I) and unfolded (U) states. The depend-
3.1.4. Fluorescence quenching IF quenching measurements by acrylamide were performed with holo-CaeSS2 at a few GuHCl concentrations, under conditions similar to those already described for apoCaeSS2. At 1.3 M GuHCl, where the concentration of the molten globule-like intermediate reaches its maximum, an increase of about 20% for the solvent accessibility was observed, compared to native state conditions (data not shown). This result contrasts with the quenching data of N-acetyl-tryptophan amide (NATA) by acrylamide, which shows a protective action of GuHCl against quenching and suggests a decreased accessibility of this model compound to the solvent [17]. This result therefore reinforces our interpretation, according to which the protein conformational change, induced by GuHCl, increases the accessibility of tryptophan residues to solvent, although this denaturant has a protective effect against acrylamide quenching.
Fig. 4. Unfolding curve of holo-CaeSS2, as obtained by plotting ANS fluorescence as a function of GuHCl. Holo-CaeSS2 1 AM plus ANS 70 AM in TRIS at 20 jC (.). Experimental data before and after the bell-shaped transition curve are similar to those for ANS 70 AM alone. Solid line: best fit to a three-state function. Inset: relative abundance of native (N), intermediate (I) and denatured (U) forms as a function of GuHCl.
3.1.5. Far UV CD The molten globule-like nature of the intermediate can also be inferred from the dependence of the mean residue molar ellipticity at 225 nm of holo-CaeSS2 on GuHCl concentration (Fig. 5). The far UV CD spectra of native and fully denatured holo-CaeSS2 are shown in Fig. 5, inset. Analysis of the native CD spectrum, according to the methods already described [12], yielded the following average values of secondary structure content: a-helix 17.4%; h-sheet 34.3%; h-turns 18.5%, random coil 29.9%. It should be noticed that our results are not in good agreement with those reported in PDB for P. interruptus and L. polyphemus Hc subunits [7,8], particularly as far as the ahelix and h-sheet contents are concerned (26 –29% for ahelix and 15– 17% for h –sheet from literature). Though several reasons may be considered to be responsible for the large discrepancy between our results and the crystallographic data—i.e., solution versus crystal conditions, different Hc subunits, different functional state of the protein (oxy and deoxy forms)—it seems somehow hard to reconcile them. Our results on holo-CaeSS2 are, however, very similar to those obtained on apo-CaeSS2, as described in our previous paper [12]. The similarity between the secondary structure content of apo-CaeSS2 and holo-CaeSS2 is not in contrast with the molten globule-like state of the apo form, since such state is characterised by maintenance of secondary structure and a considerable loss of the well-defined packing of the
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Fig. 5. Unfolding curve of holo-CaeSS2, as obtained by plotting the far UV CD intensity as a function of GuHCl. Holo-CaeSS2 1 AM in TRIS at 20 jC; (.) ellipticity data taken at 225 nm, solid line: best fit to a two-state function. Inset: far UV CD spectra of 16 AM holo-CaeSS2: (—) native and (. . .) denatured in 3 M GuHCl.
dioxygen [15]. The presence of deoxy-CaeSS2, however, does not affect the results of the unfolding experiment since this derivative does not absorb at 337 nm. It is worth noting that the plot of Fig. 6A is very similar to that derived from the maximum emission wavelength data (Fig. 3A), implying that the events monitored by these two substantially different techniques are strongly coupled. This observation implies that the protein is still able to bind oxygen at moderate GuHCl concentration, as well as that oxygen is released over a wide range of denaturant concentration, spanning from less than 1 M (i.e., before the first conformational transition) to 2 M (i.e., corresponding to global unfolding). In this context, we were interested to see how these data relate to the process of copper removal from the active site (see below). It should be mentioned, however, that the transition parameters, obtained from absorbance data, are affected by a relatively large error, considering the low concentration used and the molar extinction coefficient of the band (e c 20,000 M 1 cm 1).
native form [18,19]. At denaturant concentrations below 1.5 M, the CD spectrum of holo-CaeSS2 was only slightly modified, though the intensity of the 337-nm absorption band of oxy holo-CaeSS2 and the copper-to-protein stoichiometry were already drastically decreased (see below). These results support the idea that protein acquires a molten globule-like structure [12], similar to apo-CaeSS2, in agreement with the above-described experiments with ANS. Above 1.5 M GuHCl, the CD spectrum changes its shape, approaching the typical shape of a denaturated protein. The fact that the C1/2 derived from far UV CD data (Fig. 5) is higher than that obtained from fluorescence (Table 1) can be explained, assuming that modifications of the secondary structure, monitored by far UV CD, are only partially coupled with the loss of the tertiary structure. 3.1.6. Absorbance and copper release The absorbance band centered at 337 nm is typical of oxy holo-CaeSS2. It has been attributed to a O22 ! Cu(II) ligand-to-metal charge transfer transitions [4] and may be exploited to monitor the stability of oxy-CaeSS2 against GuHCl. However, this technique does not give any information on the possible existence of deoxy copper containing intermediate forms, since these forms are devoid of such an absorption band. The observed cooperative transition, associated with oxygen release, is shown in Fig. 6A and the corresponding best-fit C1/2 parameter, again obtained according to a simple two-state transition model, is reported in Table 1. From the inset of Fig. 6A, relative to the absorbance of the protein at 337 nm in the absence of GuHCl, holoCaeSS2 can be predicted to be present as a mixture of oxy and deoxy forms in a 60:40 ratio, respectively, as already noted in Materials and methods. The presence of a significant amount of deoxy-Hc in air equilibrated buffer depends on the rather low affinity of holo-CaeSS2 subunit for
Fig. 6. Unfolding curves of holo-HcCaeSS2, as obtained from absorbance data, as a function of GuHCl. (A) Absorbance at 337 nm (.) of 1.5 AM holo-CaeSS2 (1 cm cell pathlength). Inset: Absorption spectra at: 0 M GuHCl (—); 1.2 M GuHCl (- - -); 1.9 M GuHCl () and 3 M GuHCl ( ). (B) Copper release data from 2.7 AM holo-CaeSS2, as deduced from atomic absorbance (.). Solid line: best fit to a simple two-state function.
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Copper ions are released from the protein active site in the presence of GuHCl. As shown in Fig. 6B, this release occurred sigmoidally as a function of GuHCl concentration. There is a good overlap of these data with those relative to the 337-nm band, resulting in a very similar denaturation C1/2 value (about 1.4 M) from the two approaches (Table 1). These results suggest that under our experimental conditions, oxygen and copper are released almost concomitantly and before the step of denaturation (compare Figs. 3 and 4 with Figs. 5 and 6). 3.2. Unfolding kinetics 3.2.1. Fluorescence stopped flow measurements The unfolding kinetics of holo-CaeSS2 by GuHCl was studied by means of the stopped flow technique, with IF detection, as described under Materials and methods (excitation at 280 nm, emission above 300 nm). All measurements were performed in TRIS at 20 jC. The investigated range of denaturant concentration was 2.7– 5.4 M (below 2.7 M unfolding kinetics was too slow to be followed with this technique). The final protein concentration was always kept constant at 1 AM, while the denaturant concentration was modulated by mixing appropriate volumes of 7 M GuHCl and TRIS, each separately loaded into the other two syringes. As an example, the experimental traces obtained by exposing 1 AM oxy holo-CaeSS2 to 3.3, 4.2 and 5.1 M GuHCl (values after mixing) are shown in Fig. 7. In the whole range of denaturant concentration investigated, all kinetics were best fitted, using the Bio-kine software, by a sum of three exponentials. In the 3.9– 4.2 M GuHCl range, the presence of an additional lag phase was fitted with a fourth exponential phase of opposite amplitude. Kinetics was again three-exponential above 4.2 M. At 5.4 M, it was apparently two-exponential, because the fastest phase was completely hindered within the instrumental dead time (3– 4 ms). The observed rate constants and amplitudes of
Fig. 7. Kinetics of GuHCl induced unfolding of holo-CaeSS2, as obtained from IF data. Stopped flow traces of 1 AM, denatured at three different GuHCl concentrations: (a) 3.3 M, (b) 4.2 M, (c) 5.1 M, all in TRIS at 20 jC (1.5 mm quartz cell).
Fig. 8. Kinetic parameters of holo-CaeSS2 unfolding induced by GuHCl. Best-fit values of (a) observed rate constants and (b) amplitudes as a function of GuHCl. Experimental values were obtained from a multiexponential analysis of experimental IF stopped flow traces, such as those shown in Fig. 7.
each phase as a function of GuHCl concentration are shown in Fig. 8. All rate constants increased asymptotically at high GuHCl, with the exception of the lag phase, observed only in a narrow GuHCl range. In contrast, the corresponding amplitudes remained approximately constant in the whole GuHCl range, with the exception of the intermediate phase, which showed a slight increase between 3.5 and 4.5 M, before reaching a plateau value at higher denaturant. The total amplitude was approximately constant in the whole denaturant concentration range investigated. The rate constants of each phase were dependent on the GuHCl concentration, including the lag phase. 3.2.2. Simulation of the unfolding kinetics A series of kinetic models was considered in order to fit the observed kinetics. The general procedure used to choose the best model, among many possible ones, was as follows: each experimental kinetic trace was best fitted by an appropriate sum of exponential terms, the exact number depending on GuHCl concentration, as described above. A model, with a given number of species, was then chosen and
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the corresponding system of differential equations was numerically solved by means of KINSIM software. The corresponding traces were then fitted to the those derived experimentally, by means of the FITSIM software, leaving the quantum yields of each species as free parameters, but using the best-fit values of the rate constants and amplitudes shown in Fig. 8. Note that the total fluorescence amplitude signal is given by a sum of terms, each formed by the product of the concentration times the relative fluorescence quantum yield of each species considered in the model. These quantum yield values are calculated relative to that of the initial mixture of 60:40 oxy/deoxy forms present in TRIS. Among the several (linear, branched and circular) models considered, very few ones were found to best fit the exponential traces, as judged by the obtained v2 values. The simplest one of them consisted of a sequence of four consecutive steps, as follows:
these aggregates disappear and a highly fluorescent intermediate IF is reversibly formed. Near 1.6 M GuHCl, loss of bound oxygen and copper ions take place more or less concomitantly (cf. Fig. 6A and B) and occurs irreversibly, with formation of an APO intermediate, having some molten globule-like properties. Full denaturation finally takes place from APO at even higher GuHCl. These steps are summarised in Model 2 below. 8 9 8 9 Ao > No > > > > > > > > > > > > > > = < = < > N ! N fIF ! APOfU ðModel2Þ > > > > > > > > > > > > > > > ; > ; : : Nd Ad
The relative quantum yield best-fit values corresponding to each species are reported in Table 2.
For the sake of simplicity, the aggregation steps, triggered by Ao and Ad, have been omitted from the model. We can now attribute a nature to the species present in Model 1, on the basis of both Model 2, as follows (Model 1bis): A in Model 1 correspond to No, B to 100% Nd, C to Id, D to IF and E to APO/U (APO and U are indistinguishable at high GuHCl).
3.3. A comprehensive unfolding model
No ! Nd ! Id ! If ! ðAPO=UÞ
A!B!C!D!E
ðModel1Þ
In order to account for all the experimental results, i.e. not only those derived under kinetic conditions, but also under pseudo-equilibrium conditions, Model 1 needs to be extended. Model 1 is, in fact, quite adequate to describe the unfolding kinetics at high GuHCl, but does not take into account the behaviour of the protein at low GuHCl, where the original mixture of oxy and deoxy native forms aggregates. The new model assumes that this process, which predominates in the denaturant concentration range below 1 M, is driven by formation of the two aggregation-prone intermediates oxy (Ao) and deoxy (Ad), from the corresponding oxy (No) and deoxy (Nd) native species, respectively. The new model then considers that, near 1 M GuHCl, Table 2 IF quantum yield values of all species present in Model 1, relative to that of Aa, arbitrarily set equal to 1. These values were obtained by simulation with FITSIM GuHCl (M)
B (au)
C (au)
D (au)
E (au)
2.7 3 3.3 3.6 3.9 4.2 4.5 4.8 5.1 5.4
1.31 1.30 1.26 1.18 1.14 1.17 1.06b 1.08b 1.10b –
– – – – 1.12 1.09 0.96b 1.03b 0.81b 1.02b
1.68 1.77 1.75 1.84 1.84 1.79 1.76b 1.70b 1.74b 1.80b
1.93 2.04 2.06 2.00 2.07 2.02 2.06c 2.06c 2.07c 2.08c
a
A is holo-Hc in TRIS, i.e. a 60:40 mixture of the two oxy (No) and deoxy (Nd) native forms. b Values calculated using the extrapolated values of the last column. c Values linearly extrapolated from those at lower GuHCl concentrations.
ðModel1bisÞ
These attributions are justified by the quantum yield values of the species shown in Table 2, since they increase in the following order: No < Nd c Id < IF < APO c U.
4. Conclusions Denaturation of holo-CaeSS2 subunit of C. aestuarii appears to follow a rather complex pathway, composed of a series of steps, of which only the step leading to the apo form is irreversible. A plausible model of denaturation was obtained taking into account kinetic and pseudo-equilibrium data (Model 2). Aggregation, occurring below 1 M GuHCl, though to a much lower extent than with apo-Hc [12], is supposed to be triggered by aggregation-prone intermediates, Ao and Ad, soon formed from the corresponding native forms at very low GuHCl ( > 0.05 M). Near 1 M GuHCl, the IF intensity of holo-CaeSS2 undergoes a consistent increase, which however, remains below that of apoCaeSS2 under similar conditions [12]. This fluorescence increase cannot, however, be attributed to release either oxygen or copper ions, though they are responsible for the strong quenching observed with oxy holo-CaeSS2, since their release takes place, more or less concomitantly, well above 1 M GuHCl (C1/2 1.4 M). Rather, it can be explained with the occurrence of a protein conformational change, which relaxes structural constraints near some tryptophan residues and give rise to IF. This highly fluorescent intermediate is then irreversibly converted to the apo form, which exhibits some molten globule-like properties, as inferred from the bell-shaped ANS binding curve between 1 and 1.5 M GuHCl, with a maximum EF intensity near
R. Favilla et al. / Biochimica et Biophysica Acta 1597 (2002) 51–59
1.2 –1.3 M [20]. At higher GuHCl (>2 M), full denaturation of the apo form occurs, as detected by all spectroscopic probes investigated: IF (intensity decrease and red shift), EF (decrease due to release of bound ANS), and CD (strong decrease of intensity in the far UV, due to loss of secondary structure). This series of events is well accounted for by simulation of Model 2 and is also consistent with the observed multiphasic kinetics.
Acknowledgements This work was supported by INFM funds to R.F., two INFM research fellowships to F.D.S. and M.G. and a MURST grant (co-finanziamento prot. 9805192993_001 and 9805192993_002) to BS and M.B.
References [1] C.P. Mangum, in: C.P. Mangum (Ed.), Advances in Comparative and Environmental Physiology, Blood and Tissue Oxygen Carriers 13, Springer-Verlag, Berlin, 1992, pp. 301 – 323. [2] J.P. Truchot, in: C.P. Mangum (Ed.), Advances in Comparative and Environmental Physiology, Blood and Tissue Oxygen Carriers 13, Springer-Verlag, Berlin, 1992, pp. 377 – 410. [3] K.E. van Holde, K.I. Miller, Adv. Prot. Chem. 47 (1995) 1 – 81.
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[4] E.I. Solomon, M.J. Baldwin, M.D. Lowery, Chem. Rev. 92 (1992) 521 – 542. [5] M.E. Cuff, K.I. Miller, K.E. Holde, W.A. Hendrickson, J. Mol. Biol. 278 (1998) 855 – 870. [6] K.A. Magnus, H. Ton-That, J.E. Carpenter, Chem. Rev. 94 (1994) 727 – 735. [7] A. Volbeda, W.G.J. Hol, J. Mol. Biol. 209 (1989) 249 – 279. [8] B. Hazes, W.G.J. Hol, Protein Sci. 2 (1993) 597 – 619. [9] F. Ricchelli, B. Salvato, B. Filippi, G. Jori, Arch. Biochem. Biophys. 202 (1980) 277 – 288. [10] D.N. Georgieva, S. Stoeva, S.A. Ali, S. Abbasi, N. Genov, W. Voelter, Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc. 54 (1998) 765 – 771. [11] K. Pervanova, K. Idakieva, S. Stoeva, N. Genov, W. Voelter, Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc. 56 (2000) 615 – 662. [12] R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, Biochim. Biophys. Acta 1597/1 (2002). [13] J. Markl, H. Decker, in: C.P. Mangum (Ed.), Advances in Comparative and Environmental Physiology, Blood and Tissue Oxygen Carriers 13, Springer Verlag, Berlin, 1992, pp. 325 – 376. [14] K.A. Magnus, B. Hazes, H. Ton-That, C. Bonaventura, J. Bonaventura, W.G.J. Hol, Proteins 19 (1994) 302 – 309. [15] E. Dainese, P. Di Muro, M. Beltramini, B. Salvato, H. Decker, Eur. J. Biochem. 256 (1998) 350 – 358. [16] M. Beltramini, P. Di Muro, R. Favilla, A. La Monaca, P. Mariani, A.L. Sabatucci, B. Salvato, L. Solari, J. Mol. Struct. 475 (1999) 73 – 82. [17] M.R. Eftink, C.A. Ghiron, J. Phys. Chem. 80 (1976) 486 – 493. [18] O.B. Ptitsyn, Adv. Prot. Chem. 47 (1995) 83 – 229. [19] D.T. Haynie, E. Freire, Proteins 16 (1993) 115 – 140. [20] G.V. Semisotnov, N.A. Rodionova, O.I. Razgulyaev, V.N. Uversky, A.F. Gripas, R.I. Gilmanshin, Biopolymers 31 (1991) 119 – 128.
Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii II. Holo form Roberto Favilla a,b,*, Matteo Goldoni a,c, Fabio Del Signore a,c, Paolo Di Muro d, Benedetto Salvato d,e, Mariano Beltramini d,e,* a INFM Unit, University of Parma, Parma, Italy Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy c Department of Physics, University of Parma, Parma, Italy d CNR Center for Biomedical Technologies, Section of Padua, University of Padua, Padua, Italy e Department of Biology, University of Padua, Padua, Italy b
Received 23 April 2001; received in revised form 8 January 2002; accepted 4 February 2002
Abstract The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl < 1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C1/2 = 1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C1/2 = 1.4 M). Cooperative transitions with C1/2 values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C1/2 = 1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hemocyanin; Unfolding; Carcinus; Kinetics; Simulation; Guanidinium hydrochloride
Abbreviations: Hc(s), hemocyanin(s); holo-CaeSS2, holo (copper containing) hemocyanin subunit; oxy-CaeSS2, oxygenated holo subunit; deoxy-CaeSS2, deoxygenated subunit; apo-CaeSS2, apo (copper free) subunit; ANS, 8-anilino-1-naphtalene sulphonate; TRIS, 50 mM Tris/HCl buffer at pH 7.0; GuHCl, guanidinium hydrochloride; MG, molten globule; LS, light scattering; CD, circular dichroism; ABS, absorbance; IF, intrinsic (protein tryptophan) fluorescence; EF, extrinsic (protein bound ANS) fluorescence; SF, stopped flow; PDB, protein data bank * Corresponding authors. R. Favilla is to be contacted at Dept. of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy. Tel.: +39-0521-905488; fax: +39-0521-905223. M. Beltramini is to be contacted at Dept. of Biology, University of Padua, Via Ugo Bassi 58, 35121 Padua, Italy. Tel.: +39-049-8276337; fax: +39-049-8276300. E-mail addresses: [email protected] (R. Favilla), [email protected] (M. Beltramini). 0167-4838/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 2 ) 0 0 2 7 9 - 0
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1. Introduction The biological role of hemocyanins (Hcs)1 is based on their capability to bind molecular oxygen reversibly [1,2]. Thus, the holo protein exists in two forms, oxy and deoxyHc, the equilibrium between them depending on the oxygen partial pressure. The functionally active forms of mollusc and arthropod Hcs both contain a pair of copper ions in their active site. In all cases, each copper ion (CuA and CuB) is bound to three histidine residues. The deoxygenated form of Hcs contains the metal ions in the Cu(I) state, is colourless and diamagnetic. Binding of oxygen occurs via a reversible one-electron transfer from each copper ion to the ligand, thus the resulting complex is depicted as a [Cu(II)O22 Cu(II)] complex, where the peroxide dianion is bound in a A:D2 –D2 bridging mode. This coordination is responsible for the main peculiar optical absorption properties of oxy-Hcs: their absorption spectrum in the near UV region exhibits an intense band with maximum near 340 nm (e c 20,000 M 1 cm 1), attributed to a peroxide-to-Cu(II) charge transfer transition [3,4]. X-ray crystallography studies have shown that, although the tertiary and quaternary folds of mollusc and arthropod Hcs are markedly dissimilar, the structures of the active sites of oxy-Hcs are almost identical. The metal – metal distance is 0.35 –0.36 nm in the oxy holo-Hc from the octopod Octopus vulgaris [5] and the horseshoe crab Limulus polyphemus [6], as well as in the deoxy-Hc of the lobster Panulirus interruptus [7]. Furthermore, by superimposing the X-ray crystallographic map of the active site region of Octopus and Limulus oxy-Hcs, all residues within a radius of 0.5 nm from the Cu –Cu midpoint are not only conserved in the sequence but also occupy similar positions [8]. In addition to the functional role, the active site bound copper ions were found to play a stabilising role concerning the conformational properties of Hcs [9 –11]. On the basis of the information derived by X-ray crystallography, the two sets of histidines, each including three residues, that coordinate to the two copper ions belong to two different antiparallel a-helices [6,7]; thus, metal coordination is expected to increase the conformation rigidity. Moreover, the molecular oxygen binding mode, in the form of an equatorial bridge between the two metal ions [6,7], is possibly expected to contribute to the stabilisation of the protein conformation. Along lines similar to those exploited in our accompanying paper [12], we describe here the unfolding process of the holo form, which offered us the possibility to follow two additional parameters: the residual copper and peroxide-bound to the protein as guanidinium hydrochloride (GuHCl) concentration was increased. The affinity of Hcs for molecular oxygen is strongly modulated by protein conformation [13] and the X-ray crystallographic studies are aimed to disclose how the conformational changes occurring within one subunit are propagated through the oligomer [14]. Thus, it could be possible to observe changes in the charge transfer band
intensity at such a low denaturant concentration where the active site copper ions are still bound to the protein matrix.
2. Materials and methods 2.1. Preparation of protein samples Samples of the 5S non-reassociating Hc subunit from C. aestuarii (CaeSS2) in its functional form, i.e. containing the two active site copper ions (holo-CaeSS2), were prepared according to a procedure already described [15]. Concentrated stock samples (several mg/ml), stored in 20% sucrose at 30 jC, were dialysed at 4 jC against TRIS before measurements. Protein concentration was calculated using an extinction coefficient of 1.24 mg 1 ml cm 1 at 278 nm, at pH 7.0 and 20 jC, identical to that of apo-CaeSS2. The content of the oxy form in holo-CaeSS2 samples was determined spectrophotometrically by measuring the absorbance ratio A337/A280 and using the value of 0.21 for a 100% oxy holo-CaeSS2 solution in oxygen saturated buffer [15]. The deoxy form of holo-CaeSS2 was prepared by treatment of the oxy form with 1 mM sodium dithionite in tris(hydroxymethyl)aminomethane hydrochloride and the loss of oxygen was monitored by the disappearance of the absorbance band at 337 nm. Due to the low oxygen affinity of the isolated subunit, holo-CaeSS2 in air-equilibrated TRIS exists as a 65– 35% mixture of oxy and deoxy forms, respectively, as expected from its oxygen saturation curve [15] and as shown by the A337/A280 ratio of 0.14. 2.2. Reagents and buffer 8-Anilino-1-naphtalenesulphonate (ANS; ammonium salt), GuHCl, tris(hydroxymethyl)aminomethane hydrochloride and sodium chloride were all from Sigma Chem. Co. Solutions buffered with TRIS were used throughout. 2.3. Spectroscopic measurements All spectroscopic measurements were performed on 1 AM holo-CaeSS2, unless otherwise stated. Protein solutions were previously incubated for several hours in TRIS, containing the appropriate GuHCl concentration, in order to allow pseudo-equilibrium conditions to be reached, as manifested by the constancy of the spectroscopic signal under investigation. Actually, our spectroscopic data could not be treated as true equilibrium values, due to the irreversible loss of copper ions upon denaturation. However, refolding of the apo protein was reversible and, in fact, quite similar to that observed with apo-CaeSS2 [12]. This notwithstanding, fitting of the experimental data was performed using sigmoidal functions, in order to derive the corresponding C1/2 values (Table 1). In the case of ANS, a double sigmoidal function was used, since two consecutive transitions were observed. The protein concentration used here was 1 AM, i.e. the same
R. Favilla et al. / Biochimica et Biophysica Acta 1597 (2002) 51–59 Table 1 C1/2 values of the unfolding transitions of holo-CaeSS2, induced by GuHCl Parameter IF IF ABS337 AA EFa EFb CD detected (intensity) (kmax) oxygen (copper) (ANS) (ANS) 225 nm C1/2 (M)
1.0
1.4
1.4
1.4
1.0
1.6
53
(IF) changes were detected by excitation at 280 nm (5-nm slitwidth) and emission above 300 nm, using an appropriate cutoff filter. Other experimental and instrumental details were as previously described [12].
1.8
They are best-fit values, obtained using a two-state transition fitting function to each experimental data set. IF: intrinsic tryptophan fluorescence, ABS: absorbance, AA: atomic absorbance (for copper release), EF: (ANS) extrinsic fluorescence, CD: far UV circular dichroism. The error in all cases is within F 10%. a First transition. b Second transition.
as that used with apo-CaeSS2 [12]. At this concentration, holo-CaeSS2 exhibited only a little aggregation in the low denaturant concentration range, which however did not affect the spectroscopic measurements, as described below. The experimental error was estimated to be below 10% in all measurements. Instrumentation used for light scattering fluorescence, absorbance and circular dichroism measurements were the same as previously described [12]. The protein secondary structure percent content, derived from far UV CD spectra, was evaluated according to the methods described for apo-CaeSS2 [12]. For LS measurements a wavelength of 450 nm was used, instead of 350 nm chosen for apo-Hc [12], to avoid any absorbance effect due to the oxygen to copper charge transfer band. 2.4. Protein copper content Copper concentration in holo-CaeSS2 samples was determined by atomic absorption spectroscopy using a Perkin-Elmer Analyst 100 flame spectrophotometer. Protein samples (4 ml each), previously equilibrated in TRIS containing different concentrations of GuHCl, were ultrafiltered using Ultrafree Millipore filters (5 kDa cutoff) to separate released copper ions from the protein and washed twice with the cognate solution. Copper concentration in the ultrafiltrate and protein samples was then measured by atomic absorption, after calibration of the instrumental response with standard copper solutions (0.2, 0.4, 0.6 Ag/ml) in the same medium used for holo-CaeSS2. The concentration of holo-CaeSS2 was so low as to give negligible matrix effects on the atomic absorption measurements. The total copper concentration in the ultrafiltrate and holo-CaeSS2 solutions was found to be constant in all samples, indicating quantitative recovery of the metal. Copper concentration in each holo-CaeSS2 sample was then correlated to the protein concentration determined as described above and converted to copper-to-protein stoichiometry assuming the presence of two g-atoms of Cu per 75 kDa protein in the native sample.
3. Results and discussion 3.1. Unfolding under pseudo-equilibrium conditions 3.1.1. Light scattering The 450-nm LS intensity of 1 AM oxy holo-CaeSS2, detected at 90j with respect to the incident beam, was found to increase above 0.05 M GuHCl, reaching a maximum value, about 4-fold higher than in TRIS, between 0.2 and 0.4 M. Above 0.4 M GuHCl, the LS intensity decreased steadily and at 1.2 M GuHCl had the same value as in TRIS (Fig. 1). This effect is qualitatively similar to that observed with apo-CaeSS2, though an order of magnitude lower than with apo-CaeSS2 [12], confirming the ability of GuHCl to induce protein aggregation in the low denaturant concentration range. The smaller degree of aggregation may be attributed to the presence of copper ions at the active site of holo-CaeSS2, which probably remains in a more native-like state, in contrast to apo-CaeSS2, already present in a more labile molten globule-like conformation in TRIS [12]. It should finally be mentioned that holo-CaeSS2 is also very prone to aggregate, depending on temperature and protein concentration, as already documented in a previous SAXS study by us [16]. For instance, a 1-AM protein solution becomes slightly opalescent at 25 jC, but not at 20 jC, as well as at 20 jC a 4 AM, but not a 2 AM, solution containing 1 M GuHCl. 3.1.2. Intrinsic fluorescence IF measurements were negligibly affected by the very limited increase of LS intensity observed at low denaturant
2.5. Kinetic measurements A three-syringe stopped flow apparatus (model SFM3, Bio-Logic) was used to follow the unfolding of holoCaeSS2 induced by GuHCl. Protein intrinsic fluorescence
Fig. 1. LS intensity of holo-CaeSS2 as a function of GuHCl. Holo-CaeSS21 AM in TRIS, LS at 90j from excitation (kexc = 450 nm), 20 jC.
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Fig. 2. Fluorescence spectra of holo-CaeSS2 (—), apo-CaeSS2 (. . .) and holo-CaeSS2 denatured in 4 M GuHCl (- - - -). Inset: fluorescence spectra of holo-CaeSS2 a (60:40 mixture of oxy and deoxy forms) (- - -) and the fully deoxy form (—).
concentration. The fluorescence spectra of holo-CaeSS2 and apo-CaeSS2 in TRIS are shown in Fig. 2, together with that of holo-CaeSS2 denaturated in 4 M GuHCl. In the absence of denaturant, the IF spectrum of holo-CaeSS2 was quenched (about one third) as compared to apo-CaeSS2, while the maximum emission wavelengths were almost the same (338 and 340 nm, respectively). The much lower fluorescence quantum yield of holo-CaeSS2, with respect to that of apo-CaeSS2, already observed in several other Hcs [1], can be attributed to quenching effects by both copper ions and bound oxygen. More precisely, whereas quenching in oxy holo-CaeSS2 can be mainly attributed to energy transfer from excited trp residues to the copper –oxygen complex, quenching in deoxy holo-CaeSS2 can only be due to the presence of copper ions. Actually, this quenching of IF is already abolished in apo-CaeSS2 [12]. Furthermore, the small difference in the emission maximum (1– 2 nm) of the two forms may suggest either a little increase of solvent exposure of at least some tryptophan residues or the onset of fluorescence from a different class of fluorophores that is quenched in the native protein. The quenching effect exerted by bound oxygen can be appreciated by comparing the spectra of the holo-protein in air and of its deoxy form: the emission intensity of this latter derivative is about 40% more intense than the oxy form, but the spectral shape is similar, confirming a quantitatively relevant quenching effect by oxygen, without any appreciable increase of solvent exposure of the trp residues. It is worth noting that the fluorescence spectrum of holo-CaeSS2, shown in Fig. 2 inset (dashed line), is relative to a mixture of 65% oxy-Hc and 35% deoxy-Hc. Thus, on account of this relative abundance of the two species, the fluorescence spectrum of deoxy-Hc turned out to be 1.8-fold more intense than that of oxyCaeSS2. The fluorescence spectrum of holo-CaeSS2, denatured in 4 M GuHCl, is largely perturbed and identical to that of apo-CaeSS2 denatured under similar conditions [12], thus demonstrating that both holo- and apo-CaeSS2 con-
verge to an unfolded form exhibiting the same emissive properties. The interpretation of the emissive properties of denatured apo-CaeSS2 also applies to holo-CaeSS2, since the two copper ions are released from the active site upon denaturation. In particular, the shift of emission maximum from 338 to 355 nm is compatible with a full solvent exposure of the trp residues. Two different curves were observed when the IF intensity or maximum emission wavelength values was plotted as a function of GuHCl concentration (Fig. 3A). The corresponding best-fit values, obtained by using a two-state model for each curve, are shown in Table 1. Two transitions (midpoints near 1.0 and 1.4 M, respectively) were detected from IF intensity data when excitation was at 280 nm, whereas only the first one was detected when excitation occurred at 295 nm (Fig. 3B). This difference suggests that the transition at higher GuHCl is due to the loss of tyrosine-to-tryptophan energy transfer, resulting in a decrease of tryptophan emission intensity. Such energy transfer does not occur upon excitation at 295 nm—where only trp residues absorbed, and therefore a single transition is expected. The initial increase of IF intensity, observed at low GuHCl, can be attributed to oxygen
Fig. 3. Unfolding curves of holo-CaeSS2, as obtained by plotting the protein IF as a function of GuHCl. (A) Integrated spectrum intensity (.) and maximum emission wavelength (o) upon excitation at 280 nm; (B) Integrated spectrum intensity (n) upon excitation at 295 nm. Solid lines: best-fit curves, from a simple two-state function. Dashed line: monoexponential fit to the second half of the data points.
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55
and copper release that occur before denaturation. The observed decrease of IF intensity above 1 M GuHCl points to a more substantial unfolding, including loss of tyrosine to tryptophan energy transfer and also involves an increase of exposure of the trp residues to water. This conclusion is confirmed by similar transition obtained following the red shift of the maximum emission peak (Fig. 3A). According to these results, the two transitions are rather well separated, and therefore occur sequentially in an almost uncoupled way.
ence of the population fraction of each species as a function of GuHCl concentration was thus calculated and is shown in Fig. 4 (inset), whereas the corresponding unfolding parameter values are reported in Table 1. The intermediate Hc form detected here can be considered similar to apoCaeSS2. This interpretation is supported by the competence of the intermediate to bind ANS and by the almost coincident C1/2 value (1.6) of the two transitions (APO ! U here, apo-CaeSS2 ! U in the previous case [12]).
3.1.3. Extrinsic fluorescence (EF) In our previous paper on the unfolding of apo-CaeSS2 [12], we have shown that ANS was able to bind to apoCaeSS2 before denaturation, as demonstrated by the increase of the fluorescence intensity of the dye (EF) in the presence of protein without denaturant. Thus, based on this result, ANS was also exploited here to know as to up to what extent it is able to bind to holo-CaeSS2 as a function of denaturant concentration. In the absence of GuHCl, the EF properties of ANS in buffer did not change in the presence of holoCaeSS2, demonstrating that the dye does not bind. Hence, the presence of a properly folded active site prevents ANS binding. At intermediate GuHCl concentrations, an appreciable increase of the EF intensity was observed, with a maximum value near 1.3 M. By plotting the integrated EF intensity in the presence of holo-CaeSS2 as a function of GuHCl concentration, a bell-shaped curve was obtained (Fig. 4). The simplest interpretation of this result is that the native-like structure of oxy holo-CaeSS2 is changed into a molten globule-like intermediate (C1/2 = 1 M), before being extensively denatured at higher denaturant concentrations (at about 1.6 M) with concomitant loss of the ANS binding ability. Based on this, the overall process could be fitted by a three-state cooperative unfolding model involving native (N), intermediate (I) and unfolded (U) states. The depend-
3.1.4. Fluorescence quenching IF quenching measurements by acrylamide were performed with holo-CaeSS2 at a few GuHCl concentrations, under conditions similar to those already described for apoCaeSS2. At 1.3 M GuHCl, where the concentration of the molten globule-like intermediate reaches its maximum, an increase of about 20% for the solvent accessibility was observed, compared to native state conditions (data not shown). This result contrasts with the quenching data of N-acetyl-tryptophan amide (NATA) by acrylamide, which shows a protective action of GuHCl against quenching and suggests a decreased accessibility of this model compound to the solvent [17]. This result therefore reinforces our interpretation, according to which the protein conformational change, induced by GuHCl, increases the accessibility of tryptophan residues to solvent, although this denaturant has a protective effect against acrylamide quenching.
Fig. 4. Unfolding curve of holo-CaeSS2, as obtained by plotting ANS fluorescence as a function of GuHCl. Holo-CaeSS2 1 AM plus ANS 70 AM in TRIS at 20 jC (.). Experimental data before and after the bell-shaped transition curve are similar to those for ANS 70 AM alone. Solid line: best fit to a three-state function. Inset: relative abundance of native (N), intermediate (I) and denatured (U) forms as a function of GuHCl.
3.1.5. Far UV CD The molten globule-like nature of the intermediate can also be inferred from the dependence of the mean residue molar ellipticity at 225 nm of holo-CaeSS2 on GuHCl concentration (Fig. 5). The far UV CD spectra of native and fully denatured holo-CaeSS2 are shown in Fig. 5, inset. Analysis of the native CD spectrum, according to the methods already described [12], yielded the following average values of secondary structure content: a-helix 17.4%; h-sheet 34.3%; h-turns 18.5%, random coil 29.9%. It should be noticed that our results are not in good agreement with those reported in PDB for P. interruptus and L. polyphemus Hc subunits [7,8], particularly as far as the ahelix and h-sheet contents are concerned (26 –29% for ahelix and 15– 17% for h –sheet from literature). Though several reasons may be considered to be responsible for the large discrepancy between our results and the crystallographic data—i.e., solution versus crystal conditions, different Hc subunits, different functional state of the protein (oxy and deoxy forms)—it seems somehow hard to reconcile them. Our results on holo-CaeSS2 are, however, very similar to those obtained on apo-CaeSS2, as described in our previous paper [12]. The similarity between the secondary structure content of apo-CaeSS2 and holo-CaeSS2 is not in contrast with the molten globule-like state of the apo form, since such state is characterised by maintenance of secondary structure and a considerable loss of the well-defined packing of the
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Fig. 5. Unfolding curve of holo-CaeSS2, as obtained by plotting the far UV CD intensity as a function of GuHCl. Holo-CaeSS2 1 AM in TRIS at 20 jC; (.) ellipticity data taken at 225 nm, solid line: best fit to a two-state function. Inset: far UV CD spectra of 16 AM holo-CaeSS2: (—) native and (. . .) denatured in 3 M GuHCl.
dioxygen [15]. The presence of deoxy-CaeSS2, however, does not affect the results of the unfolding experiment since this derivative does not absorb at 337 nm. It is worth noting that the plot of Fig. 6A is very similar to that derived from the maximum emission wavelength data (Fig. 3A), implying that the events monitored by these two substantially different techniques are strongly coupled. This observation implies that the protein is still able to bind oxygen at moderate GuHCl concentration, as well as that oxygen is released over a wide range of denaturant concentration, spanning from less than 1 M (i.e., before the first conformational transition) to 2 M (i.e., corresponding to global unfolding). In this context, we were interested to see how these data relate to the process of copper removal from the active site (see below). It should be mentioned, however, that the transition parameters, obtained from absorbance data, are affected by a relatively large error, considering the low concentration used and the molar extinction coefficient of the band (e c 20,000 M 1 cm 1).
native form [18,19]. At denaturant concentrations below 1.5 M, the CD spectrum of holo-CaeSS2 was only slightly modified, though the intensity of the 337-nm absorption band of oxy holo-CaeSS2 and the copper-to-protein stoichiometry were already drastically decreased (see below). These results support the idea that protein acquires a molten globule-like structure [12], similar to apo-CaeSS2, in agreement with the above-described experiments with ANS. Above 1.5 M GuHCl, the CD spectrum changes its shape, approaching the typical shape of a denaturated protein. The fact that the C1/2 derived from far UV CD data (Fig. 5) is higher than that obtained from fluorescence (Table 1) can be explained, assuming that modifications of the secondary structure, monitored by far UV CD, are only partially coupled with the loss of the tertiary structure. 3.1.6. Absorbance and copper release The absorbance band centered at 337 nm is typical of oxy holo-CaeSS2. It has been attributed to a O22 ! Cu(II) ligand-to-metal charge transfer transitions [4] and may be exploited to monitor the stability of oxy-CaeSS2 against GuHCl. However, this technique does not give any information on the possible existence of deoxy copper containing intermediate forms, since these forms are devoid of such an absorption band. The observed cooperative transition, associated with oxygen release, is shown in Fig. 6A and the corresponding best-fit C1/2 parameter, again obtained according to a simple two-state transition model, is reported in Table 1. From the inset of Fig. 6A, relative to the absorbance of the protein at 337 nm in the absence of GuHCl, holoCaeSS2 can be predicted to be present as a mixture of oxy and deoxy forms in a 60:40 ratio, respectively, as already noted in Materials and methods. The presence of a significant amount of deoxy-Hc in air equilibrated buffer depends on the rather low affinity of holo-CaeSS2 subunit for
Fig. 6. Unfolding curves of holo-HcCaeSS2, as obtained from absorbance data, as a function of GuHCl. (A) Absorbance at 337 nm (.) of 1.5 AM holo-CaeSS2 (1 cm cell pathlength). Inset: Absorption spectra at: 0 M GuHCl (—); 1.2 M GuHCl (- - -); 1.9 M GuHCl () and 3 M GuHCl ( ). (B) Copper release data from 2.7 AM holo-CaeSS2, as deduced from atomic absorbance (.). Solid line: best fit to a simple two-state function.
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Copper ions are released from the protein active site in the presence of GuHCl. As shown in Fig. 6B, this release occurred sigmoidally as a function of GuHCl concentration. There is a good overlap of these data with those relative to the 337-nm band, resulting in a very similar denaturation C1/2 value (about 1.4 M) from the two approaches (Table 1). These results suggest that under our experimental conditions, oxygen and copper are released almost concomitantly and before the step of denaturation (compare Figs. 3 and 4 with Figs. 5 and 6). 3.2. Unfolding kinetics 3.2.1. Fluorescence stopped flow measurements The unfolding kinetics of holo-CaeSS2 by GuHCl was studied by means of the stopped flow technique, with IF detection, as described under Materials and methods (excitation at 280 nm, emission above 300 nm). All measurements were performed in TRIS at 20 jC. The investigated range of denaturant concentration was 2.7– 5.4 M (below 2.7 M unfolding kinetics was too slow to be followed with this technique). The final protein concentration was always kept constant at 1 AM, while the denaturant concentration was modulated by mixing appropriate volumes of 7 M GuHCl and TRIS, each separately loaded into the other two syringes. As an example, the experimental traces obtained by exposing 1 AM oxy holo-CaeSS2 to 3.3, 4.2 and 5.1 M GuHCl (values after mixing) are shown in Fig. 7. In the whole range of denaturant concentration investigated, all kinetics were best fitted, using the Bio-kine software, by a sum of three exponentials. In the 3.9– 4.2 M GuHCl range, the presence of an additional lag phase was fitted with a fourth exponential phase of opposite amplitude. Kinetics was again three-exponential above 4.2 M. At 5.4 M, it was apparently two-exponential, because the fastest phase was completely hindered within the instrumental dead time (3– 4 ms). The observed rate constants and amplitudes of
Fig. 7. Kinetics of GuHCl induced unfolding of holo-CaeSS2, as obtained from IF data. Stopped flow traces of 1 AM, denatured at three different GuHCl concentrations: (a) 3.3 M, (b) 4.2 M, (c) 5.1 M, all in TRIS at 20 jC (1.5 mm quartz cell).
Fig. 8. Kinetic parameters of holo-CaeSS2 unfolding induced by GuHCl. Best-fit values of (a) observed rate constants and (b) amplitudes as a function of GuHCl. Experimental values were obtained from a multiexponential analysis of experimental IF stopped flow traces, such as those shown in Fig. 7.
each phase as a function of GuHCl concentration are shown in Fig. 8. All rate constants increased asymptotically at high GuHCl, with the exception of the lag phase, observed only in a narrow GuHCl range. In contrast, the corresponding amplitudes remained approximately constant in the whole GuHCl range, with the exception of the intermediate phase, which showed a slight increase between 3.5 and 4.5 M, before reaching a plateau value at higher denaturant. The total amplitude was approximately constant in the whole denaturant concentration range investigated. The rate constants of each phase were dependent on the GuHCl concentration, including the lag phase. 3.2.2. Simulation of the unfolding kinetics A series of kinetic models was considered in order to fit the observed kinetics. The general procedure used to choose the best model, among many possible ones, was as follows: each experimental kinetic trace was best fitted by an appropriate sum of exponential terms, the exact number depending on GuHCl concentration, as described above. A model, with a given number of species, was then chosen and
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the corresponding system of differential equations was numerically solved by means of KINSIM software. The corresponding traces were then fitted to the those derived experimentally, by means of the FITSIM software, leaving the quantum yields of each species as free parameters, but using the best-fit values of the rate constants and amplitudes shown in Fig. 8. Note that the total fluorescence amplitude signal is given by a sum of terms, each formed by the product of the concentration times the relative fluorescence quantum yield of each species considered in the model. These quantum yield values are calculated relative to that of the initial mixture of 60:40 oxy/deoxy forms present in TRIS. Among the several (linear, branched and circular) models considered, very few ones were found to best fit the exponential traces, as judged by the obtained v2 values. The simplest one of them consisted of a sequence of four consecutive steps, as follows:
these aggregates disappear and a highly fluorescent intermediate IF is reversibly formed. Near 1.6 M GuHCl, loss of bound oxygen and copper ions take place more or less concomitantly (cf. Fig. 6A and B) and occurs irreversibly, with formation of an APO intermediate, having some molten globule-like properties. Full denaturation finally takes place from APO at even higher GuHCl. These steps are summarised in Model 2 below. 8 9 8 9 Ao > No > > > > > > > > > > > > > > = < = < > N ! N fIF ! APOfU ðModel2Þ > > > > > > > > > > > > > > > ; > ; : : Nd Ad
The relative quantum yield best-fit values corresponding to each species are reported in Table 2.
For the sake of simplicity, the aggregation steps, triggered by Ao and Ad, have been omitted from the model. We can now attribute a nature to the species present in Model 1, on the basis of both Model 2, as follows (Model 1bis): A in Model 1 correspond to No, B to 100% Nd, C to Id, D to IF and E to APO/U (APO and U are indistinguishable at high GuHCl).
3.3. A comprehensive unfolding model
No ! Nd ! Id ! If ! ðAPO=UÞ
A!B!C!D!E
ðModel1Þ
In order to account for all the experimental results, i.e. not only those derived under kinetic conditions, but also under pseudo-equilibrium conditions, Model 1 needs to be extended. Model 1 is, in fact, quite adequate to describe the unfolding kinetics at high GuHCl, but does not take into account the behaviour of the protein at low GuHCl, where the original mixture of oxy and deoxy native forms aggregates. The new model assumes that this process, which predominates in the denaturant concentration range below 1 M, is driven by formation of the two aggregation-prone intermediates oxy (Ao) and deoxy (Ad), from the corresponding oxy (No) and deoxy (Nd) native species, respectively. The new model then considers that, near 1 M GuHCl, Table 2 IF quantum yield values of all species present in Model 1, relative to that of Aa, arbitrarily set equal to 1. These values were obtained by simulation with FITSIM GuHCl (M)
B (au)
C (au)
D (au)
E (au)
2.7 3 3.3 3.6 3.9 4.2 4.5 4.8 5.1 5.4
1.31 1.30 1.26 1.18 1.14 1.17 1.06b 1.08b 1.10b –
– – – – 1.12 1.09 0.96b 1.03b 0.81b 1.02b
1.68 1.77 1.75 1.84 1.84 1.79 1.76b 1.70b 1.74b 1.80b
1.93 2.04 2.06 2.00 2.07 2.02 2.06c 2.06c 2.07c 2.08c
a
A is holo-Hc in TRIS, i.e. a 60:40 mixture of the two oxy (No) and deoxy (Nd) native forms. b Values calculated using the extrapolated values of the last column. c Values linearly extrapolated from those at lower GuHCl concentrations.
ðModel1bisÞ
These attributions are justified by the quantum yield values of the species shown in Table 2, since they increase in the following order: No < Nd c Id < IF < APO c U.
4. Conclusions Denaturation of holo-CaeSS2 subunit of C. aestuarii appears to follow a rather complex pathway, composed of a series of steps, of which only the step leading to the apo form is irreversible. A plausible model of denaturation was obtained taking into account kinetic and pseudo-equilibrium data (Model 2). Aggregation, occurring below 1 M GuHCl, though to a much lower extent than with apo-Hc [12], is supposed to be triggered by aggregation-prone intermediates, Ao and Ad, soon formed from the corresponding native forms at very low GuHCl ( > 0.05 M). Near 1 M GuHCl, the IF intensity of holo-CaeSS2 undergoes a consistent increase, which however, remains below that of apoCaeSS2 under similar conditions [12]. This fluorescence increase cannot, however, be attributed to release either oxygen or copper ions, though they are responsible for the strong quenching observed with oxy holo-CaeSS2, since their release takes place, more or less concomitantly, well above 1 M GuHCl (C1/2 1.4 M). Rather, it can be explained with the occurrence of a protein conformational change, which relaxes structural constraints near some tryptophan residues and give rise to IF. This highly fluorescent intermediate is then irreversibly converted to the apo form, which exhibits some molten globule-like properties, as inferred from the bell-shaped ANS binding curve between 1 and 1.5 M GuHCl, with a maximum EF intensity near
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1.2 –1.3 M [20]. At higher GuHCl (>2 M), full denaturation of the apo form occurs, as detected by all spectroscopic probes investigated: IF (intensity decrease and red shift), EF (decrease due to release of bound ANS), and CD (strong decrease of intensity in the far UV, due to loss of secondary structure). This series of events is well accounted for by simulation of Model 2 and is also consistent with the observed multiphasic kinetics.
Acknowledgements This work was supported by INFM funds to R.F., two INFM research fellowships to F.D.S. and M.G. and a MURST grant (co-finanziamento prot. 9805192993_001 and 9805192993_002) to BS and M.B.
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