Anion-Induced Folding of Rabbit Muscle Pyruvate Kinase: Existence of Multiple Intermediate Conformations at Low pH

Archives of Biochemistry and Biophysics Vol. 381, No. 1, September 1, pp. 99 –110, 2000 doi:10.1006/abbi.2000.1968, available online at http://www.idealibrary.com on

Anion-Induced Folding of Rabbit Muscle Pyruvate Kinase: Existence of Multiple Intermediate Conformations at Low pH F. Edwin* ,1 and M. V. Jagannadham* *Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India

Received March 13, 2000, and in revised form June 14, 2000

Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using enzymatic activity measurements and a combination of optical methods, such as circular dichroism, fluorescence, and ANS binding. At low pH and low ionic strength, pyruvate kinase exists in a partially unfolded state (U A state) retaining half of the secondary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na 2SO 4, to the acidunfolded state induces refolding, resulting structural propensities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and decrease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to the intermolecular association. The anion-refolded state is more stable than the U A state, and its stability is nearly equal to that of native protein toward chemical-induced unfolding by Gu–HCl and urea. Moreover, at low concentrations, Gu–HCl behaves like an anion, by inducing refolding of the acid-unfolded state with structural features equivalent to that of native molecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability. © 2000 Academic Press Key Words: pyruvate kinase; acid-unfolded state; molten globule; circular dichroism; ANS binding; intrinsic fluorescence; salt-induced folding; Gu–HCl unfolding.

1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Graduate Research Center, University of Massachusetts, Amherst, MA 01003. Fax: (413) 5453291. E-mail: [email protected].

0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Most folding studies focus on the monomeric proteins as model systems and have established the existence of transient and equilibrium intermediates in the folding pathway (1– 6). However, very few proteins have been found to show very cooperative folding behavior (see for review [7]). Substantial evidence has accumulated to support a model of protein folding where partially folded conformations are the key intermediates in the formation of most aggregates (see for review [8]). The role of quaternary interactions and the structures of transient species in the folding pathways of oligomeric proteins is not well understood. Renaturation of oligomeric proteins is generally a complex process that involves a number essentially uncoupled folding and association steps, some of them in competition with nonproductive aggregation (9 –12). The propensity for association or aggregation is a well-known general characteristic of non-native conformations of proteins (1, 8, 13). However, in the case of several dimers folding and association are coupled and conform to two-state transitions, with only denatured monomers and folded dimeric proteins significantly populated at equilibrium (14). Proteins, such as tetrameric tumor suppressor p53, fold through a transient dimeric intermediate and through a dimeric transition state (15). Proteins can be unfolded by a decrease in pH due to Coulombic repulsion from the net positive charges of the polypeptide chain (16). This pH effect can be offset by the presence of anions, which bind to the positively charged sites and screen them, leading to the formation of partially folded intermediates known as A states (17–20). Efficiency of such screening by anions depends on the net positive charges of the acid-unfolded polypeptide chain and in the amount of structure as well as the compactness of the partially folded protein (20, 21). Pyruvate kinase (ATP-pyruvate 2-O-phosphotransferase) is a glycolytic enzyme that plays an important role in regulating the flux from fructose-1,6-biphos99

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phate to pyruvate. In general, the enzyme exists as tetramer of identical subunits (molecular mass of each subunit about 53 kDa) and displays a cooperative binding to phosphoenolpyruvate (PEP). Presence of both monovalent (K ⫹) and divalent (Mg 2⫹) cations is essential for its activity. PK 2 was the first enzyme for which dependence of activity on monovalent cation was documented, though the monovalent cation requirement can be eliminated by substitution of lysine for glutamate 117 (22). The crystal structure of rabbit muscle PK, complexed with monovalent and divalent ions and with various substrates, was solved recently by Larsen et al. (23, 24). The subunit structure of rabbit muscle PK during urea denaturation has been examined by Steinmetz and Deal (25), and various intermediates in the unfolding pathway have been detected. These workers deduced from the sedimentation measurements that the native tetramer dissociates, with increasing urea concentration, into compact and active dimers, compact but inactive monomers, and disordered monomers. Dissociation and deactivation of this enzyme by urea and guanidine hydrochloride (Gu– HCl) were shown to be overall reversible (26). But, Doster and Hess (27) reported a different pathway of rabbit muscle PK unfolding by urea and Gu–HCl. In this investigation, the first intermediate, with increasing concentration of denaturant, is a less compact and inactive tetramer, which can be renatured if substrates are added. Dissociation of the inactive tetramer, upon further increase in the concentration of denaturant, results in an expanded dimer with a partial loss of secondary structure. The final state is a completely disordered monomer. In view of the different models of unfolding as described above, the unfolding of PK at acidic conditions and the effect of anions are investigated to better understand the intermediate conformations and their role in the folding of PK. Studies in this direction will give insight into the presence of partially folded intermediates and their role in the unfolding of rabbit muscle PK in light of its subunit and domain structure. We present here a detailed investigation of the presence of partially folded intermediates of rabbit muscle PK, under acidic conditions and in the equilibrium unfolding pathway. These equilibrium intermediates are characterized by various physicochemical methods. Further, it is established that rabbit muscle pyruvate kinase exists in a molten globule-like conformation at acidic pH and low ionic strength while salt induces association of this partially folded state to “near-native-like” state with 2 Abbreviations used: Gu–HCl, guanidine hydrochloride; PK, rabbit muscle pyruvate kinase; ANS, 8-anilino-1-naphthalenesulfonic acid; CD, circular dichroism; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; U A, acid-unfolded; I A, anion-refolded (anioninduced); PDI, protein disulfide isomerase.

all the structural characteristic of native protein. Anions not only induce the structure but also stabilize the acidunfolded state against denaturants. Studies on the structure, characteristics, and unfolding behavior of these intermediate states have been carried out. The significance of these results in the unfolding of rabbit muscle PK is discussed in view of its subunit and domain structure. EXPERIMENTAL PROCEDURES Materials. Rabbit muscle pyruvate kinase (PK; EC 2.7.140) and lactate dehydrogenase (LDH) were obtained from Boehringer Mannheim GmbH (Mannheim, Germany), and the homogeneity was checked by SDS–polyacrylamide gel electrophoresis (28) and gel filtration (29). The PK concentration was determined spectrophotometrically using the extinction coefficient of ␧ 11%cm ⫽ 5.1 at 280 nm (30). 8-Anilino-1-naphthalenesulfonic acid (ANS) was purchased from Sigma-Aldrich (St. Louis, MO). Guanidine hydrochloride (Gu– HCl) was obtained from Serva Fine Biochemica GmbH (Germany). Ultrapure urea was purchased from Sigma Chemicals (St. Louis, MO). Concentrations of Gu–HCl and urea in solutions were determined from refractive index measurements (31). All other reagents used were of analytical grade and highest quality available commercially. All measurements were made at room temperature (23°C) unless mentioned otherwise. Absorbance, fluorescence, and circular dichroism. Absorbance measurements were carried out on a Beckman DU-640B spectrophotometer equipped with a constant temperature cell holder. Protein concentration for all absorbance measurements was between 4 and 10 ␮M. Fluorescence measurements were carried out on Perkin– Elmer LS-5B and LS-50B spectrofluorimeters equipped with a constant temperature cell holder. Protein concentration was between 1 and 6 ␮M. For tryptophan fluorescence of the protein, excitation was at 292 nm and emission was recorded from 300 to 400 nm with 10and 5-nm slit widths for excitation and emission, respectively. Circular dichroism (CD) measurements were done on a JASCO 500A spectropolarimeter equipped with a 500N data processor. The instrument was calibrated with a 0.1% d-10-camphorsulfonic acid solution (32). Conformational changes in the secondary structure of the protein were monitored between 200 and 260 nm with a protein concentration of 0.1 mg/ml in a 1-mm path length cuvette. Changes in the tertiary structure were measured with a 10 mm path length cuvette in the region of 260 to 320 nm with a protein concentration of 1 mg/ml. Respective baseline spectra were subtracted from each spectra. Each spectrum represents the average of three scans. The results are expressed as the mean residue molar ellipticity [⌰] (deg 䡠 cm 2 䡠 dmol ⫺1), which is defined as [⌰] ⫽ ⌰ obs ⫻ MRW/(10lc), where ⌰ obs is the observed ellipticity in degrees, c is the concentration in g/ml, and l is the length of light path in centimeters. A mean residue weight (MRW) of 112 is used. Samples for all spectroscopic measurements were filtered through 0.45-␮m membrane filters, and the exact concentration of the protein was determined by absorbance. All spectra were recorded at 23°C unless otherwise mentioned. ANS binding. A stock solution of ANS was prepared in methanol, and the concentration of ANS was determined using an extinction coefficient of ␧ ⫽ 5000 M ⫺1 cm ⫺1 at 350 nm (33). After ANS stock was added to the protein solutions, samples were kept in dark and the measurements were made within an hour. For ANS fluorescence, excitation was at 380 nm and the emission spectra were scanned between 400 and 600 nm with slit widths of 10 and 5 nm for excitation and emission, respectively. The molar ratio of protein and ANS was approximately 1:100 in all experiments. Quenching of Trp fluorescence. Quenching of Trp fluorescence in PK under acidic conditions by acrylamide was measured to estimate the exposure of the buried aromatic amino acids. Increasing amounts

ANION-INDUCED FOLDING OF PYRUVATE KINASE

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FIG. 1. pH-dependent conformational changes in rabbit muscle pyruvate kinase as monitored by (a) ellipticity at 222 nm, (b) fluorescence intensity, (c) ANS intensity, and (d) by enzymatic activity. All measurements were carried out at 23°C. Protein concentration was between 0.05 and 0.1 mg/ml. Solutions were preincubated for 2 h prior to measurements.

of freshly prepared acrylamide solution were added to the samples of PK under different conditions. The solutions were incubated at 23°C in dark for 30 min before measurements. The Trp fluorescence quenchable fractions were calculated from the Stern–Volmer plot using the equation F 0 /F ⫽ 1 ⫹ K SV关Q兴,

[1]

where F and F 0 are the maximum fluorescence intensities in the presence and absence of quencher, respectively. K SV is the Stern– Volmer quenching constant, and [Q] is the quencher concentration. Acid denaturation of PK. Acid denaturation of PK was carried out in a wide range of pH using the following buffers: KCl–HCl (pH 1.0 –1.5), Gly–HCl (pH 2.0 –3.5), acetate (pH 3.5–5.0), citrate–phosphate (pH 5.5–7.5), and Tris–HCl (pH 7.5–9.0). Concentrations of all buffers were 50 mM. A stock solution of protein was added to the appropriate buffer, and the samples were incubated for 30 min at 23°C. Final pH and concentration of protein are measured before spectroscopic measurements. Chemical-induced and thermal unfolding of PK. In the case of unfolding by Gu–HCl or urea, protein samples were incubated at the given concentration of denaturant for 24 h at room temperature. Final concentration of the denaturant was measured by refractive index before measurement. For thermal unfolding studies, protein samples were incubated at the desired temperature for 15 min and different spectroscopic parameters were measured. Actual temperature of the solution inside the cuvette was measured with a thermocouple connected to a digital multimeter. Enzymatic activity of PK. The enzyme activity was determined by a coupled assay procedure with lactate dehydrogenase (27). Standard assays were run in the following medium: 10 mM ADP, 100 mM KCl, 20 mM MgSO 4, 10 mM phosphoenolpyruvate, 0.3 mM NADH, and 0.05 mg/ml LDH in 50 mM Tris–HCl buffer at pH 7.6, 30°C. The reaction was initiated by the addition of 10 ␮l of pyruvate kinase to the cuvette containing 1 ml of the above medium and was monitored by the decrease in absorbance at 340 nm for 5 min. The rate of reaction was measured from the initial linear region of the curve.

Results are presented as percentage of activity with activity at pH 7.0 considered to be 100%.

RESULTS

pH-Induced Conformational Changes in Pyruvate Kinase The effect of pH on various structural parameters of PK is studied by far- and near-UV CD spectra, intrinsic fluorescence, ANS fluorescence, and enzymatic activity measurements. Figure 1 shows the changes in different parameters as a function of pH. PK retains typical structural characteristics of native protein and activity in the pH range from 6.0 to 8.0. Upon acidification, the enzyme slowly loses its structural integrity and activity, as seen from changes in intrinsic fluorescence, secondary structure, and activity measurements. Interestingly, the loss of enzymatic activity and secondary structure is non-coincidental, suggesting the presence of a partially folded intermediate with substantial secondary structure. As seen from Fig. 1a and b, a decrease in pH below 2.0 induces some secondary structure along with an increase in the fluorescence intensity, probably due to the effect of increased concentration of Cl ⫺ ions in the HCl at low pH. During the acid denaturation of PK the extent of ANS binding upon unfolding was also monitored to determine whether any hydrophobic groups were exposed to solvent as is seen in the case of other proteins upon acidification. Pyruvate kinase does not have any interaction with ANS above pH 5.5 but starts binding ANS at lower pH with a maximum binding around pH 2.0

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that a large conformational change occurs in PK upon acidification. Solvent Accessibility of Tryptophan in the U A State Intrinsic fluorescence maximum (␭ max) is an excellent parameter to monitor the polarity of Trp environment in the protein, and is sensitive to the protein conformation (34). The solvent accessibility of tryptophan residues in the acid-unfolded state (U A state) was assessed using fluorescence emission as a measure (Fig. 3a). In unfolded state (in 6 M Gu–HCl), where all the tryptophan residues are exposed to the solvent, PK shows a fluorescence emission maximum at 356 nm while it is 338 nm in native state. In the U A state emission maximum is in the vicinity of 345 nm with the fluorescence intensity between that of native and unfolded states suggesting that the Trp environment in the U A state is different from the unfolded or native protein. It is well known that ANS binds only to the solvent-exposed hydrophobic clusters that are buried inside the protein in the native state (35). Upon ANS binding to the U A state, the emission maximum of ANS is shifted by 30 nm from 515 nm to 485 nm while in the case of native and denatured states the maximum is around 515 nm (Fig. 3b). The exposure of protein inteFIG. 2. Tertiary and secondary structural conformations of PK under different conditions. (a) Near- and (b) far-UV CD spectra of native (—), acid-unfolded (– – –), and completely unfolded (䡠 䡠 䡠) PK at 23°C. Protein concentration used for near-UV CD was 1 mg/ml with 10 mm cell pathlength, while for far-UV CD the protein concentration was 0.1 mg/ml and the path length was 1 mm.

along with a shift of 30 nm in the emission maximum. An increase in far-UV CD and a decrease in ANS binding below pH 2.0 also suggest the induction of ordered structure at lower pH. Characterization of Acid-Induced Unfolded State (U A State) PK retains some amount of secondary structure with a stable hydrophobic core at low pH as seen from far-UV CD and ANS binding. This state was further characterized and compared with the native molecule at neutral conditions. Figure 2a and b show the near-UV and far-UV CD spectra of PK at pH 2.0, pH 7.0, and in the unfolded state in 6 M Gu–HCl. At acidic pH, the protein loses all the tertiary interactions and the spectra are similar to those of completely unfolded protein. The protein concentration used for near-UV CD was 1 mg/ml; however, a higher protein concentration did not have any effect on the spectra (data not shown). Whereas protein retains around 50% of the secondary structure at acidic pH, the spectrum loses the typical negative peaks. These observations suggest

FIG. 3. Fluorescence properties of PK. (a) Intrinsic fluorescence and (b) ANS fluorescence spectra of PK at pH 7.0 (—), pH 2.0 (– – –), and at 6 M Gu–HCl (䡠 䡠 䡠). Protein concentration was 0.05 mg/ml. For ANS fluorescence, ANS and protein used were in 100:1 ratio.

ANION-INDUCED FOLDING OF PYRUVATE KINASE

FIG. 4. Quenching of PK Trp fluorescence by acrylamide. Stern– Volmer plot of the quenching of tryptophan fluorescence of PK at pH 7.0 (■) and pH 2.0 (F). The intercept, on the y axis, of the linear fit represents the accessible fraction of tryptophan fluorescence quenched by acrylamide.

rior was seen further by acrylamide quenching of Trp fluorescence. Figure 4 shows the quenching of PK Trp fluorescence in the acid-unfolded state (filled squares) and in the native state (filled circles). The data can well fit Eq. [1] and show a linear relationship with the quencher concentration. The Stern–Volmer plot indicates that aromatic amino acids of PK in the acidunfolded state are more exposed to the solvent than the native molecule, and consequently the fluorescence was quenched more in the U A state.

103

and low ionic strength, pyruvate kinase exists in a partially unfolded state, while it exists in a partially folded conformation in presence of anions with increased amount of secondary structure. The secondary and tertiary structural propensities of different conformations of PK are presented in Table I. Similarly, the effect of anions on PK in the acidunfolded state was also studied by intrinsic fluorescence (Fig. 5c and d) and ANS fluorescence (Fig. 5e and f). Increasing concentrations of anion increases the intrinsic fluorescence along with a shift in emission maximum toward lower wavelength, suggesting refolding of the molecule (Fig. 5c). The molecule in native and U A states have emission maxima at 338 and 345 nm, respectively, while that of completely denatured protein is at 356 nm. Increasing the concentration of anions not only restores the fluorescence intensity but also shifts the emission maximum toward the native value (Fig. 5c), whereas addition of anions does not affect the fluorescence properties of native PK (filled circles, Fig. 5d). The effect of anions on the extent of ANS binding to the U A state was monitored by ANS fluorescence. In the U A state PK binds to ANS very strongly and the fluorescence intensity increases sev-

Effect of Anions on Acid-Unfolded State Induction of secondary structure. The effect of monovalent cations, like KCl and NaCl, on the conformation of PK in the U A state was studied by near- and far-UV CD, intrinsic fluorescence, and ANS binding. There was a significant increase in the secondary structure of the molecule in U A state at a higher ionic strength. Figure 5a shows the far-UV CD spectra of pyruvate kinase under different conditions. At pH 2.0 the protein loses around 50% of the secondary structure with the absence of typical characteristic peaks. Increasing anion concentration leads to an increase in the negative molar ellipticity at 222 nm in the U A state, indicating the induction of secondary structure (Fig. 5a and b). Such an increase in the secondary structure with the addition of anions reaches a maximum around 0.7 M KCl, and a further addition of salt leads to sudden collapse of the structure resulting a drop in secondary structure (Fig. 5b), whereas such an effect of anion was not seen in PK under neutral conditions (Fig. 5b, filled circles). Interestingly, the increase in anion concentration, leading to the formation of additional secondary structure, does not restore tertiary structure, as seen by the absence of signal in the near-UV CD spectra (data not shown). Thus, at low pH

FIG. 5. Anion-induced folding of PK at pH 2.0 monitored by (a, b) far-UV CD, (c, d) fluorescence intensity, and (e, f) ANS intensity. Open and filled circles represent PK at pH 2.0 and 7.0, respectively. The lines represent PK in N state at pH 7.0 (—); U A state at pH 2.0 (– – –); I A state at pH 2.0, 0.25 M (– 䡠 –); and in completely unfolded state in 6 M Gu–HCl (䡠 䡠 䡠). For CD measurements the protein concentration was 0.1 mg/ml with 1 mm cell pathlength while for fluorescence measurements protein concentration was 0.05 mg/ml. All measurements were made at 23°C.

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EDWIN AND JAGANNADHAM TABLE I

Structural Propensities of Pyruvate Kinase Intermediates by CD State

Conditions

Native state (N) Acid-unfolded state (U A) Anion-refolded state (I A) Gu–HCl-refolded state (I A) Gu–HCl-unfolded state (U)

pH pH pH pH pH

7.0, 2.0, 2.0, 2.0, 7.0,

23°C 23°C 23°C, 0.25–0.7 M KCl 23°C, 0.4–1.0 M Gu–HCl 23°C, 6 M Gu–HCl

% tertiary a

[␪] 222

% secondary b

100 0 0 0 0

⫺9266 ⫺4633 ⫺8927 c ⫺7546 d ⫺1214

100 50.5 96.3 81.4 0

a

Ellipticity at 278 nm in the native state is considered as 100%. Ellipticity at 222 nm in the native state is considered as 100%. c Value at 0.5 M KCl is shown. d Value at 1.0 M Gu–HCl is shown. b

eral fold along with a shift in the emission maximum (Fig. 5e). If the presence of anion induces any secondary structure to the acid-unfolded state as seen by CD, it must lead to the burial of some exposed hydrophobic groups into the molecule. As expected, the ANS fluorescence decreases in a linear fashion with increase in anion concentration and attains a plateau around 0.3 M KCl (Fig. 5e and f). However, an increase in anion concentration beyond 0.7 M leads to a sudden increase in ANS fluorescence (data not shown) along with a decrease in secondary structure and fluorescence properties. Thus, PK under acidic conditions in the absence (U A state) and presence (I A state) of salt exists in partially unfolded states with characteristics of a molten globule state, with the latter being a more ordered one. The fluorescence properties of PK in different states are summarized in Table II. From the observations above it can be concluded that around 0.25 M KCl the acid-unfolded PK attains the native-like properties, and in all studies reported below 0.25 M KCl was used. Gu–HCl-induced unfolding of U A and I A states of PK. If the conformation and stability of U A and I A states are different, it must reflect in the chemical-induced unfolding. Figure 6 shows the Gu–HCl-induced unfolding of PK as monitored by ellipticity in amide region, fluorescence intensity, and emission maximum. An increase in the secondary structure is seen with Gu–HCl

up to 1 M, while further increases in denaturant concentration lead to unfolding of the molecule (Fig. 6a and b), indicating that the protein in the U A state unfolds through some intermediate state. It appears that, at lower concentrations, Gu–HCl has a similar effect as anions, and the amount of secondary structure at 1 M Gu–HCl is almost equal to that as seen in the presence of added salt (Fig. 6a). Moreover, the unfolding transition of the U A state beyond 1 M Gu–HCl is not very cooperative, suggesting the presence of intermediates, while the anion-refolded state (I A state) (0.25 M KCl in all the unfolding studies at pH 2.0) also unfolds non-cooperatively, with a higher transition midpoint compared to that of native protein under neutral conditions. In the I A state, the secondary structure decreases marginally up to 3.0 M Gu–HCl and thereafter a sudden decrease in the ellipticity with increasing concentration of the denaturant is seen. At the same time, chemical-induced unfolding of the native protein was non-cooperative with less stability than the I A state and the overall transition was complete at lower denaturant concentration (Fig. 6b, open triangle). A similar phenomenon was seen with fluorescence emission maximum and intensity upon Gu–HCl-induced unfolding. The molecule in the U A state refolds at lower concentrations of denaturant as seen by the

TABLE II

Fluorescence Properties of Various PK Intermediates State Native state (N) Acid-unfolded state (U A) Anion-refolded state (I A) Gu–HCl-refolded state (I A) Gu–HCl-unfolded state (U) a b

Conditions pH pH pH pH pH

7.0, 2.0, 2.0, 2.0, 7.0,

Fluorescence of native state as 100%. ANS binding to U A state as 100%.

23°C 23°C 23°C, 0.25–0.7 M KCl 23°C, 1.0 M Gu–HCl 23°C, 6 M Gu–HCl

% Native

␭ max, nm

ANS binding

ANS ␭ max, nm

100 a 46.8 97 98 73.7

338 ⫾ 1 345 ⫾ 1 339 ⫾ 1 341 ⫾ 1 356 ⫾ 1

5 100 b 20 25 5

515 ⫾ 2 485 ⫾ 1 495 ⫾ 1 490 ⫾ 1 515 ⫾ 1

ANION-INDUCED FOLDING OF PYRUVATE KINASE

105

enzymatic activity at 50°C (Fig. 7c). These observations indicate that the acid-unfolded state is less stable while the stability of anion-refolded state (IA state) is almost similar to that of native molecule. As it is seen, Gu–HCl stabilizes the U A state at lower concentrations, and such an effect is attributed to the ionic character of the denaturant. In general, the effect of denaturation of proteins by Gu–HCl and urea is different. In view of the ionic effect attributed to Gu– HCl, it has to be seen whether urea, a nonionic denaturant, behaves similarly or differently. Thus, it will give insight into whether the denaturant-induced stabilization of the U A state is due to denaturant or the inherent character of the protein in the U A state. Ureainduced unfolding of PK in different states was carried out and compared to the native molecule. Figure 8 shows the urea-induced unfolding of PK in different

FIG. 6. Gu–HCl-induced unfolding of PK in U A state at pH 2.0 (E), I A state at pH 2.0, 0.25 M KCl (F), and N state at pH 7.0, 23°C (‚). Unfolding transition monitored by (a, b) far-UV CD, (c, d) fluorescence intensity, and (e) by emission maximum. All other conditions were the same as those described for Fig. 5.

reversal of ␭ max from 345 to 340 nm, which is nearly equivalent to the emission maximum of native molecule (Fig. 6c, d, and e). A further increase in Gu–HCl concentration leads to the unfolding of the molecule. However, the unfolding was non-cooperative beyond 1 M Gu–HCl, suggesting the presence of at least one partially folded intermediate in the unfolding pathway. Whereas the I A state unfolds at higher concentration of denaturant with a gradual loss of structure initially up to 3 M Gu–HCl and proceeded by unfolding at higher concentration of the denaturant, the unfolding of PK under neutral conditions is complete at lower concentrations of denaturant and the presence of any salt does not affect the transition (open triangles, Fig. 6e). The decrease in enzymatic activity of PK as a function of Gu–HCl concentration at neutral pH occurs in a monophasic nature and the transitions is complete at 1.5 M Gu–HCl (Fig. 7a). Table III lists the transition midpoints of PK under various conditions. As in the case Gu–HCl-induced unfolding of PK, the stability of U A and I A states must be different toward temperature. Native PK unfolds with a transition midpoint of 54.9°C while the molecule in the UA state unfolds at much lower temperature with a transition midpoint of 37.3°C (Fig. 7b). Surprisingly, PK in the IA state unfolds with a transition midpoint of 53.8°C almost similar to that of native state. The native molecule loses complete

FIG. 7. (a) Gu–HCl-induced unfolding transition of PK at pH 7.0 was monitored by enzymatic activity. (b) Thermal unfolding of PK in the I A state (F), U A state (E), and in native state (Œ) was monitored by far-UV ellipticity at 222 nm. (c) Enzymatic activity of PK at pH 7.0 was measured as a function of temperature. All other conditions were the same as described in Experimental Procedures.

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EDWIN AND JAGANNADHAM TABLE III

Unfolding Transition Midpoints of Pyruvate Kinase State Native state (N)

Conditions pH 7.0, 23°C

Denaturant

Method

Cm

Completion

Gu–HCl

CD [␪] 222 Fluo. Activity CD [␪] 222 Fluo. CD [␪] 222 Activity CD [␪] 222 Fluo. CD [␪] 222 Fluo. CD [␪] 222 CD [␪] 222 Fluo. CD [␪] 222 Fluo. CD [␪] 222

1.00 ⫾ 0.05 M 0.90 ⫾ 0.05 M 0.86 ⫾ 0.05 M 2.65 ⫾ 0.15 M 2.55 ⫾ 0.10 M 54.9 ⫾ 0.3°C 39.1 ⫾ 0.3°C 2.60 ⫾ 0.20 M 2.05 ⫾ 0.20 M 2.25 ⫾ 0.15 M 0.82 ⫾ 0.10 M 37.3 ⫾ 0.4°C 3.25 ⫾ 0.20 M 2.65 ⫾ 0.11 M 3.35 ⫾ 0.26 M 3.22 ⫾ 0.23 M 53.8 ⫾ 0.4°C

2.12 ⫾ 0.10 M 1.75 ⫾ 0.15 M 1.45 ⫾ 0.10 M 4.15 ⫾ 0.20 M 4.45 ⫾ 0.25 M 65.0 ⫾ 0.4°C 50.0 ⫾ 0.3°C 4.10 ⫾ 0.51 M 3.45 ⫾ 0.35 M 3.22 ⫾ 0.20 M 2.00 ⫾ 0.15 M 50.0 ⫾ 0.5°C 4.10 ⫾ 0.30 M 4.05 ⫾ 0.25 M 4.55 ⫾ 0.24 M 4.54 ⫾ 0.35 M 65.0 ⫾ 0.5°C

Urea Temp. Acid-unfolded state (U A)

pH 2.0, 23°C

Gu–HCl Urea

Anion-refolded state (I A)

pH 2.0, 23°C, 0.25 M KCl

Temp. Gu–HCl Urea Temp.

states as monitored by fluorescence intensity, emission maximum, and ellipticity at 222 nm. The unfolding of the U A state by urea is non-cooperative, and the transition is complete with a lower transition midpoint relative to both native and I A state. The stabilization of the U A state seen at lower concentrations of Gu–HCl was not seen with urea, confirming the anionic effect of Gu–HCl at lower concentration. However, the unfolding of the I A state occurs at a higher concentration of urea, and the transition midpoints are very high. It is interesting to note that the urea-induced unfolding curves of I A and native states are similar, indicating that the stability induced by salt to the U A state is almost equal to that of native molecule. As expected, addition of salt did not have any effect on the ureainduced unfolding transition of native PK. The unfolding curves are non-cooperative and confirm the presence of an intermediate state in the unfolding pathway. The transition displays two discrete steps, each involving loss of some amount of secondary structure before proceeding to the next state. These observations indicate that anions induce and stabilize the structure only in the acid-unfolded state of PK but do not have any effect on the protein under neutral conditions. The results of Gu–HCl and urea-induced unfolding transitions are summarized in Table III. Figure 9 shows the urea-induced unfolding of I A and U A states of PK monitored by ANS binding. In the absence of anion, ANS intensity falls very sharply in the U A state and attains a minimum at 1 M urea, while in the I A state some amount of binding was observed with increasing denaturant concentration, suggesting stabilization of some hydrophobic clusters throughout. Moreover, unfolding of PK in I A state is non-cooperative and the presence of intermediate is imminent.

DISCUSSION

The results presented here establish that rabbit muscle pyruvate kinase exists in a partially unfolded state at acidic pH and low ionic strength with characteristics of molten globule state. Addition of the anions to the acid-unfolded state induces refolding resulting a near-native-like intermediate (I A). Further, the chemical- and temperature-induced unfolding reveals that the anion-induced state is stable and has stability almost equivalent to that of the native molecule. Acid-Unfolded (U A) State The acid-unfolded state of rabbit muscle pyruvate kinase is populated under conditions of low pH (between pH 2.0 and 2.5) and low ionic strength due to the charge– charge interactions (repulsion) from the net positive charge on the protein molecule. This state does not possess any tertiary interaction but retains around 50% of the native secondary structure. The tryptophan residues in the U A state are considerably exposed to the solvent as the fluorescence emission maximum redshifted around 6 – 8 nm along with some decrease in the fluorescence intensity. In the U A state the protein binds to ANS preferentially and the emission maximum of ANS fluorescence blue shifts by 35 nm, suggesting considerable exposure of hydrophobic groups to the solvent as ANS binds only to the solvent-exposed hydrophobic clusters and not to the unfolded state or native molecule (35). From these observations it appears that the U A state of pyruvate kinase possess some amount globular conformation and has characteristics of the molten globule state as observed in the case of some other small monomeric proteins.

ANION-INDUCED FOLDING OF PYRUVATE KINASE

107

charge and the effective increased size of the charged residues, caused by the presence of the associated counter ions on the protein molecule, which may prevent the formation of native tertiary structure in the extreme pH region and thus stabilize the intermediate conformational state (17). Thus the resulting conformation must be one of the native-like conformations possessing almost all the native secondary structure in the absence of any tertiary interactions. The stabilizing effect of salts on protein structure follows the Hofmeister series (36) as sulfate ⬎ phosphate ⬎ fluoride ⬎ chloride ⬎ bromide ⬎ iodide ⬎ perchlorate ⬎ thiocyanate. In this series the ions to the left of chloride stabilize the native structure of proteins, whereas the anions to the right of chloride cause destabilization. Arakawa and Timasheff (37) have shown that preferential hydration of proteins occurs in the presence of salts such as NaCl, sodium acetate, and Na 2SO 4. The resulting unfavorable free energy of the unfolded state is related to the stabilizing effects of these salts on the proteins. Solvent Accessibility of Tryptophan

Anion-Refolded Acid-Unfolded State

Rabbit muscle pyruvate kinase has three tryptophan residues in its subunit structure, and they are buried inside the molecule in the native state. Fluorescence measurements are expected to give an idea on the exposure of the interior to the solvent during unfolding. Solvent accessibility of the three tryptophans was studied by intrinsic fluorescence emission with the excitation being done exclusively at 292 nm. The fluorescence of PK in the U A state gives an emission maximum around 346 nm with a substantial decrease in the fluorescence intensity, suggesting that the acid-unfolded state has substantial exposure of the interior core to the solvent. Addition of anions in this state

The acid-unfolded state is populated in many proteins under conditions of low pH and low ionic strength due to Coulombic repulsion from the net positive charge on the protein molecule (16). This effect can be offset by the presence of anions, which bind to the positively charged sites and screen them, leading to formation of partially folded intermediates known as A states (17–20). Addition of anions to the U A state of PK induces a large amount of secondary structure along with restoration of fluorescence properties to an extent equivalent to the native molecule. Secondary structure gradually increases up to 0.25 M KCl with increase in salt concentration and the ellipticity reaches values that are similar to those of the native molecule along with a substantial increase in fluorescence intensity and a blue shift in the emission maximum. Nevertheless, the increase in secondary structure observed does not correspond to a concurrent restoration of tertiary structure. This behavior may be due the high net

FIG. 9. Urea-induced unfolding of PK in the U A (E) and I A (F) states monitored by ANS fluorescence. Protein concentration was 0.05 mg/ml. Protein to ANS concentration was in the ratio 1:100. All measurements were carried out at 23°C.

FIG. 8. Urea-induced unfolding of PK at pH 7.0 (squares) and pH 2.0 (circles) monitored by (a) far-UV CD at 222 nm, (b) emission maximum, and (c) fluorescence intensity. Open and filled symbols represent the measurements in the absence and presence of 0.25 M KCl, respectively. All other conditions were the same as those described for Fig. 5. All measurements were made at 23°C.

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restores all the fluorescence properties, both the emission maximum and the intensity, to be nearly equivalent to native molecule. Exposure of aromatic residues was also seen by acrylamide quenching studies, which revealed that around 60% of aromatic groups are exposed to the solvent environment under acidic conditions (Fig. 4). ANS Binding to U A and I A States The extent of ANS binding to a protein is an excellent method for monitoring the presence of molten globule-like state or any other compact intermediate conformations (5, 17, 35, 38, 39). We have earlier shown that papain under acidic conditions exists in a molten globule conformation with strong binding to ANS molecules relative to native as well as denatured states (5). Similarly, the U A state of pyruvate kinase binds to ANS very strongly, which was absent either in the native or in the completely unfolded states. On addition of KCl, the ANS fluorescence decreases and attains a plateau at around 0.3 M KCl (Figs. 3c and 4d). Nevertheless, it retains some amount of binding above 0.3 M KCl, suggesting the exposure of some amount of hydrophobic cluster in the anion-refolded state. If the salt-induced conformational transitions of PK in the U A state seen here are the results of the changes in the water structure, then the effects of various ions should follow the Hofmeister series. According to this series Na 2SO 4 is much more effective than KCl in stabilizing hydrophobic interactions and increasing the stability of proteins. Thus, it is expected that Na 2SO 4 is much more effective than KCl or NaCl and the amount of salt required to induce the structure may be less. Altogether, the spectroscopic characterization of the acid-induced unfolded state and anionrefolded state suggests that the two states are distinct intermediates having molten globule-like characteristics with the latter having native-like properties. Equilibrium Denaturation of U A and I A Intermediates Understanding the thermodynamical stability of partially folded intermediates will give a clue in the understanding of the role of these conformers in the folding and oligomerization process of multimeric proteins. Gu–HCl-induced unfolding of the U A state shows a stabilizing effect, at low concentrations of denaturant, where the secondary structure and fluorescence properties are restored to the level of anion-refolded or native state. This behavior is probably due to salt effect of Gu–HCl which was observed in the case of some other proteins (40 – 42). On the other hand, Gu–HClinduced unfolding curves of I A state show complex shapes consistent with the presence of two stages in the unfolding process. Though there was no clear bi-

phasicity in the transition, it appears that presence of intermediate state is imminent. The interesting observation is that the anion-refolded state unfolds at a higher concentration of denaturant in comparison to the protein in the native state. This is surprising because most of the proteins in the A state are thermodynamically less stable than their native counterparts (1, 2, 43). However, a few proteins, such as barstar, unfold at a higher concentration of denaturant in the molten globule state (44) though the physical basis of this stability is not known. Intersubunit or interdomain interactions are expected to play a major role in this unusual stability in the case of mutidomain/multimeric proteins. In addition, the non-coinciding unfolding curves of both U A and I A states suggest the presence of intermediates in their unfolding. Stabilizing interactions between KCl and low concentrations of denaturants such as Gu–HCl with the native and the partially folded states is noted previously. Pace et al. (45) found that Gu–HCl increases the stability of native state of ribonuclease T1, while Havel et al. (46) reported Gu–HCl-induced dimerization of bovine growth factor that occurs at a much lower concentration of denaturant. Similarly, Gu–HCl stabilizes protein disulfide isomerase (PDI) at lower concentrations due to specific stabilizing interactions between the intermediate and denaturant through binding, from an effect of Gu–HCl on the electrostatic shielding (41) or from an effect of Gu–HCl on the structure of water (19). The observed stabilization of PK at low concentrations of Gu–HCl in the U A state is most likely due to the result of electrostatic shielding because the inclusion of 0.25 M KCl has significant effect on the Gu–HCl-induced denaturation (cf. Fig. 6). This confirms that the stabilizing action of Gu–HCl at lower concentration is due to the anionic effect of the denaturant, i.e., the electrostatic screening effect. Anions stabilize the molten globule state of cytochrome c and apomyoglobin at low pH, where the protein is positively charged and the intermediate is more positively charged than the native state (19). Stabilization by anions and ionic denaturants could be fairly common, because proteins at low pH frequently contain ion-binding sites of varying affinity and specificity. Salts affect mainly the electrostatic and hydrophobic interactions in the protein molecules. Conformational transitions, in the presence of anions at acidic or alkaline pH, from a largely unfolded state to an intermediate state have been reported for several small globular proteins (17, 18), but very few reports on the effects of salts on the subunit assembly of multimeric proteins are available (20, 47). The data on the urea-induced unfolding of PK in the U A and I A states show that both the intermediate states have different unfolding behavior with different transition midpoints, suggesting different thermody-

ANION-INDUCED FOLDING OF PYRUVATE KINASE

namic stability. The I A state has stability almost similar to that of the native protein. Studies on the thermodynamic stability of PK in the U A and I A states upon thermal unfolding yielded interesting observations. The U A state is less stable as expected compared to the native state, while the protein in the I A state has stability similar to that of native molecule. This observation unambiguously confirms that anions not only induce the structure but also stabilize the induced structure even more than that of the native molecule. Such unusual stability might be due to the strong interdomain or intersubunit interactions caused by the increased hydrophobic stability. In general, the native state of a protein is energetically more stable than its intermediate and denatured states (if we ignore any kinetic traps). The unusual stability of the I A state under acidic conditions suggests that the intermediate might be one of the on-pathway energy trap (barrier) in the folding pathway of PK and that the barrier may be crossed when the folding conditions were manipulated. When it fails to fold to its native state from this trap, it starts to aggregate as seen from the light-scattering studies (data not shown). The I A state can be readily refolded to the active tetrameric state when exchanged with the native pH buffer (data not presented), suggesting indeed that the I A state is an on-pathway intermediate in the folding of PK. It has been shown that PK exists in multiple intermediate conformations possessing substantial secondary structure in the unfolding pathway at neutral conditions (27). The intermediate conformations observed in the present study at low pH in the absence and presence of salt show a good correlation with existence of intermediates with some differences. Doster and Hess (27) reported a good correlation between the amount of secondary structure and the subunit conformation. Thus, the UA state has secondary structure similar to an expanded dimer, which is functionally inactive. The data on the Gu–HCl and urea unfolding of the I A state confirm the existence of an equilibrium intermediate between this more ordered intermediate and the completely unfolded states. The results of denaturantinduced unfolding of PK can be explained in view of its domain structure. The subunits of PK consist of four domains, and three of them are in contact with the neighboring subunits of the oligomer (23, 48). Out of the four domains, the main intersubunit contact comes from C-terminal domain along with a minor contribution from A and N-terminal domains. Two of the three Trp residues in the PK subunit are located in the C-terminal domain while the third is in the B domain. As the B domain is located in the central part of the subunit structure and C-terminal domain has most of the subunit contacts, the three Trp are buried well inside the molecule in the native conditions. Thus the dissociation process of tetramer into dimer and then

109

into monomer would involve large changes in structural conformation along with shift in the fluorescence emission maximum. Our results suggest that the Cterminal domain, which consists of five ␣-helices and five ␤-strands, is the more stable one, where the unfolding of secondary structure and the red shift in fluorescence maximum occur in parallel. The data presented here also suggest a sequential unfolding of the domains because each dissociation step involves a substantial decrease in secondary structure along with red shift in emission maximum. The conformational changes in the tetramer should also involve the B domain where the third Trp is located. Mechanism of Unfolding of PK It is known that anions differ in their neutralizing effect on the net positive charges in the acid-unfolded polypeptide chain and in the amount of structure and compactness they induce (18, 19). It has been widely accepted that equilibrium partially folded conformations of a protein molecule can be good models for transient kinetic intermediates in protein folding (49 – 51). Such equilibrium counterparts can facilitate considerably the description of the structural properties of kinetic (transient) intermediates. Our data are consistent with the general belief that the observed equilibrium partially folded intermediates will be specific to a

FIG. 10. Schematic representation of the proposed folding/unfolding pathway of rabbit muscle pyruvate kinase (PK)-induced by salt and denaturants. N and U represent the native tetramer and unfolded monomer, respectively. I 1 and I 2 indicate the dimeric and tetrameric intermediates of PK, respectively.

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given protein rather than be general intermediates. Based on the results presented above, a model of folding of rabbit muscle PK can be represented as U 3 I1 3 I2 3 N

[2]

where I 1 and I 2 represent the acid-unfolded dimeric intermediate and native-like intermediate, respectively. U and N correspond to the completely unfolded and native states, respectively. The above model can be represented schematically, as shown in Fig. 10. The native tetrameric PK dissociates into compact dimer (I1) at low pH, while the addition of anions induces structure to the molecule resulting an inactive native-like tetramer (I2). Further, unfolding of I 1 by denaturant resulted in complete unfolded monomers (U), while I2 unfolds through an I1-like intermediate finally to the U state. ACKNOWLEDGMENTS Authors acknowledge Deepika Lal for some preliminary work. One of the authors (F.E.) acknowledges UGC, Government of India, for financial assistance in the form of a fellowship. The authors also thank DBT, Government of India, for financial assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20.

Ptitsyn, O. B. (1995) Adv. Protein Chem. 47, 83–229. Kuwajima, K. (1989) Proteins: Struct. Funct. Genet. 6, 87–103. Dobson, C. M. (1992) Curr. Opin. Struct. Biol. 2, 6 –12. Fink, A. L. (1995) Ann. Rev. Biophys. Biomol. Struct. 24, 495–522. Edwin, F., and Jagannadham, M. V. (1998) Biochem. Biophys. Res. Commun. 252, 654 – 660. Kundu, S., Sundd, M., and Jagannadham, M. V. (1999) Biochem. Biophys. Res. Commun. 264, 635– 642. Jackson, S. E. (1998) Folding Design 3, R81–R91. Fink, A. L. (1998) Folding Design 3, R9 –15. Jaenicke, R. (1987) Prog. Biophys. Mol. Biol. 49, 117–237. Jaenicke, R. (1999) Prog. Biophys. Mol. Biol. 71, 155–241. Garel, J. R. (1992) in Protein Folding (Creighton, T. E., Ed.), pp. 405– 454, Freeman Co., New York. Price, N. C. (1994) in Mechanisms of Protein Folding (Pain, R. H., Ed.), pp. 160 –193, IRL Press, Oxford. Semisotnov, G. V., Kihara, H., Kotova, N. V., Kimura, K., Amemiya, Y., Wakabayashi, K., Serdynk, I. N., Timchenko, A. A., Chiba, K., Nikaido, K., Ikura, I., and Kuwajima, K. (1996) J. Mol. Biol. 262, 559 –574. Neet, K. E., and Timm. D. E. (1994) Protein Sci. 3, 2167–2174. Mateu, M. G., Del Pino, M. M. S., and Fersht, A. R. (1999) Nat. Struct. Biol. 6, 191–198. Tanford, C. (1968) Adv. Protein Chem. 23, 121–282. Goto, Y., and Fink, A. L. (1989) Biochemistry 28, 945–952. Goto, Y., Calciano, L. J., and Fink, A. L. (1990a) Proc. Natl. Acad. Sci. U.S.A. 87, 573–577. Goto, Y., Takahashi, N., and Fink, A. L. (1990b) Biochemistry 29, 3480 –3488. Uversky, V. N., Karnoup, A. S., Segel, D. J., Seshadri, S., Doniach, S., and Fink, A. L. (1998) J. Mol. Biol. 278, 875– 894.

21. Uversky, V. N., Karnoup, A. S., Khurana, R., Segel, D. J., Doniach, S., and Fink, A. L. (1999) Protein Sci. 8, 161–173. 22. Laughlin, L. T., and Reed, G. H. (1997) Arch. Biochem. Biophys. 348, 262–267. 23. Larsen, T. M., Laughlin, L. M., Holden, H. M., Rayment, I., and Reed, G. H. (1994) Biochemistry 33, 6301– 6309. 24. Larsen, T. M., Benning, M. W., Wesenberg, G. E., Rayment, I., and Reed, G. H. (1997) Arch. Biochem. Biophys. 345, 199 –206. 25. Steinmetz, M. A., and Deal, W. C. (1966) Biochemistry 5, 1399 – 1405. 26. Johnson, G. S., Steinmetz, M. A., Kayne, M., and Deal, W. C. (1969) Biochemistry 8, 2455–2462. 27. Doster, W., and Hess, B. (1981) Biochemistry 20, 772–780. 28. Laemmli, U. K. (1970) Nature 227, 680 – 685. 29. Fish, W., Mann, K. G., and Tanford, C. (1969) J. Biol. Chem. 244, 4989 – 4994. 30. Boyer, D. (1969) Biochem. Biophys. Res. Commun. 34, 702–706. 31. Pace, C. N., Shirley, B. A., and Thomson, J. A. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., Ed.), pp. 311– 330, IRL Press, Oxford. 32. Cassim, J. Y., and Yang, J. T. (1969) Biochemistry 8, 1947–1951. 33. Stryer, L. (1965) J. Mol. Biol. 13, 482– 495. 34. Stryer, L. (1968) Science 162, 526 –540. 35. Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991) Bioploymers 31, 119 –128. 36. Von Hipple, P. H., and Wong, K.-Y. (1965) J. Biol. Chem. 240, 3909 –3923. 37. Arakawa, T., and Timasheff, S. N. (1982) Biochemistry 21, 6545– 6552. 38. Rodionova, N. A., Semisotnov, G. V., Kutyshenko, V. P., Uversky, V. N., Bolotina, I. A., Bychkova, V. E., and Ptitsyn, O. B. (1989) Mol. Biol. (Moscow) 23, 683– 692. 39. Engelhard, M., and Evans, P. E. (1995) Protein Sci. 4, 1553–1562. 40. Mayr, L. M., and Schmid, F. X. (1993) Biochemistry 32, 7994 – 7998. 41. Morjana, N. A., McKeone, B. J., and Gibert, H. F. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2107–2111. 42. Morjana, N. A., and Tal, R. (1998) Biotechnol. Appl. Biochem. 28, 7–17. 43. Baldwin, R. L. (1995) J. Biomol. NMR 5, 103–109. 44. Khurana, R., and Udgaonkar, J. B. (1994) Biochemistry 33, 106 – 115. 45. Pace, C. N., Laurents, D. V., and Thomson, J. A. (1990) Biochemistry 29, 2564 –2572. 46. Havel, H. A., Kauffman, E. W., Plaisted, W. M., and Brems, D. N. (1986) Biochemistry 25, 6533– 6538. 47. Prajapati, S., Bhakuni, V., Babu, K. R., and Jain, S. K. (1998) Eur. J. Biochem. 255, 178 –184. 48. Muirhead, H., Clayden, D. A., Barford, D., Lorimer, C. G., Fothergill-Gilmore, L. A., Schiltz, E., and Schmitt, W. (1986) EMBO J. 5, 475– 481. 49. Ikeguchi, M., Kuwajima, K., Mitani, M., and Sugai, S. (1986) Biochemistry 25, 6965– 6972. 50. Hughson, F. M., Wright, P. E., and Baldwin, R. L. (1990) Science 249, 1544 –1548. 51. Jennings, P. A., and Wright, P. E. (1993) Science 262, 892– 896.