Role of the intersubunit disulfide bond in the unfolding pathway of dimeric red kidney bean purple acid phosphatase

BB ELSEVIER

Biochi~ic~a et BiophysicaA~ta

Biochimica et Biophysica Acta 1296 (1996) 76-84

Role of the intersubunit disulfide bond in the unfolding pathway of dimeric red kidney bean purple acid phosphatase Anil G. Cashikar, N. Madhusudhana Rao

*

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received 14 March 1996; accepted 27 March 1996

Abstract Quantitative equilibrium denaturation studies on oligomeric proteins have the potential to provide information on the role of subunit interactions in protein function and structure. We studied the equilibrium denaturation of red kidney bean purple acid phosphatase (KBPAP), a homodimer with a single disulfide bond between the two subunits, with an objective to understand the role of the intersubunit disulfide bond in KBPAP structure. Binding of 8-anilino-l-naphthalenesulfonic acid, enzymatic activity, size-exclusion chromatography, tryptophan fluorescence and circular dichroism studies revealed that the protein undergoes unfolding through at least three intermediates. Susceptibility of KBPAP for denaturation increases on reduction of the disulfide and aggregation was the predominant product of denaturation. In terms of stability, an intersubunit disulfide bond contributes to 25% of the overall stability of the dimer. Keywords: Phosphatase; Purple acid phosphatase; Denaturation; Intersubunit disulfide bond; Unfolding intermediate; (Red kidney bean)

1. Introduction Unfolding and refolding studies using chaotropic agents have given a wealth of information on the structure and biogenesis of proteins [1]. Solvent denaturation studies on monomeric proteins have been extremely rewarding in our understanding of protein structure. Similar information is not available on dimeric or oligomeric proteins, where additional modes of stabilization are available at the quatemary level [2]. Such studies would help in our understanding the rationale for the formation of oligomers and assess the strength of tertiary and quaternary level interactions in protein biogenesis [2]. Subunit dissociation leading to native monomers, followed by unfolding of the monomer (3-state denaturation) has been demonstrated in the case of k repressor [3] and aspartate aminotransferase [4]. Partially unfolded dimeric intermediates were detected in the case of alkaline phosphatase [5] and phosphoglucose isomerase

Abbreviations: KBPAP, red kidney bean purple acid phosphatase; gdmC1, guanidinium chloride; 13ME, 13-mercaptoethanol; RFI, relative fluorescence intensity; ANS, 8-anilino-l-naphthalenesulfonic acid; SEC, size-exclusion chromatography; CD, circular dichroism. * Corresponding author. Fax: + 91 40 671195; e-mail: [email protected].

[6]. Subunit dissociation leading directly to unfolded monomers (2-state denaturation) has been observed in the case of the arc repressor protein [7]. In case of neurotrophins various homologous structures could be distinguished by comparing their denaturation data [8]. None of the oligomers on which equilibrium denaturation studies were done have an intersubunit disulfide, to assess the influence of a disulfide on stability of the oligomer. To understand the role of the intersubunit disulfide in the unfolding pathway of the protein and also its contribution to the stability of a protein, we have studied the unfolding pathway of the red kidney bean purple acid phosphatase (KBPAP), a homodimeric glycoprotein with only one disulfide bond, which is between the Cys-345 residues of the two subunits [9,10]. KBPAP ( M r 111 000) hydrolyzes monoesters and anhydrides of phosphate in the pH range from 4 to 7. The amino-acid sequence of the protein has been established [11]. The crystal structure of KBPAP reveals an N-terminal 120 amino-acid domain and a C-terminal 312 amino-acid domain [12]. Amino acids ligating the metal ions and those involved in active site were located entirely in the C-terminal domain and the N-terminal domain is absent in the mammalian counterpart, uteroferrin. Limited subunit interactions were observed between et 5 helix and amino-acid residues from 2 5 3 - 2 6 0 apart from the intersubunit disul-

0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S 0 1 6 7 - 4 8 3 8 ( 9 6 ) 0 0 0 5 5 - 6

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

fide bond at Cys-345. KBPAP has a 13et13a13 motif and has similar amino acids ligating the metal ions as in uteroferrin [13]. Despite their wide occurrence and abundance, plant phosphatases, except few, do not show substrate specificity and their in vivo function is still unclear [14]. However, several acid phosphatases have been clearly shown to be inducible on phosphate starvation and are intimately involved in phosphate mobilization [14]. A 30fold enhancement in acid l?hosphatase activity during germination of the seeds and a consequent decrease in seed organic phosphate reserves, impart a crucial role for this protein in seed development [15]. Despite the crystal structure, very little is known on the relationships between the structure and function of KBPAP. We initiated studies on the stability of this protein in the presence of denaturants and substrates/inhibitors to understand the relationships between structure and function. Significant protection to solvent denaturation was observed in KBPAP upon binding the active-site ligand, phosphate and phosphate binding alters the unfolding pathway from a multistate to a threestate transition [16]. In this communication we present data on the structural stability of KBPAP in the presence of guanidinium chloride (gdmC1) and the contribution of the intersubunit disulfide bond to the structural stability.

2. Materials and methods

2.1. Materials Guanidinium chloride (gdmC1) and twice crystallized acrylamide were purchased from Serva, Germany; 8anilino-l-naphthalenesulfonic acid (ANS) was from Aldrich, USA; p-nitrophenyl phosphate (pNPP) was from Sisco, India; Superose 12 gel-filtration column and the FPLC system were from Pharmacia LKB, Sweden. Water used for the experiments was purified over a Milli-Q water purification system from Millipore, USA. KBPAP was purified from overnight soaked red kidney beans by the procedure described earlier [10].

2.2. Denaturation of protein GdmC1 stock solutions were made in Milli-Q water and the concentration was determined by measuring the refractive index by means of a Schmidt-Haensch-DUR refractometer thermostated at 25°C. The denaturation mix contained 10 mM Tris buffer (pH 7.4), 500 mM NaC1 and various concentrations of gdmC1 with or without 50 mM 13-mercaptoethanol (13ME). The protein was incubated in the denaturing mix for 6 h at 25°C. Data from activity, circular dichroic spectroscopy and tryptophan fluorescence studies are represented as the fraction of denatured form according to the equation: fo

X-X. x~-x.

(1)

77

where X is the measured value at any given denaturant concentration and X n and Xd are values for the native and denatured states of various parameters such as activity, [t91220, tryptophan fluorescence hrnax and F34o//F35o ratios of tryptophan fluorescence emission spectra.

2.3. Measurement of enzymatic activity Enzyme activity was measured by diluting an aliquot of the enzyme denatured at the appropriate concentration of gdmC1 into reaction mix containing 50 mM sodium acetate buffer (pH 5), 500 mM NaC1 and 10 mM pNPP. The reaction was stopped by addition of 2 volumes of 0.5 N NaOH. Absorbance measured at 410 nm were corrected with respective blanks. All the activity measurements were done in the absence of 13ME. For reducing the disulfide, [3ME (50 mM) was present along with gdmC1 during denaturation.

2.4. Circular dichroism CD spectra were recorded on a Jobin-Yvon Autodichrograph Mark V spectropolarimeter. A protein concentration of 0.1 m g / m l in the denaturing mix and path length of 1 mm were used. Baseline corrections were done by subtracting the buffer spectra from the sample spectra. The assumed mean residue molecular weight (116 Da) was calculated from the amino-acid composition of the protein [10].

2.5. Fluorimetry 2.5.1. Tryptophan fluorescence Fluorescence spectra were recorded on a Hitachi F-4010 spectrofluorimeter. Intrinsic tryptophan fluorescence spectra were recorded by setting excitation wavelength at 295 nm and excitation and emission slit widths at 3 nm. Spectra were corrected for variations in wavelength-dependent lamp intensities. Baseline corrections were done with the spectra obtained without the protein. Acrylamide quenching studies were done at each concentration of gdmC1 by addition of small aliquots of acrylamide from a stock solution made in buffer containing the respective concentration of gdmC1. The fluorescence intensities were corrected for increase in volume as required and for inner filter effect of acrylamide according to Eftink and Ghiron [171.

2.5.2. ANS fluorescence Protein was denatured prior to the addition of ANS. ANS spectra were obtained by adding ANS to KBPAP at a final concentration of 20 IxM. Excitation wavelength was set at 350 nm with 10 nm slit width. Emission spectra were recorded between 450 and 570 nm with a slit width of 5 nm. ANS fluorescence spectra in buffer alone was subtracted from the sample spectra.

78

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

2.6. Size-exclusion chromatography SEC studies were performed on Superose 12 column using the FPLC system. Elution characteristics and the reproducibility of the standard proteins was routinely verified with the manufacturer's instructions and the coefficient of variation associated with the elution volumes was less than 5%. The column was equilibrated with three bed volumes of the required buffer containing various concentrations of gdmC1. The protein was denatured by preincubating in corresponding gdmCl at a concentration of 1.2 m g / m l and loaded on to the column. A 280 nm filter was used to detect the peaks. The elution profiles were normalized for total area under the peaks. SEC was also performed on protein denatured in the presence of [3ME. Proportionate areas in each of the peaks were calculated from the elution profiles from a commercial software (Jandel)

2.7. Calculation of Stokes' radii Stokes' radii (R s) were calculated according to Uversky [18] using the equations: For native proteins (dimer: MW = 120 000 Da) log(Rs) = - (0.254 + 0.002) + (0.369 + 0.001)log MW

(2) For proteins denatured in gdmC1 (MW dimer: 120 000 Da and monomer: 60 000 Da) log Rs = - ( 0 . 5 4 3 + 0.004) + (0.502 _ 0.001)logMW

(3)

3. Results

of reducing agent. The overlapping activity profiles (data not presented) indicate that denaturation was not of the 'N 2 ~ 2 N / 2 D ' type and that subunit dissociation did not occur. Earlier we have demonstrated by SDS-PAGE that the protein monomerises only in the presence of reducing agents [2].

3.1.2. Tryptophan fluorescence Tryptophan fluorescence was very sensitive to its surroundings and was used effectively to monitor structural changes in the protein. KBPAP has 13 tryptophans per subunit. Emission wavelength maximum of tryptophan in the native protein was at 340 nm, which shifts to 351 nm in the unfolded protein, indicative of exposure of the tryptophan residues to a relatively polar environment upon denaturation. Relative fluorescence intensity (RFI) at 340 nm in denatured protein was about half that of the native protein. The sigmoid behaviour of both the intensity and the hma x o n denaturation were similar to the activity profile (Fig. 2A and B). However, the reduced protein denatures earlier than the normal protein. The ratio of fluorescence intensities at 340 nm and 350 nm (F34o/F35o) was used to measure the extent of 'nativeness' of the protein sample (Fig. 2B). This parameter was very sensitive to the structural changes and was used to follow the denaturation in KBPAP in gdmCl. A red-shift associated with a decrease in RFI indicative of unfolded protein molecule was further analyzed by studying the collisional quenching of tryptophan fluorescence by an apolar quencher, acrylamide (Fig. 3). The slopes of the SternVolmer plots ( K s v ) at various gdmC1 concentrations indicate the extent of solvent accessibility of tryptophan. When Ksv was plotted against gdmC1 concentration a transition

1.2. 1.0"

3.1. Denaturation of KBPAP in gdmCl 0.8-

3.1.1. Activity The activity remaining after 6 h of incubation in various concentrations of gdmC1 in the presence and absence of 13ME, shows a typical sigmoid profile for denaturation (Fig. 1). The transition was broad (3 M) in the absence of 13ME but much narrower in the presence of [3ME. The enzyme was inactive by 4.5 M gdmCl in the absence and by 2.5 M gdmCl in the presence of 13ME. A marginal but consistent increase (10-15%) in the activity was observed below 2 M gdmCl, shown as negative values in the unfolded fraction. The D1/2, denaturant concentration required for 50% denaturation, has been observed to increase with increasing protein concentrations during denaturation of non-covalently linked dimeric proteins [19]. To verify whether the D~/2 in the case of KBPAP was dependent on protein concentration, we repeated experiments in Fig. 1 over 100-fold range of protein concentration in the absence

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GdmCl fM) Fig. 1. Activity of KBPAP as a function of denaturant concentration: Fraction denatured (fD) was calculated (see Section 2) from activity profiles of KBPAP denaturation in gdmC1. Relative activity was calculated using the activity value in the absence of gdmCl as one. The enzyme (12 b g / m l ) was incubated for 6 h in specified gdmC1 and the activity was determined in the absence of gdmCl and I3ME in the reaction mix. ( O ) Denaturation without ~ME, and ( O ) with I3ME. Mid point of denaturation as estimated by the loss of activity, i.e. D I/ 2, was 3.15 + 0.15 M of gdmC1 (n = 3).

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

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Fig. 2. Changes in protein tryptophan fluorescence as a function of denaturant concentration: The protein (0.12 m g / m l ) was incubated as specified in Fig. 1 and protein fluorescence spectra were recorded in gdmC1 in the presence ( 0 ) and absence ((3) of 13ME. f o was calculated using emission wavelength maximum (~-max) in panel A and ~:atio of fluorescence intensities at 340 nm and 350 nm (F34o/F35 o) in panel B. Coefficient of variation associated with the fluorescence data was 2.4% (n = 4).

between 2.5 M and 4 M gdmC1 was observed, which is similar to the data in Fig. 2. We obtained similar susceptibility of tryptophan fluorescence to a polar quencher, potassium iodide (data not shown). 3.1.3. ANS fluorescence Unfolding entails opening up of a protein and thus exposing occluded nonpolar pockets to bulk water. Such conformational changes result in excess binding of hydrophobic probes to the protein [20]. Binding of ANS to protein is known to be dependent on the surface hydrophobicity of the protein and has been effectively used in solvent denaturation studie:s [20]. The h, max of ANS fluorescence was 490 nm and 477 nm in the absence and presence of 13ME, respectively. Binding of ANS to the native protein has two peaks at 1.5 M and 4 M concentra-

tions of gdmCl (Fig. 4). Marginal increase in the binding of ANS was observed in the presence of 13ME as a function of gdmC1 concentration and the second peak at 4 M was absent in the presence of [3ME. The enhancement in RFI of ANS at 1.5 M gdmC1 was 4.5-fold as compared to a 2-fold enhancement at 4 M gdmC1. Both the position and the fold enhancements in RFI were reproducible. 3.1.4. Circular dichroism Far-UV circular dichroism spectra of KBPAP was used to monitor the changes in the secondary structure of the protein as a function of gdmC1. CD studies were performed only in the absence of [3ME. Data in Fig. 5A shows a loss of secondary structure with increase in the gdmC1 concentration. Molar ellipticity at 220 nm ([O]220),

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GdmCl (M) Fig. 3. Quenching efficiency of tryptophan fluorescence by the nonpolar quencher, acrylamide, during solvent denaturation of KBPAP: SternVolmer quenching constant (Ksv), derived as slope from a detailed acrylamide concentration-dependent quenching of tryptophan fluorescence of KBPAP, was plotted against the gdmC1 concentration• The protein concentration was 0.1 m g / m l . The error associated with this data was identical to that in Fig. 2.

2

4

6

8

OdmC1 ( ~ Fig. 4. Surface hydrophobicity of KBPAP, as probed by ANS, in gdmCh Protein was incubated in specified gdmC1 for 6 h at 25°C and ANS (20 ~M) was added to the mix and the RFI at 490 nm (F49 o) was plotted against the gdmC1 concentration. Open circles indicate denaturation without 13ME and filled circles indicate denaturation in the presence of [3ME. In both cases, the F490 values were normalized by taking the RFI of ANS in the absence of gdmC1 as one. The coefficient of variation associated with this data was < 5%.

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

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Fig. 5. CD spectra of KBPAP during solvent denaturation using gdmCl: Spectra were obtained on protein at 0.1 m g / r n l concentration in a 0.1 cm path length cuvette. The samples were incubated in specified gdmCl for 6 h at 25°C before the measurements were taken. The spectra were corrected for the baseline. (A) CD spectra in the presence of only 0 M (native), 3 M, 4 M, 4.5 M and 6 M gdmC1 were given for clarity. (B) The [O]220 was plotted as the fraction denatured (fD) as a function of the gdmC1 concentration. The coefficient of variation associated with this data was 4.6% (n = 4).

indicative of helical content in the protein, when replotted as a function of gdmC1 decreases sharply above 3 M gdmC1 (Fig. 5B). Transition from the native to the denatured protein, was much sharper with a D~/2 at 3.5 M gdmC1. CD spectrum in 6 M gdmCl indicates presence of residual structure in the protein.

3.1.5. Size-exclusion chromatography SEC, especially with Superose 12, is a simple and versatile method to study the slow exchange intermediates during solvent denaturation of proteins besides obtaining them in the pure form. SEC with Superose 12 is an inert

method and does not influence the equilibrium between various intermediates [18]. Fig. 6 shows elution profiles of KBPAP at various gdmCl concentrations in the absence (Fig. 6A) and presence (Fig. 6B) of reducing agent. The native protein has an elution volume of 10.96_ 0.14 ml (R s = 41.7 A) and the completely unfolded molecule in the presence of reducing agent (6 M gdmC1 + 13ME) - i.e., a denatured monomer - - has 8.57 _ 0.07 ml elution volume (R s = 71.7 A). In the presence of 6 M gdmCl and in the absence of I~ME i.e., a denatured dimer, has an elution volume of 7.34 _ 0.05 ml (R s = 101.6 ,~). Molecular radii were calculated based on the relation given in the

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Fig. 6. Elution profiles of KBPAP on Superose 12 column preequilibrated in buffer containing the corresponding gdmCl concentration in the absence (A) and presence (B) of 50 mM 13ME: The protein was preincubated for 6 h in gdmC1 before injection. The column and instrument were calibrated as per the manufacturer specifications. The errors associated with this data was calculated to be < 2% with respect to elution volumes and < 5% with respect to areas under the peaks (see Section 3.1.5).

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84 120

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Fig. 7. SEC data of KBPAP in various concentrations of gdmCl in the absence of ~3ME. (A) Relative areas of peak l, 2 and 3 (see Fig. 6A) were plotted as a function of gdmCl. The relative areas were calculated using a graphic routine available from Jandel Corporation. (B) Elution volume of peak 2 vs. gdmCl concentration.

methods. Areas under the peaks were calculated using a commercial software. The denatured fraction (peak 2 in Fig. 6A) begins to appear from 2 M gdmC1 onwards (Fig. 7A) and both the denatured and native proteins tend to elute earlier with an increase in the gdmC1 concentration. This suggests a steady increase in the molecular volume of native and denatured protein (Fig. 7B). A fraction of the protein was observed in ~te void volume between 1.5 M and 4.5 M gdmC1 and may represent an aggregated fraction (peak 1 in Fig. 6A artd B) of the protein. Individual fractions were collected ~ d their properties were studied immediately. Only the native peak (peak 3 in Fig. 6A and peak 4 in Fig. 6B) had activity. Decrease in the elution volumes suggest a steady increase in the volume of these fractions. The Fig. 7B shows a denaturant dependent decrease in the native dimer, which was reflected in the activity profiles in gdmC1 (see Fig. 1). At 2 M gdmC1, peak 2 appears at 9.3 ml artd steadily shifts to 7.34 ml in 6 M gdmCl. On plotting the elution volume of peak 2 as a

120.

100

function of the gdmC1 (Fig. 7B), the profile shows a clear break at 4 M, suggesting that the fractions appearing before and after 4 M gdmC1 may be different. In the presence of 13ME, t h e D1/2 of the native fraction (peak 4) decreased from 3 M to 1.75 M gdmC1 (Fig. 8A). Reduction of the intersubunit disulfide resulted in aggregation, which was the major fraction between 1.5 to 3.5 M gdmC1 (peak 1). A third fraction begins to appear from 2 M gdmC1 with elution characteristics typical of the denatured dimer (peak 2). Beyond 3.5 M gdmC1, both the aggregated and native fraction were absent and protein exists predominantly ( > 90%) as a denatured monomer (peak 3) with less than 10% protein as denatured dimer. Both these peaks were inactive. By 6 M gdmC1 only the denatured monomer fraction was present. Elution volumes of the denatured dimer and denatured monomer decrease with increasing gdmC1 concentrations indicating that these fractions continuously increase in molecular size with increasing denaturant concentration (Fig. 8B).

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Fig. 8. SEC data of KBPAP in various concentrations of gdmC1 in the presence of 50 mM 13ME. (A) Relative areas of peaks 1, 2, 3 and 4 (see Fig. 6B) plotted as a function of gdmCl. (I3) Elution volumes of peaks 2 and 3 as a function of gdmCl concentration.

82

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

f

4. D i s c u s s i o n

We have monitored KBPAP denaturation by functional, spectroscopic and gel permeation methods as a model to understand the unfolding pathway of disulfide linked dimeric proteins. Denaturation of KBPAP in gdmC1 was a reversible process, indicated by the complete recovery of the protein tryptophan fluorescence upon dilution of the denaturant. SEC, SDS-PAGE and also independence of D1/a on protein concentration demonstrate that monomers do not form on denaturation of native KBPAP. In the presence of 13ME, the D~/2 of activity, h . . . . RFI of tryptophan, and native protein fraction versus gdmCl concentration profiles shifted to lower concentration of gdmC1 indicating that the protein was more susceptible to denaturation when the disulfide was reduced. SEC of reduced protein indicated aggregation was the predominant product of denaturation ( > 1 M gdmC1). Stabilizing properties of disulfide bonds, especially intrachain, were elegantly shown in the case of RNase TI [21]. Disulfides were proposed to increase the stability of the protein by virtue of their ability to decrease the degree of freedom (decreasing the entropic contribution), of the chain fold. Compared to the native dimer (R S = 41.7 A) the denatured monomer and denatured dimer were larger by 1.8- and 2.5-times, respectively. The denatured dimer was less than the twice the size of the denatured monomer, which suggests there may be some secondary structure remaining. The CD spectrum at 6 M gdmC1 shows presence of residual secondary structure in the denatured dimer. Using activity or tryptophan RFI we obtained a AG(H20) of 5.2 kcal/mol in 0.5 M NaC1 and at pH of 7.4, which was at the lower end of the spectrum of reported values for a dimeric protein [21]. The stabilization energy (AG(H20)) decreases to a value of 3.8 kcal/mol in the presence of 13ME suggesting that the contribution of the intersubunit disulfide to the stability of KBPAP was 1.4 kcal/mol. This value is less than the reported value of 3.4 kcal/mol for an intrachain disulfide [21]. From the crystal structure it is apparent that the monomers interact to a limited extent involving the a5 helix and a stretch of amino-acid residues from 253-260 [12]. The intersubunit disulfide link probably plays an essential role of keeping the monomers together thus maintaining the structural integrity of the protein. The data obtained on the denaturation of KBPAP by various parameters suggests a multistage unfolding pathway, populated with at least three intermediates (Fig. 9). Between 0-1.5 M gdmC1 KBPAP has an interesting property of enhanced ANS binding, indicative of exposed hydrophobic surfaces. No significant change occurs in any of the other structural parameters. The exposure of hydrophobic surfaces may have resulted in aggregation to some extent ( < 10% of the total protein), resulting in appearance of a peak outside the resolving limit of the column (i.e., 300 kDa) (see Fig. 6A and Fig. 7). This aggregation may be unstable since these aggregates disap-

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GdmC1 (M) Fig. 9. Minimal denaturation pathway of KBPAP: 'N' is the native protein. 'I t ' is the first intermediate which shows increased hydrophobic surfaces, but shows no loss in enzymatic activity, tertiary or secondary structures and denaturation may be limited to the N-terminal domain. 'I 2' is the second intermediate with partial loss in secondary and tertiary structure and may be similar to a 'partially folded state' [25]. 'A' is the weakly aggregated intermediate. Do is the denatured dimer, rDo is the denatured dimer with the intersubunit disulfide reduced. Mo is the denatured monomerobserved only during denaturation in the presence of [3ME. Do could be convertedto Mo by addinga disulfide reductant such as [3ME[10]. pear upon increasing the gdmC1 concentration in the medium. Loss in enzymatic activity is generally the first noticeable change upon denaturation of enzymes and followed by changes in other structural parameters [22,23]. KBPAP may be a unique example wherein the changes in some other parameter - - i.e., ANS binding - - were observed earlier than loss in activity upon denaturation. Similarity studies with the mammalian acid phosphatase homologues and crystal structure of KBPAP, suggest that the N-terminal domain is not necessary for the activity and does not have any intersubunit interactions [13]. Structural changes observed in gdmCl upto 1.5 M gdmC1 may be limited to the N-terminal region where structural rearrangements led to an exposure of hydrophobic sites. Despite the enhanced ANS binding, which is one of the specific criteria for identifying a molten globule intermediate [24], we could not observe any change in hma x of tryptophan fluorescence. Exposure of hydrophobic sites leading to limited nonspecific aggregation, in the absence of attendant secondary structural and molecular volume changes, suggests that partial unfolding of domains may be the first step, leading to an intermediate (I l) in the pathway of unfolding. In the presence of [3ME the activity was reduced by 10% and a marginal increase in ANS binding (rll). On further denaturation - - i.e., in 1.5-3.5 M gdmCl range - - the partially opened dimer shows a steady change in all the parameters such as exposure of tryptophan residues to polar environment and partial loss of secondary structure. This range of gdmCl concentration was characterized by the marginal decrease in activity, significant loss in ANS binding and also disappearance of the aggregated fraction. The data in Fig. 7B suggests that the fraction appearing in the peak 2 upto 3.5 M gdmC1 may be different from the completely denatured dimer. The most

A.G. Cashikar, N.M. Rao / Biochimica et Biophysica Acta 1296 (1996) 76-84

significant change being ne,arly half of the protein appears as a denatured dimer. Tei'fiary and quaternary structural rearrangements with a partial loss in secondary structure, could be ascribed to an increase in the molecular volume resulting in dissolution of ANS binding sites and disaggregation (12). This state may be compared with the 'partially folded state' described by Uversky and Ptitsyn [25]. Reduction of the intersubunit disulfide bond led to extensive aggregation of the protein and loss of activity. Aggregation was the first consequence of denaturation in the presence of reducing agent (rA). Elution of a fraction of the protein as a denatured dimer in this denaturant range suggests that following the aggregation the protein tends to disaggregate but still exist as dimers (rl)D). The elution volume of this peak suggests a steady increase in the molecular volume of the denatured dimer. Presence of denatured dimer upon breaking the disulfide bond suggests intersubunit interactions other than disulfide, :for example interaction between et 5 helices of the subunits, were sufficient to retain the dimer structure. Structural consequences of denaturation of native protein in 3.5 to 5 M gdmC1 region were aggregation and appearance of new ANS binding sites (A). Absence of aggregates abow; 5 M gdmC1 suggests that the aggregation was weak and may exist in equilibrium with both I: and the unfolded diimer (DD). We could not detect any change in the turbidity of identically treated samples, which suggests that the aggregation may be self limiting and probably weak. Near complete loss of secondary structure may have resulted in exposure of hydrophobic pockets leading to weak aggregates, which were in equilibrium with the denatured dimers. Though the aggregation involved more than half of the protein, the limited increase in RFI of ANS suggests that hydrophobic pockets exposed at this stage of denaturation were different from the ANS binding sites observed at 1.5 M gdmC1. A distinctive decrease in the concentration of protein appearing in peak 2 after 3 M of gdmC1 and subsequent increase after 4 M gdmC1 suggests that the fraction appearing in peak 2 before and after 4 M gdrnCl may be different. This was also supported by the observed break at 4 M gdmC1 in the elution volume of peak 2 (Fig. 7B). In the presence of reducing agent the protein completely disaggregates. Interactions other than the disulfide bond, such as ionic and hydrophobic interactions, iholding the subunits weaken and the dimer begins to monomerise. Beyond 5 M gdmC1 only denatured monomers (M D) in the presence of 13ME and only denatured dimers in the absence of 13ME were observed. Observed multistate denaturation pathway of KBPAP suggests that the two domains in a subunit unfold independently resulting in at leas~Lthree intermediates. Due to the low energy barriers, these intermediates could be in equilibrium, as suggested by the overlapping elution profiles. Further, denaturation both at tertiary/quaternary and at secondary structural level yielded different intermediates, which have similar prolx;rties of enhanced ANS binding

83

and nonspecific aggregation. Experiments performed in the presence of a reducing agent clearly demonstrate that the protein becomes less stable and aggregation was the primary product of denaturation. To keep the subunits intact interactions other than the disulfide may be involved, such as between the ot 5 helices. Equilibrium denaturation of monomeric globular proteins demonstrated that a simplistic cooperative two-state transition is an exception [26]. Non-coincidence of denaturation profiles observed by different techniques proves the presence of equilibrium intermediates populating the unfolding pathway. Generally, unfolding studies performed on oligomeric proteins indicate that their denaturation was either two-state or three-state transition depending on the extent of stabilization of tertiary structure by the quaternary structure - - i.e., whether the structure of the subunit was stabilized by intersubunit interactions [2]. Oligomeric proteins on which most of the unfolding studies were done either do not have any intersubunit disulfide bonds or the experiments were done under reducing conditions. Denaturation studies on KBPAP demonstrates that an intact disulfide alters the unfolding pathway significantly. Since the disulfides restrict the freedom of various domains in the intersubunit region of the protein, the unfolding resulted in at least three intermediates. Further, the unfolding was not complete as judged by circular dichroic spectrum of the protein at highest gdmC1. Presence of residual structure even in 6 M gdmC1 and non-cooperative transitions may be responsible for the low AG(H20) obtained for this protein. Contribution of the intersubunit disulfide in the stabilization of the native structure was only to an extent of about 25% of the overall stability of the protein. The stability of KBPAP in the presence of 10 mM phosphate, an inhibitor of KBPAP increases by 3.8 k c a l / m o l [16]. Phosphate-bound KBPAP was less prone to the destabilizing effects of [3ME [16]. Mammalian purple acid phosphatases are monomeric and have similar active site and metal ligating amino acids as dimeric KBPAP [13]. Dimeric enzymes may have an advantage of regulation of catalytic activity through the cooperativity of the subunits. However, no cooperative type of kinetics were reported with KBPAP with any substrates/ligands. The dimeric nature of KBPAP may serve a yet unknown function.

Acknowledgements G.A.C. was a recipient of the Senior Research Fellowship from the Council for Scientific and Industrial Research (CSIR), Government of India.

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