- Email: [email protected]
Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation
Biochimica et Biophysica Acta 1478 (2000) 221^231 www.elsevier.com/locate/bba
Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation Guillermo Mendoza-Herna¨ndez *, Fernando Minauro, Juan L. Rendo¨n Departamento de Bioqu|¨mica, Facultad de Medicina, Universidad Nacional Auto¨noma de Me¨xico, Apdo. postal 70-159, Me¨xico, D.F. 04510, Mexico Received 8 July 1999; received in revised form 14 December 1999; accepted 1 February 2000
Abstract The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid. At slightly higher urea concentrations (0.75^2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Glucose dehydrogenase; Urea; Denaturation; Bacillus megaterium
1. Introduction Glucose dehydrogenase (L-D-glucose:NAD(P) 1oxido-reductase, EC 1.1.1.47) from Bacillus megaterium is a member of the short-chain family of alcohol dehydrogenases [1]. The enzyme catalyzes the oxidation of L-D-glucose to D-L-glucono-1,5-lactone using Abbreviations: CD, circular dichroism; Rs , Stokes' radius; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis; Bis-ANS, 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid 1-anilinonaphthalene-8-sulfonic acid * Corresponding author. Fax: +52-5-6162419; E-mail: [email protected]
NAD or NADP as a coenzyme. Glucose dehydrogenase is produced during bacterial sporulation [2^ 4] and it has been reported that it plays an important role in spore germination [5,6]. At neutral pH, the enzyme is a tetrameric protein with a molecular mass of 120 000 and is constituted by identical subunits of 262 amino acid residues each [7], with known amino acid sequence [8]. The protein lacks cysteine and contains four tryptophan residues. This dehydrogenase is readily dissociated into inactive monomers by shifting the pH to 8.5^9.5. Complete reassociation and reactivation has been reported by readjustment to neutral pH [9]. The changes in structure and functional properties of glucose dehydrogenase upon al-
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 0 2 5 - X
BBAPRO 36109 10-5-00
222
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
kaline dissociation have been investigated by several spectroscopic methods. All changes which occur in the course of the alkaline dissociation were found to be fully reversible by readjusting the pH [9^11]. No changes in protein structure other than dissociation were observed. The lack of irreversible conformational changes upon alkaline dissociation of glucose dehydrogenase prompted us to an examination of the denaturing behavior of this enzyme to determine the occurrence of unfolding/folding intermediates. In the present work, changes in enzymatic activity, far-UV circular dichroism (CD), size exclusion chromatography, protein cross-linking, transverse urea gradient gels, intrinsic protein £uorescence and 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid 1-anilinonaphthalene-8sulfonic acid (bis-ANS) £uorescence were used to study the denaturation behavior of glucose dehydrogenase from B. megaterium by urea.
appeared homogeneous by polyacrylamide gel electrophoresis under denaturing conditions using the discontinuous bu¡er system of Laemmli [12], on separation gels containing 15% acrylamide. The purity of the preparation was also checked by N-terminal amino acid sequencing using a Beckman LF 3000 protein sequencer. Protein concentrations were determined by the Coomassie blue G dye binding assay of Bradford [13].
2. Materials and methods
2.4. Inactivation by urea
2.1. Reagents
Triplicate inactivation experiments were performed at room temperature by incubation of glucose dehydrogenase (0.2 nmol subunit/ml) in urea solutions of di¡erent concentrations (in the range of 0.1^8 M) for 18 h in a ¢nal volume of 1 ml of 100 mM sodium phosphate bu¡er containing 1 mM EDTA (pH 7.0). After the incubation period, the residual activity was determined by adding a small aliquot of 5 M D-glucose (20 Wl) and 100 mM NAD (10 Wl) stock solutions in the same bu¡er. The reversibility of the inactivation was studied in triplicate samples of enzyme incubated in urea solutions of various concentrations as above but in a ¢nal volume of 30 Wl. After 18 h, the samples were diluted by adding 1 ml of reactivation bu¡er: 100 mM sodium phosphate, 50 mM EDTA (pH 7.0) [14]. The diluted solutions were incubated for a further 24 h and assayed for enzyme activity.
Glucose dehydrogenase from B. megaterium M1286 with an activity of 207 U/mg was purchased from Sigma. Urea (ultrograde) and gel ¢ltration calibration proteins were obtained from Pharmacia LKB. Bis-ANS (dipotassium salt) was from Molecular Probes. Electrophoresis reagents, including calibration standards, were from Bio-Rad. Glutaraldehyde and D-glucose were products from Merck. NAD was obtained from Boehringer Mannheim. All other reagents were of analytical grade and used without further puri¢cation. Reverse osmosis puri¢ed water was used in the preparation of solutions. 2.2. Protein puri¢cation Glucose dehydrogenase was further puri¢ed from the commercial preparation according to the procedure described by Pauly and P£eiderer [7]. The puri¢ed enzyme had a speci¢c enzyme activity of 400 U/ mg using 3 mM NAD, 140 mM glucose as substrates in the assay conditions described by Maurer and P£eiderer [10]. The puri¢ed glucose dehydrogenase
2.3. Enzymatic assays The glucose dehydrogenase activity was determined at room temperature in a Cecil 5000 double beam spectrophotometer by following the initial linear increase in absorbance at 340 nm due to the reduction of NAD by glucose. The reaction mixtures were prepared in a 1 ml cuvette containing 1 mM NAD, 100 mM D-glucose, 1 mM EDTA and 100 mM sodium phosphate (pH 7.0).
2.5. Size exclusion chromatography The structural changes such as oligomerization/dissociation and the presence of unfolded states of glucose dehydrogenase were studied by zonal size exclusion chromatography at di¡erent urea concentrations
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
using a Beckman high performance liquid chromatography (HPLC) system with a 7.5U300 mm Ultraî (Mr Spherogel-SEC 3000 column, pore size of 230 A 3 5 range: 5U10 ^7U10 ). The mobile phase contained 1 mM EDTA, 150 mM NaCl, 100 mM sodium phosphate (pH 7.0) and the required concentration of urea. Before injection, samples were ¢ltered using a 0.22 Wm ¢lter and eluted at a £ow rate of 1 ml/min. The protein pro¢le was monitored at 280 nm. Stokes' radius (Rs ) determinations were performed using well characterized globular protein standards of known Rs as described by Ackers [15]. 2.6. Cross-linking experiments The aggregation state of the enzyme during denaturation with urea was also determined by cross-linking with glutaraldehyde, followed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^ PAGE). Protein samples were cross-linked with glutaraldehyde, according to Jaenicke et al. [16]. A small volume of glutaraldehyde (25%, w/v) was added to enzyme^urea solutions in standard bu¡er (10 Wg of protein) at 1% (v/v) of the ¢nal mixture. After incubation for 30 s, the reaction was quenched by the addition of solid NaBH4 to give a ¢nal molar ratio of NaBH4 /glutaraldehyde of 2. After 20 min incubation at room temperature, the protein was co-precipitated with sodium deoxycholate by adding trichloroacetic acid (50%, v/v), resolubilized in 20 Wl of 1.5 M Tris^HCl bu¡er (pH 8.8) containing 1% (w/v) SDS and 50 mM DTE and immediately analyzed by SDS^ PAGE. The SDS^PAGE was performed at room temperature with a 4^20% acrylamide gradient slab gel using the Tris^glycine bu¡er system described by Laemmli [12]. The gels were stained with Coomassie blue R and destained using conventional procedures. 2.7. Transverse urea gradient gel electrophoresis The unfolding/folding transitions of glucose dehydrogenase were analyzed by transverse urea gradient electrophoresis, according to Creighton [17]. Polyacrylamide slab gels (100U80 mm) containing a 0^8 M urea concentration gradient were casted with an inverse gradient of acrylamide concentration of 11^7% to ensure uniformity of migration with respect to urea viscosity. The method used for the preparation
223
of gels was adapted from Goldenberg [18] and has been described previously [19]. To avoid the pH-induced dissociation displayed by glucose dehydrogenase in mild alkaline conditions (pH 9), the 100 mM sodium phosphate bu¡er system (pH 7.0) described by Weber and Osborn [20] (without SDS) was used in the casting procedure and as the running bu¡er. Gels were pre-electrophoresed towards the anode for 1 h at 70 V. Either native or previously incubated enzyme in 8 M urea (75 Wg protein) were applied across the top of the gel and electrophoresed at a constant 100 V for 4 h. The gels were stained with Coomassie blue R. 2.8. CD experiments Far-UV CD measurements were carried out on an Aviv 62DS spectropolarimeter interfaced to a computer controlled by Aviv software. The thermostated cell holder was maintained at 25³C. CD spectra were recorded from 200 to 260 nm using strength free quartz cells with 0.1 cm optical path-length. Enzyme samples were scanned after at least 18 h incubation in the appropriate bu¡er^urea solution. Five repetitive scans were performed for each urea concentration at 1 nm intervals, with a time constant of 5 s and 1 nm bandwidth. At 8 M urea, the CD spectra could be recorded only as far as 210 nm. Data were corrected for the baseline contribution of phosphate bu¡er and urea. The residue ellipticities were calculated from the average data obtained in this way for two independent experiments. The mean residue weight was calculated from the amino acid composition of the enzyme [8]. The ellipticity at 220 nm was taken to indicate changes in the protein secondary structure. 2.9. Fluorescence studies Fluorescence measurements were made on an Aminco SPF-500 spectro£uorimeter with a semimicro 1 cm light path cell. Samples were excited at 295 nm to analyze selectively the tryptophan residues of the protein, and emission was recorded from 300 to 540 nm; bandwidths were 4 nm for excitation and 5 nm for the emission monochromator. For experiments in the presence of bis-ANS, the £uorescence emission spectra were obtained by using
BBAPRO 36109 10-5-00
224
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
an excitation wavelength of 380 nm. The excitation and emission spectral bandwidths were 4 and 8 nm, respectively. The concentration of bis-ANS was determined spectrophotometrically at 387 nm based in a molar extinction coe¤cient of 17 000 M31 cm31 [21]. The £uorescence intensity of the dye bound to the enzyme was assessed by adding in a Wl volume, a ¢xed concentration of bis-ANS (3.5 WM, ¢nal) to a cuvette containing 1 ml of native or pre-incubated enzyme^urea solution. Protein was 0.4 WM. Corrections were made by subtracting the £uorescence intensity of the dye in bu¡er at the same concentration of urea. Quenching of tryptophan £uorescence of glucose dehydrogenase by bis-ANS was studied as above, but the excitation wavelength was 290 nm and emission was monitored from 300 to 650 nm. 3. Results 3.1. Activity of glucose dehydrogenase during denaturation with urea Results of the changes of enzymatic activity of glucose dehydrogenase after incubation for 18 h in urea solutions of di¡erent concentration are shown in Fig. 1. The enzyme is stable in urea only up to 0.5
Fig. 2. Size exclusion HPLC elution pro¢les of B. megaterium glucose dehydrogenase at selected urea concentrations. Protein samples (1 mg/ml) were incubated with di¡erent concentrations of urea for 18 h; then, a 50 Wl aliquot was chromatographed on a UltraSpherogel-SEC 3000 column pre-equilibrated with the required concentration of urea. Other conditions were as described in Section 2.
M, losing its activity with increasing concentrations of the denaturant. The smooth inactivation curve has a midpoint at 1.25 M urea. On the other hand, complete reactivation was attained after a 35-fold dilution of the pre-incubation mixtures, even at an urea concentration of 8 M (Fig. 1, inset). 3.2. Size exclusion chromatography
Fig. 1. Relative activity changes of glucose dehydrogenase as a function of urea concentration. The enzyme was incubated at the indicated denaturant concentrations for 18 h; thereafter, residual activity was determined in the same urea solution as described under Section 2. (Inset) Time course of reactivation of an enzyme sample pre-incubated for 18 h in 8 M urea after a 35-fold dilution with phosphate, EDTA bu¡er. In both cases, a ¢nal concentration of 0.2 nmol subunit/ml was used. Each point is the average of three independent determinations.
In order to investigate the changes in the oligomeric state of glucose dehydrogenase, its chromatographic behavior on a gel ¢ltration column was analyzed under both native conditions and in the presence of di¡erent concentrations of urea. Fig. 2 shows the elution pro¢les of native and urea-treated enzyme at di¡erent concentrations of denaturing reagent. In native conditions, a single chromatoî graphic peak was detected with a Rs of 44.5 A (Mr = 120 000). At 1 M urea, a second minor peak, eluting in the void volume of the column, was observed, suggesting the formation of polymeric species. The relative proportions of these two peaks changed with increasing urea concentrations. Be-
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
225
Fig. 3. Rs values and relative abundance of glucose dehydrogenase species as a function of urea concentration. The size exclusion chromatographic data from Fig. 2 were used to determine: (A) the Rs and (B) the relative abundance of glucose dehydrogenase speî ) were used for calibration: thyroglobulin, cies as a function of urea concentration. Protein standards with the following Rs values (A 85.0; ferritin, 61.0; catalase, 52.2; aldolase, 48.1; albumin, 35.5; ovalbumin, 30.5; chymotrypsinogen A, 20.9; ribonuclease A; 16.4. The relative abundances of included and aggregated forms were determined by the peak area using the Beckman system Gold software.
tween 1 and 2 M urea, the relative abundance of the excluded peak sharply increased at expense of the tetrameric protein. At the later urea concentration, î, a minor new peak, corresponding to a Rs of 26.5 A appeared. The Rs value strongly suggests that this new species corresponds to the monomeric protein. At 2.5 M urea and over, this was the only peak detected; however, its elution volume was diminishing with increasing urea concentrations. At 8 M urea, î . The its elution volume corresponds to a Rs of 49 A Rs values of the included species and the relative abundance of included and aggregated forms determined by the peak area are shown in Fig. 3A,B. 3.3. Cross-linking with glutaraldehyde and SDS^PAGE analysis Cross-linking experiments can be performed in a su¤ciently short time to freeze the states of association during either unfolding or folding process [22]. Fig. 4 shows the electrophoretic species pattern of the enzyme after covalent cross-linking in the presence of di¡erent concentrations of urea. In the absence of urea, glucose dehydrogenase was almost entirely cross-linked, and only the tetrameric form could be detected. Up to 1.5 M urea, tetramer still represents the main species, but now coexist with both monomeric and aggregated small fractions of
Fig. 4. SDS^PAGE of glucose dehydrogenase after covalent cross-linking in the presence of various concentrations of urea. Enzyme samples (20 Wg) were pre-incubated 18 h at the selected urea concentration and then mixed with glutaraldehyde and NaBH4 as described in Section 2. Protein was co-precipitated with sodium deoxycholate, dissolved in SDS sample treatment bu¡er and electrophoresed on a 4^20% acrylamide gradient SDS slab gel. Standard markers (lane 1); glucose dehydrogenase, control (lane 2); glucose dehydrogenase after cross-linking without urea (lane 3). Enzyme incubated in urea at 1, 1.5, 2.0, 2.5, 3.0, 5.0 and 7.0 M (lanes 4^10, respectively) before crosslinking.
BBAPRO 36109 10-5-00
226
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
the enzyme. At 2.0 M urea, higher aggregates, which could not be resolved on the 4^20% acrylamide gradient gel, were the only species detected on top of the gel. The multimeric species are present in a short range of urea concentrations, so at 2.5 M urea, their relative abundances sharply decrease. The disappearance of the multimeric species is concomitant with the presence of a band corresponding to the monomeric protein. From 3 to 8 M urea, the monomeric species was the only band detectable. The cross-linking experiments con¢rm the data obtained by HPLC gel ¢ltration and provide evidence that the denaturation of glucose dehydrogenase with urea involves an initial stage of unfolding with the formation of a more expanded tetrameric enzyme, production of multimeric species, dissociation to the partially unfolded monomer, followed by further unfolding of the latter. 3.4. Transverse urea gradient gel electrophoresis Electrophoresis through polyacrylamide gels with a perpendicular gradient of urea is a sensitive method of resolving di¡erent conformational states of a protein and has also been extensively used to identify unfolding intermediates [23]. In order to more fully characterize the unfolding transitions induced by urea of glucose dehydrogenase, the electrophoretic behavior of the enzyme was analyzed in transverse urea gradient gels (0^8 M). From the electrophoretic pro¢les obtained starting with either native (Fig. 5A) or previously unfolded enzyme (Fig. 5B), it is clear that the enzyme does not follow a simple two-state cooperative unfolding transition, but suggest the presence of multiple species that interconvert and unfold with di¡erent rates. When native glucose dehydrogenase was applied to the gel, a pattern consisting of two well de¢ned bands, parallel to the urea concentration gradient, was obtained. Besides these two bands, there is a transition zone as is seen by a shared, faint, di¡use, vertical band in a short interval of intermediate urea concentrations. The upper band showed three distinct mobilities regions; at low urea concentrations (0^2 M), the protein had a mobility corresponding to the tetrameric enzyme in the native conformation, followed by a well de¢ned in£exion point to species with decreased mobility. The latter species were detectable up to 3.5 M urea. This pat-
Fig. 5. Transverse urea gradient gel electrophoresis of B. megaterium glucose dehydrogenase. One hundred Wl samples containing 75 Wg of native (A) or previously incubated enzyme in 8 M urea for 18 h (B) were layered across the top of a slab gel containing a 0^8 M transverse gradient of urea and subjected to electrophoresis toward the anode at pH 7.0. Other conditions were as described under Section 2.
tern is consistent with a rapid transition from the native to the aggregated forms of the enzyme. At low urea concentrations (0.5^1 M), the lower band was detectable as a di¡use band with an electrophoretic mobility corresponding to the native monomer, followed by a similarly di¡use transition zone, while at higher urea concentrations (2.5^8 M), the gel displayed a sharply de¢ned band with a reduced electrophoretic mobility. These species, however, move faster than the native tetramer. The existence of a heterogeneous population of unfolded forms in the folding/assembly pathway of glucose dehydrogenase was also evident from the pattern obtained when electrophoresis was initiated with the unfolded protein (Fig. 5B). The gel displayed now one continuous and predominant band extending through the complete urea gradient, a band of oligomers at the top of the gel in the region of 1.5^3.0 M urea, and a shared, faint band extending from the oligomers to the faster mobility region
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
on the continuous protein band. Species with an electrophoretic mobility corresponding to that of the native tetrameric protein were not detected, suggesting the presence of a slow assembly step on the time scale of the electrophoresis. These results suggest that during the folding of glucose dehydrogenase, an heterogeneous population of intermediates, including high molecular mass oligomers, with di¡erent interconversion rates is present. 3.5. CD studies CD spectroscopy was used to monitor the changes in the secondary structure content of glucose dehydrogenase upon urea-induced unfolding. The far-UV CD spectra of the native and pre-incubated enzyme in urea solutions of di¡erent concentrations are depicted in Fig. 6. The spectrum of the native enzyme was characterized by an almost £at region between two negative minima around 210 and 222 nm. The spectrum crosses the base line from negative to positive at 198^199 nm. Incubation of the enzyme in
Fig. 6. Far-UV CD spectra of B. megaterium glucose dehydrogenase in urea. Enzyme samples were incubated for 18 h at selected concentrations of urea before spectra were recorded. Concentration of urea (M): 0 (b); 1.2 (R); 1.5 (O); 1.75 (a); 3.0 (7); 8 (8). For clarity, the spectra obtained with other concentrations of urea are not shown. The inset is the transition curve of glucose dehydrogenase as measured by changes in ellipticity at 222 nm.
227
urea solutions results in both changes in the shape of the CD spectrum and in an urea concentrationdependent loss in ellipticity. Between 0.75 and 1.2 M urea, the CD spectra showed a negative minima peak at 213 nm with an increase in the ratio of [3]213 / [3]222 , reaching a maximum at 1 M urea. A red shift of the wavelength at which signals cross from negative to positive ellipticity values (204^205 nm) was also observed, as compared with the native protein. Similar changes in the CD spectra have been observed previously with other oligomeric proteins [24,25], and are consistent with conformational changes that a¡ect the state of oligomerization of glucose dehydrogenase. On the other hand, the molar ellipticity at 222 nm changes sharply toward more positive values in the range 1^1.75 M urea, followed by a smoother change up to 8 M urea (inset). 3.6. Fluorescence studies of glucose dehydrogenase Intrinsic £uorescence is an excellent spectroscopic probe to investigate conformational changes in tertiary structure of proteins. The conformational transitions of glucose dehydrogenase were also monitored by following the changes in intensity and position of the maximum £uorescence emission in the presence of urea. As shown in Fig. 7, the maximum of the tryptophan £uorescence emission of enzyme was at 340 nm in the native state. Upon denaturation by 8 M urea, the maximum emission was shifted to 360 nm. The induced urea red shift was accompanied by an increase in £uorescence intensity. During the denaturation process, an initial stage of £uorescence changes takes place at low urea concentrations involving changes in both emission intensity and a gradual red shift in the wavelength of maximum emission. From 0.25 M to 1 M urea, an increase in the emission intensity with slight changes in the wavelength of the tryptophan maximum emission was observed. At urea concentrations higher than 1 M, a decrease in emission intensity and a marked red shift of the maximum emission takes place, leading to a completion at 2 M urea. Further changes in the tryptophan environment occur at urea concentrations higher than 2 M, as was evident from a new gradual increase in the emission intensity up to 8 M urea. The changes in the maximum emission wavelength and in £uorescence intensity at 360 nm as a
BBAPRO 36109 10-5-00
228
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
3.8. Quenching of tryptophan £uorescence of glucose dehydrogenase by bis-ANS
Fig. 7. Fluorescence emission spectra of B. megaterium glucose dehydrogenase under native conditions and in the presence of 8 M urea. The native protein (- - -) was in 0.1 M phosphate bu¡er (pH 7.0); the unfolded sample (9) was pre-incubated for 18 h and contained 8 M urea in addition. The £uorescence of the native enzyme at 340 nm was taken as 100%. Other conditions were as described in Section 2. The inset shows the red shift in the wavelength of the maximum £uorescence emission (b), and the relative changes in £uorescence emission intensity at 360 nm (a) as a function of urea molarity.
Bis-ANS is an aromatic compound that interact with aromatic amino acids in proteins through hydrophobic interactions [30]. To obtain further information on the environment of the tryptophan residues under native and denaturing conditions, quenching experiments were performed. In the dyefree state, native glucose dehydrogenase £uoresces with a maximum near 340 nm due to tryptophan emission (Fig. 7). Upon the addition of increasing concentrations of bis-ANS to the enzyme solution, the bis-ANS £uorescence emission (500 nm) increases with a concurrent decrease in the enzyme tryptophan emission. At 4 WM bis-ANS, quenching of enzyme £uorescence was 50% (Fig. 9). These data indicate that, in fact, tryptophan residues on the surface of the native enzyme are accessible to this quencher. When bis-ANS was added to pre-incubated enzyme solutions of increasing urea concentrations, the changes in the bis-ANS £uorescence were as described in the individual studies (Fig. 8). That the
function of urea concentration are depicted in Fig. 7 (inset). 3.7. Binding of bis-ANS to glucose dehydrogenase Anilinonaphthalene sulfonates have widely been used as sensitive reporters of apolar regions in proteins and to probe protein conformational changes [26^29]. Fig. 8 shows the £uorescence emission spectra of free and native enzyme-bound bis-ANS. On excitation at 380 nm, free bis-ANS in the aqueous bu¡er solution was non-£uorescent. The addition of the dye to the native glucose dehydrogenase in the bu¡er solution produced a marked enhancement of £uorescence intensity, indicating the binding of bisANS to the enzyme. The maximum emission of this complex was at 500 nm. The interaction of bis-ANS with glucose dehydrogenase was a¡ected by urea (Fig. 8, inset). Upon addition to pre-incubated enzyme^urea mixtures, the bis-ANS £uorescence intensity was further increased at very low urea concentrations, reaching a maximum at 0.25 M urea. Higher concentrations of denaturant were accompanied by a sharply decrease in bis-ANS £uorescence.
Fig. 8. Binding of bis-ANS to glucose dehydrogenase under native conditions and in the presence of di¡erent concentrations of urea. Fluorescence emission spectra of 3.5 WM of bis-ANS alone in 0.1 M Na-phosphate bu¡er (lower spectrum) or in enzyme solutions under native conditions and in the presence of urea. Excitation was at 380 nm. Numbers above the curves refer to the concentrations of urea at which enzyme was pre-incubated. The inset shows the relative bis-ANS £uorescence intensity at 500 nm in enzyme solutions as a function of urea concentration. The maximum of £uorescence that appeared at 0.25 M urea was taken as 100%.
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
Fig. 9. Quenching of tryptophan £uorescence of B. megaterium glucose dehydrogenase by bis-ANS. Fluorescence emission spectra of samples with glucose dehydrogenase (0.4 WM) in the presence of (1) 0, (2) 1, (3) 2, (4) 4, (5) 6, (6) 10 and (7) 20 WM bis-ANS. All the spectral measurements were carried out in 0.1 M Na-phosphate bu¡er (pH 7), using an excitation wavelength of 290 nm. The peaks near 340 nm represent tryptophan £uorescence of the enzyme, while those at 500 nm represent bis-ANS £uorescence.
bis-ANS £uorescence emission decreases at low urea concentrations in the protein titration with a concomitant increase in the tryptophan £uorescence emission is not surprising; Horowitz and Butler [31] have previously suggested that for the binding of bis-ANS is important not only having individual residues exposed, but having those residues be part of an organized hydrophobic surface. Urea disrupts hydrophobic interactions. Thus, above 0.25 M urea, bis-ANS reports the progressive loss of organized hydrophobic surfaces of glucose dehydrogenase. 4. Discussion The unfolding of proteins in denaturing compounds has been widely investigated. In the case of oligomeric proteins, inactivation, dissociation and unfolding have often been described as the structural events that occur in succession during the denaturation process. The present paper reports the activity, conformational and aggregation changes of tetrameric glucose dehydrogenase during unfolding in urea. The denaturation of this enzyme takes place in several stages. The initial changes in secondary and tertiary structure are overlapped with the formation of
229
highly aggregated species that eventually leads ¢rst to a partially unfolded, inactive monomer and then to a complete unfolded state of the molecule. It is interesting that, at very low urea concentrations (0.1^ 0.5 M), where enzyme is still completely active and tetrameric, the binding of bis-ANS is increased, reaching its maximum extent at 0.25 M urea. This increase in the bis-ANS £uorescence is accompanied by a gradual increase in emission intensity of the intrinsic £uorescence of protein; however, little changes are discernible by CD measurements. On the other hand, gel ¢ltration chromatography reveals a slight increase in Rs of the protein. These data suggest that a ¢rst stage in the urea-induced unfolding of glucose dehydrogenase is associated with a modi¢ed state of the native species to give exposed organized hydrophobic surfaces. This intermediate state retains a high degree of secondary structure. Although it is tempting to propose a molten globule like structure for this intermediate [32,33], we must take into account several experimental facts: (a) the chromatographic behavior of glucose dehydrogenase did not reveal a signi¢cant increase in its Rs at the urea concentration at which the £uorescence of enzyme-bound bis-ANS is maximal; (b) the increase of this £uorescence above the basal value in the absence of urea is not as great as expected for a molten globule intermediate; (c) the retention of full enzymatic activity at low urea concentrations. Based on the above evidence, we propose a model in which a localized region of the polypeptide chain undergoes a minor conformational change, thus exposing an organized hydrophobic surface [34]. The next stage in the unfolding of glucose dehydrogenase occurs at urea concentrations in the range of 0.75^2.0 M urea. This transition, that leads to a complete loss of activity, is concomitant with the presence of sticky species that form large aggregates. These high molecular mass forms are eluted in the exclusion volume of the HPLC gel ¢ltration column. Glutaraldehyde cross-linking reveals aggregated protein species at the top of the gel. A major conformational transition is discernible under these conditions by both CD and £uorescence, involving changes in the shape of CD spectrum, a signi¢cant decrease in the amplitude of its negative ellipticity and a gradual red shift of the maximum emission of the intrinsic protein £uorescence. Electrophoresis in a transverse
BBAPRO 36109 10-5-00
230
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
urea gradient, starting with native enzyme, shows a well de¢ned transition from the native tetrameric protein to species with decreased mobility. Concomitant with this electrophoretic transition, a less intense protein band, with a mobility corresponding to the native monomer, is observed. The latter protein band also undergoes a well de¢ned transition to slowly migrating species, but becomes more intense and sharply de¢ned at expense of the aggregated protein, which progressively decreased until it is no longer detectable. That the latter protein band represents, in the lower urea concentration side of the gel, the partially unfolded monomeric species is supported by the appearance at 2.5 M urea of a new î and in cross-linking exspecies with a Rs of 26.5 A periments, by a protein band with a molecular mass of 30 000. These results are consistent with a sharp aggregation occurring at 1.0^1.5 M urea, followed by a similarly drastic dissociation to give a monomeric protein unfolded in some extent at 2.0^2.5 M urea. Finally, a new stage takes place at urea concentrations above 2 M. In this stage, a fully unfolded state of the molecule is reached, as is evident from a further progressive decrease in the amplitude of negative ellipticity, a marked increase of the red-shifted maximum emission of its intrinsic £uorescence and a î. gradual increase in the Rs up to 49 A It is a well known fact that in the refolding and assembly of polypeptide chains of some oligomeric proteins, the observed reactivation will be a function of protein concentration due to a kinetic competition between folding and aggregation [35]. In order to dilucidate the existence of such a phenomenon in the refolding pathway of glucose dehydrogenase, the e¡ect of protein concentration on the reactivation of the enzyme was analyzed. Enzyme samples in the range of 0.001^10 WM subunit were incubated at 8 M urea, diluted and assayed for enzyme activity as described in Section 2.4. No decrease in the e¤ciency of reactivation was observed (data not shown), thus discarding the kinetic competence in the folding mechanism of glucose dehydrogenase as the rate-limiting step. On the basis of the above, a model that summarized the results is as follows: Tn3Tn0 3A3Mn0 3Mu
In this model, Tn represents native tetrameric enzyme that, at low urea concentrations, forms a fully active intermediate (TnP) with a high degree of secondary structure but with more hydrophobic surfaces exposed to the solvent. The exposed hydrophobic surfaces are still organized, since they are able to bind bis-ANS [31]. As the urea concentration is increased, there is further exposure of hydrophobic interactive area that leads to the formation of aggregated species (A). However, these newly exposed hydrophobic surfaces are not organized. Similar results have been reported previously with human placental 17 L estradiol dehydrogenase, but in this case the irreversible formation of aggregates during the refolding process leads to the inactivation of the enzyme [19]. The aggregation of glucose dehydrogenase induced by urea is fully reversible. Finally, in the region of 2.5^3 M urea, the highly aggregated species dissociate into partly unfolded monomers (MnP) that eventually are completely unfolded (Mu) accompanied by a further increase in the intrinsic maximum £uorescence emission. The reversible denaturation of glucose dehydrogenase has been taken as an example that the native conformation of an oligomeric protein is a result of its primary structure [9]. Folding studies of small monomeric proteins often are described by a simple two-state transition. In the case of large multidomain monomeric and oligomeric proteins, the folding process is further complicated by the occurrence of a heterogeneous population of intermediates that folds with di¡erent rates [36^38]. The irreversible formation of aggregates is a well documented fact in oligomeric proteins. It is now accepted that the aggregated forms are generated through wrong interactions of local structures present in folding intermediates; these interactions act as kinetic traps and result in irreversible aggregation. Recently, the occurrence of transient multimeric species during the refolding of a monomeric protein has been reported [39]. In summary, this paper describes some aspects of the denaturation behavior of tetrameric glucose dehydrogenase in urea. The results show that in the folding/assembly pathway of the enzyme, a stable population of highly aggregated species is generated. While aggregation is generally irreversible, these ag-
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
gregated species are transient, since complete reactivation and native quaternary structure is obtained by dilution of the denaturing compound. Acknowledgements This work was supported by Research Grant IN202397 from DGAPA, UNAM. References [1] H. Jo«rnvall, H. Von Bahr-Lindstro«m, K.-D. Jany, W. Ulmer, M. Fro«schle, FEBS Lett. 165 (1984) 190^196. [2] B.D. Church, H. Halvorson, J. Bacteriol. 73 (1957) 470^476. [3] R. Doi, H. Halvorson, B. Church, J. Bacteriol. 77 (1959) 43^ 54. [4] Y. Fujita, R. Ramaley, E. Freese, J. Bacteriol. 132 (1977) 282^293. [5] M. Otani, N. Ihara, C. Umezawa, K. Sano, J. Bacteriol. 167 (1986) 148^152. [6] K. Sano, M. Otani, R. Uehara, M. Kimura, C. Umezawa, Microbiol. Immunol. 32 (1988) 877^885. [7] H.E. Pauly, G. P£eiderer, Hoppe-Seyler's Z. Physiol. Chem. 356 (1975) 1613^1623. [8] K.-D. Jany, W. Ulmer, M. Fro«schle, G. P£eiderer, FEBS Lett. 165 (1984) 6^10. [9] H.E. Pauly, G. P£eiderer, Biochemistry 16 (1977) 4599^4604. [10] E. Maurer, G. P£eiderer, Biochim. Biophys. Acta 827 (1985) 381^388. [11] E. Maurer, G. P£eiderer, Z. Nat.forsch. 42 (1987) 907^915. [12] U.K. Laemmli, Nature 227 (1970) 680^685. [13] M.M. Bradford, Anal. Biochem. 72 (1976) 248^254. [14] W.C. Deal, Biochemistry 8 (1969) 2795^2805. [15] G.K. Ackers, J. Biol. Chem. 242 (1967) 3227^3228. [16] Jaenicke, R. and Rudolph, R., in: T.E. Creighton (Ed.), Protein Structure a Practical Approach, IRL Press, Oxford, 1989, pp. 191^223.
231
[17] T.E. Creighton, J. Mol. Biol. 129 (1979) 235^264. [18] Goldenberg, D.P., in: T.E. Creighton (Ed.), Protein Structure a Practical Approach, IRL Press, Oxford, 1989, pp. 225^250. [19] G. Mendoza-Herna¨ndez, J.L. Rendo¨n, Biochim. Biophys. Acta 1297 (1996) 219^227. [20] K. Weber, M. Osborn, J. Biol. Chem. 244 (1969) 4406^4412. [21] F.J. Farris, G. Weber, C.C. Chiang, I.C. Paul, J. Am. Chem. Soc. 100 (1978) 4469^4474. [22] R. Hermann, R. Rudolph, R. Jaenicke, Nature 277 (1979) 243^245. [23] D.P. Goldenberg, T.E. Creighton, Anal. Biochem. 138 (1984) 1^18. [24] W.C. Johnson Jr., Proteins Struct. Funct. Genet. 7 (1990) 205^214. [25] J.P. Hennessey Jr., W.C. Johnson Jr., C. Bahler, H.G. Wood, Biochemistry 21 (1992) 642^646. [26] L. Stryer, J. Mol. Biol. 13 (1965) 482^495. [27] P.M. Horowitz, V. Prasad, R.F. Luduena, J. Biol. Chem. 259 (1984) 14647^14650. [28] A. Kollmann-Koch, H. Eggerer, Eur. J. Biochem. 185 (1989) 441^447. [29] J.L. Silva, C.F. Silveira, A. Correia Jr., L. Pontes, J. Mol. Biol. 223 (1992) 545^555. [30] F.Y.-H. Wu, C.-W. Wu, Biochemistry 17 (1978) 138^144. [31] P.M. Horowitz, M. Butler, J. Biol. Chem. 268 (1993) 2500^ 2504. [32] O.B. Ptitsyn, R.H. Pain, G.V. Semisotnov, E. Zerovnik, O.I. Razgulyaev, FEBS Lett. 262 (1990) 20^24. [33] C.R. Matthews, Annu. Rev. Biochem. 62 (1993) 653^683. [34] D.T. Haynie, E. Freire, Proteins Struct. Funct. Genet. 16 (1993) 115^140. [35] Garel, J.R., in: T.E. Creighton (Ed.), Protein Folding, Freeman, New York, pp. 405^454, 1992. [36] A. Mitraki, B. Fane, C. Haase-Pettingel, J. Sturtevant, J. King, Science 253 (1991) 54^58. [37] R. Jaenicke, Biochemistry 30 (1991) 3147^3161. [38] M. Nozais, J.-J. Be¨chet, M. Houadjeto, Biochemistry 31 (1992) 1210^1215. [39] F. Pecorari, P. Minard, M. Desmadril, J.M. Yon, J. Biol. Chem. 271 (1996) 5270^5276.
BBAPRO 36109 10-5-00
Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation Guillermo Mendoza-Herna¨ndez *, Fernando Minauro, Juan L. Rendo¨n Departamento de Bioqu|¨mica, Facultad de Medicina, Universidad Nacional Auto¨noma de Me¨xico, Apdo. postal 70-159, Me¨xico, D.F. 04510, Mexico Received 8 July 1999; received in revised form 14 December 1999; accepted 1 February 2000
Abstract The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid. At slightly higher urea concentrations (0.75^2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Glucose dehydrogenase; Urea; Denaturation; Bacillus megaterium
1. Introduction Glucose dehydrogenase (L-D-glucose:NAD(P) 1oxido-reductase, EC 1.1.1.47) from Bacillus megaterium is a member of the short-chain family of alcohol dehydrogenases [1]. The enzyme catalyzes the oxidation of L-D-glucose to D-L-glucono-1,5-lactone using Abbreviations: CD, circular dichroism; Rs , Stokes' radius; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis; Bis-ANS, 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid 1-anilinonaphthalene-8-sulfonic acid * Corresponding author. Fax: +52-5-6162419; E-mail: [email protected]
NAD or NADP as a coenzyme. Glucose dehydrogenase is produced during bacterial sporulation [2^ 4] and it has been reported that it plays an important role in spore germination [5,6]. At neutral pH, the enzyme is a tetrameric protein with a molecular mass of 120 000 and is constituted by identical subunits of 262 amino acid residues each [7], with known amino acid sequence [8]. The protein lacks cysteine and contains four tryptophan residues. This dehydrogenase is readily dissociated into inactive monomers by shifting the pH to 8.5^9.5. Complete reassociation and reactivation has been reported by readjustment to neutral pH [9]. The changes in structure and functional properties of glucose dehydrogenase upon al-
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 0 2 5 - X
BBAPRO 36109 10-5-00
222
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
kaline dissociation have been investigated by several spectroscopic methods. All changes which occur in the course of the alkaline dissociation were found to be fully reversible by readjusting the pH [9^11]. No changes in protein structure other than dissociation were observed. The lack of irreversible conformational changes upon alkaline dissociation of glucose dehydrogenase prompted us to an examination of the denaturing behavior of this enzyme to determine the occurrence of unfolding/folding intermediates. In the present work, changes in enzymatic activity, far-UV circular dichroism (CD), size exclusion chromatography, protein cross-linking, transverse urea gradient gels, intrinsic protein £uorescence and 4,4P-dianilino-1,1P-binaphthyl-5.5P-disulfonic acid 1-anilinonaphthalene-8sulfonic acid (bis-ANS) £uorescence were used to study the denaturation behavior of glucose dehydrogenase from B. megaterium by urea.
appeared homogeneous by polyacrylamide gel electrophoresis under denaturing conditions using the discontinuous bu¡er system of Laemmli [12], on separation gels containing 15% acrylamide. The purity of the preparation was also checked by N-terminal amino acid sequencing using a Beckman LF 3000 protein sequencer. Protein concentrations were determined by the Coomassie blue G dye binding assay of Bradford [13].
2. Materials and methods
2.4. Inactivation by urea
2.1. Reagents
Triplicate inactivation experiments were performed at room temperature by incubation of glucose dehydrogenase (0.2 nmol subunit/ml) in urea solutions of di¡erent concentrations (in the range of 0.1^8 M) for 18 h in a ¢nal volume of 1 ml of 100 mM sodium phosphate bu¡er containing 1 mM EDTA (pH 7.0). After the incubation period, the residual activity was determined by adding a small aliquot of 5 M D-glucose (20 Wl) and 100 mM NAD (10 Wl) stock solutions in the same bu¡er. The reversibility of the inactivation was studied in triplicate samples of enzyme incubated in urea solutions of various concentrations as above but in a ¢nal volume of 30 Wl. After 18 h, the samples were diluted by adding 1 ml of reactivation bu¡er: 100 mM sodium phosphate, 50 mM EDTA (pH 7.0) [14]. The diluted solutions were incubated for a further 24 h and assayed for enzyme activity.
Glucose dehydrogenase from B. megaterium M1286 with an activity of 207 U/mg was purchased from Sigma. Urea (ultrograde) and gel ¢ltration calibration proteins were obtained from Pharmacia LKB. Bis-ANS (dipotassium salt) was from Molecular Probes. Electrophoresis reagents, including calibration standards, were from Bio-Rad. Glutaraldehyde and D-glucose were products from Merck. NAD was obtained from Boehringer Mannheim. All other reagents were of analytical grade and used without further puri¢cation. Reverse osmosis puri¢ed water was used in the preparation of solutions. 2.2. Protein puri¢cation Glucose dehydrogenase was further puri¢ed from the commercial preparation according to the procedure described by Pauly and P£eiderer [7]. The puri¢ed enzyme had a speci¢c enzyme activity of 400 U/ mg using 3 mM NAD, 140 mM glucose as substrates in the assay conditions described by Maurer and P£eiderer [10]. The puri¢ed glucose dehydrogenase
2.3. Enzymatic assays The glucose dehydrogenase activity was determined at room temperature in a Cecil 5000 double beam spectrophotometer by following the initial linear increase in absorbance at 340 nm due to the reduction of NAD by glucose. The reaction mixtures were prepared in a 1 ml cuvette containing 1 mM NAD, 100 mM D-glucose, 1 mM EDTA and 100 mM sodium phosphate (pH 7.0).
2.5. Size exclusion chromatography The structural changes such as oligomerization/dissociation and the presence of unfolded states of glucose dehydrogenase were studied by zonal size exclusion chromatography at di¡erent urea concentrations
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
using a Beckman high performance liquid chromatography (HPLC) system with a 7.5U300 mm Ultraî (Mr Spherogel-SEC 3000 column, pore size of 230 A 3 5 range: 5U10 ^7U10 ). The mobile phase contained 1 mM EDTA, 150 mM NaCl, 100 mM sodium phosphate (pH 7.0) and the required concentration of urea. Before injection, samples were ¢ltered using a 0.22 Wm ¢lter and eluted at a £ow rate of 1 ml/min. The protein pro¢le was monitored at 280 nm. Stokes' radius (Rs ) determinations were performed using well characterized globular protein standards of known Rs as described by Ackers [15]. 2.6. Cross-linking experiments The aggregation state of the enzyme during denaturation with urea was also determined by cross-linking with glutaraldehyde, followed by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^ PAGE). Protein samples were cross-linked with glutaraldehyde, according to Jaenicke et al. [16]. A small volume of glutaraldehyde (25%, w/v) was added to enzyme^urea solutions in standard bu¡er (10 Wg of protein) at 1% (v/v) of the ¢nal mixture. After incubation for 30 s, the reaction was quenched by the addition of solid NaBH4 to give a ¢nal molar ratio of NaBH4 /glutaraldehyde of 2. After 20 min incubation at room temperature, the protein was co-precipitated with sodium deoxycholate by adding trichloroacetic acid (50%, v/v), resolubilized in 20 Wl of 1.5 M Tris^HCl bu¡er (pH 8.8) containing 1% (w/v) SDS and 50 mM DTE and immediately analyzed by SDS^ PAGE. The SDS^PAGE was performed at room temperature with a 4^20% acrylamide gradient slab gel using the Tris^glycine bu¡er system described by Laemmli [12]. The gels were stained with Coomassie blue R and destained using conventional procedures. 2.7. Transverse urea gradient gel electrophoresis The unfolding/folding transitions of glucose dehydrogenase were analyzed by transverse urea gradient electrophoresis, according to Creighton [17]. Polyacrylamide slab gels (100U80 mm) containing a 0^8 M urea concentration gradient were casted with an inverse gradient of acrylamide concentration of 11^7% to ensure uniformity of migration with respect to urea viscosity. The method used for the preparation
223
of gels was adapted from Goldenberg [18] and has been described previously [19]. To avoid the pH-induced dissociation displayed by glucose dehydrogenase in mild alkaline conditions (pH 9), the 100 mM sodium phosphate bu¡er system (pH 7.0) described by Weber and Osborn [20] (without SDS) was used in the casting procedure and as the running bu¡er. Gels were pre-electrophoresed towards the anode for 1 h at 70 V. Either native or previously incubated enzyme in 8 M urea (75 Wg protein) were applied across the top of the gel and electrophoresed at a constant 100 V for 4 h. The gels were stained with Coomassie blue R. 2.8. CD experiments Far-UV CD measurements were carried out on an Aviv 62DS spectropolarimeter interfaced to a computer controlled by Aviv software. The thermostated cell holder was maintained at 25³C. CD spectra were recorded from 200 to 260 nm using strength free quartz cells with 0.1 cm optical path-length. Enzyme samples were scanned after at least 18 h incubation in the appropriate bu¡er^urea solution. Five repetitive scans were performed for each urea concentration at 1 nm intervals, with a time constant of 5 s and 1 nm bandwidth. At 8 M urea, the CD spectra could be recorded only as far as 210 nm. Data were corrected for the baseline contribution of phosphate bu¡er and urea. The residue ellipticities were calculated from the average data obtained in this way for two independent experiments. The mean residue weight was calculated from the amino acid composition of the enzyme [8]. The ellipticity at 220 nm was taken to indicate changes in the protein secondary structure. 2.9. Fluorescence studies Fluorescence measurements were made on an Aminco SPF-500 spectro£uorimeter with a semimicro 1 cm light path cell. Samples were excited at 295 nm to analyze selectively the tryptophan residues of the protein, and emission was recorded from 300 to 540 nm; bandwidths were 4 nm for excitation and 5 nm for the emission monochromator. For experiments in the presence of bis-ANS, the £uorescence emission spectra were obtained by using
BBAPRO 36109 10-5-00
224
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
an excitation wavelength of 380 nm. The excitation and emission spectral bandwidths were 4 and 8 nm, respectively. The concentration of bis-ANS was determined spectrophotometrically at 387 nm based in a molar extinction coe¤cient of 17 000 M31 cm31 [21]. The £uorescence intensity of the dye bound to the enzyme was assessed by adding in a Wl volume, a ¢xed concentration of bis-ANS (3.5 WM, ¢nal) to a cuvette containing 1 ml of native or pre-incubated enzyme^urea solution. Protein was 0.4 WM. Corrections were made by subtracting the £uorescence intensity of the dye in bu¡er at the same concentration of urea. Quenching of tryptophan £uorescence of glucose dehydrogenase by bis-ANS was studied as above, but the excitation wavelength was 290 nm and emission was monitored from 300 to 650 nm. 3. Results 3.1. Activity of glucose dehydrogenase during denaturation with urea Results of the changes of enzymatic activity of glucose dehydrogenase after incubation for 18 h in urea solutions of di¡erent concentration are shown in Fig. 1. The enzyme is stable in urea only up to 0.5
Fig. 2. Size exclusion HPLC elution pro¢les of B. megaterium glucose dehydrogenase at selected urea concentrations. Protein samples (1 mg/ml) were incubated with di¡erent concentrations of urea for 18 h; then, a 50 Wl aliquot was chromatographed on a UltraSpherogel-SEC 3000 column pre-equilibrated with the required concentration of urea. Other conditions were as described in Section 2.
M, losing its activity with increasing concentrations of the denaturant. The smooth inactivation curve has a midpoint at 1.25 M urea. On the other hand, complete reactivation was attained after a 35-fold dilution of the pre-incubation mixtures, even at an urea concentration of 8 M (Fig. 1, inset). 3.2. Size exclusion chromatography
Fig. 1. Relative activity changes of glucose dehydrogenase as a function of urea concentration. The enzyme was incubated at the indicated denaturant concentrations for 18 h; thereafter, residual activity was determined in the same urea solution as described under Section 2. (Inset) Time course of reactivation of an enzyme sample pre-incubated for 18 h in 8 M urea after a 35-fold dilution with phosphate, EDTA bu¡er. In both cases, a ¢nal concentration of 0.2 nmol subunit/ml was used. Each point is the average of three independent determinations.
In order to investigate the changes in the oligomeric state of glucose dehydrogenase, its chromatographic behavior on a gel ¢ltration column was analyzed under both native conditions and in the presence of di¡erent concentrations of urea. Fig. 2 shows the elution pro¢les of native and urea-treated enzyme at di¡erent concentrations of denaturing reagent. In native conditions, a single chromatoî graphic peak was detected with a Rs of 44.5 A (Mr = 120 000). At 1 M urea, a second minor peak, eluting in the void volume of the column, was observed, suggesting the formation of polymeric species. The relative proportions of these two peaks changed with increasing urea concentrations. Be-
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
225
Fig. 3. Rs values and relative abundance of glucose dehydrogenase species as a function of urea concentration. The size exclusion chromatographic data from Fig. 2 were used to determine: (A) the Rs and (B) the relative abundance of glucose dehydrogenase speî ) were used for calibration: thyroglobulin, cies as a function of urea concentration. Protein standards with the following Rs values (A 85.0; ferritin, 61.0; catalase, 52.2; aldolase, 48.1; albumin, 35.5; ovalbumin, 30.5; chymotrypsinogen A, 20.9; ribonuclease A; 16.4. The relative abundances of included and aggregated forms were determined by the peak area using the Beckman system Gold software.
tween 1 and 2 M urea, the relative abundance of the excluded peak sharply increased at expense of the tetrameric protein. At the later urea concentration, î, a minor new peak, corresponding to a Rs of 26.5 A appeared. The Rs value strongly suggests that this new species corresponds to the monomeric protein. At 2.5 M urea and over, this was the only peak detected; however, its elution volume was diminishing with increasing urea concentrations. At 8 M urea, î . The its elution volume corresponds to a Rs of 49 A Rs values of the included species and the relative abundance of included and aggregated forms determined by the peak area are shown in Fig. 3A,B. 3.3. Cross-linking with glutaraldehyde and SDS^PAGE analysis Cross-linking experiments can be performed in a su¤ciently short time to freeze the states of association during either unfolding or folding process [22]. Fig. 4 shows the electrophoretic species pattern of the enzyme after covalent cross-linking in the presence of di¡erent concentrations of urea. In the absence of urea, glucose dehydrogenase was almost entirely cross-linked, and only the tetrameric form could be detected. Up to 1.5 M urea, tetramer still represents the main species, but now coexist with both monomeric and aggregated small fractions of
Fig. 4. SDS^PAGE of glucose dehydrogenase after covalent cross-linking in the presence of various concentrations of urea. Enzyme samples (20 Wg) were pre-incubated 18 h at the selected urea concentration and then mixed with glutaraldehyde and NaBH4 as described in Section 2. Protein was co-precipitated with sodium deoxycholate, dissolved in SDS sample treatment bu¡er and electrophoresed on a 4^20% acrylamide gradient SDS slab gel. Standard markers (lane 1); glucose dehydrogenase, control (lane 2); glucose dehydrogenase after cross-linking without urea (lane 3). Enzyme incubated in urea at 1, 1.5, 2.0, 2.5, 3.0, 5.0 and 7.0 M (lanes 4^10, respectively) before crosslinking.
BBAPRO 36109 10-5-00
226
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
the enzyme. At 2.0 M urea, higher aggregates, which could not be resolved on the 4^20% acrylamide gradient gel, were the only species detected on top of the gel. The multimeric species are present in a short range of urea concentrations, so at 2.5 M urea, their relative abundances sharply decrease. The disappearance of the multimeric species is concomitant with the presence of a band corresponding to the monomeric protein. From 3 to 8 M urea, the monomeric species was the only band detectable. The cross-linking experiments con¢rm the data obtained by HPLC gel ¢ltration and provide evidence that the denaturation of glucose dehydrogenase with urea involves an initial stage of unfolding with the formation of a more expanded tetrameric enzyme, production of multimeric species, dissociation to the partially unfolded monomer, followed by further unfolding of the latter. 3.4. Transverse urea gradient gel electrophoresis Electrophoresis through polyacrylamide gels with a perpendicular gradient of urea is a sensitive method of resolving di¡erent conformational states of a protein and has also been extensively used to identify unfolding intermediates [23]. In order to more fully characterize the unfolding transitions induced by urea of glucose dehydrogenase, the electrophoretic behavior of the enzyme was analyzed in transverse urea gradient gels (0^8 M). From the electrophoretic pro¢les obtained starting with either native (Fig. 5A) or previously unfolded enzyme (Fig. 5B), it is clear that the enzyme does not follow a simple two-state cooperative unfolding transition, but suggest the presence of multiple species that interconvert and unfold with di¡erent rates. When native glucose dehydrogenase was applied to the gel, a pattern consisting of two well de¢ned bands, parallel to the urea concentration gradient, was obtained. Besides these two bands, there is a transition zone as is seen by a shared, faint, di¡use, vertical band in a short interval of intermediate urea concentrations. The upper band showed three distinct mobilities regions; at low urea concentrations (0^2 M), the protein had a mobility corresponding to the tetrameric enzyme in the native conformation, followed by a well de¢ned in£exion point to species with decreased mobility. The latter species were detectable up to 3.5 M urea. This pat-
Fig. 5. Transverse urea gradient gel electrophoresis of B. megaterium glucose dehydrogenase. One hundred Wl samples containing 75 Wg of native (A) or previously incubated enzyme in 8 M urea for 18 h (B) were layered across the top of a slab gel containing a 0^8 M transverse gradient of urea and subjected to electrophoresis toward the anode at pH 7.0. Other conditions were as described under Section 2.
tern is consistent with a rapid transition from the native to the aggregated forms of the enzyme. At low urea concentrations (0.5^1 M), the lower band was detectable as a di¡use band with an electrophoretic mobility corresponding to the native monomer, followed by a similarly di¡use transition zone, while at higher urea concentrations (2.5^8 M), the gel displayed a sharply de¢ned band with a reduced electrophoretic mobility. These species, however, move faster than the native tetramer. The existence of a heterogeneous population of unfolded forms in the folding/assembly pathway of glucose dehydrogenase was also evident from the pattern obtained when electrophoresis was initiated with the unfolded protein (Fig. 5B). The gel displayed now one continuous and predominant band extending through the complete urea gradient, a band of oligomers at the top of the gel in the region of 1.5^3.0 M urea, and a shared, faint band extending from the oligomers to the faster mobility region
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
on the continuous protein band. Species with an electrophoretic mobility corresponding to that of the native tetrameric protein were not detected, suggesting the presence of a slow assembly step on the time scale of the electrophoresis. These results suggest that during the folding of glucose dehydrogenase, an heterogeneous population of intermediates, including high molecular mass oligomers, with di¡erent interconversion rates is present. 3.5. CD studies CD spectroscopy was used to monitor the changes in the secondary structure content of glucose dehydrogenase upon urea-induced unfolding. The far-UV CD spectra of the native and pre-incubated enzyme in urea solutions of di¡erent concentrations are depicted in Fig. 6. The spectrum of the native enzyme was characterized by an almost £at region between two negative minima around 210 and 222 nm. The spectrum crosses the base line from negative to positive at 198^199 nm. Incubation of the enzyme in
Fig. 6. Far-UV CD spectra of B. megaterium glucose dehydrogenase in urea. Enzyme samples were incubated for 18 h at selected concentrations of urea before spectra were recorded. Concentration of urea (M): 0 (b); 1.2 (R); 1.5 (O); 1.75 (a); 3.0 (7); 8 (8). For clarity, the spectra obtained with other concentrations of urea are not shown. The inset is the transition curve of glucose dehydrogenase as measured by changes in ellipticity at 222 nm.
227
urea solutions results in both changes in the shape of the CD spectrum and in an urea concentrationdependent loss in ellipticity. Between 0.75 and 1.2 M urea, the CD spectra showed a negative minima peak at 213 nm with an increase in the ratio of [3]213 / [3]222 , reaching a maximum at 1 M urea. A red shift of the wavelength at which signals cross from negative to positive ellipticity values (204^205 nm) was also observed, as compared with the native protein. Similar changes in the CD spectra have been observed previously with other oligomeric proteins [24,25], and are consistent with conformational changes that a¡ect the state of oligomerization of glucose dehydrogenase. On the other hand, the molar ellipticity at 222 nm changes sharply toward more positive values in the range 1^1.75 M urea, followed by a smoother change up to 8 M urea (inset). 3.6. Fluorescence studies of glucose dehydrogenase Intrinsic £uorescence is an excellent spectroscopic probe to investigate conformational changes in tertiary structure of proteins. The conformational transitions of glucose dehydrogenase were also monitored by following the changes in intensity and position of the maximum £uorescence emission in the presence of urea. As shown in Fig. 7, the maximum of the tryptophan £uorescence emission of enzyme was at 340 nm in the native state. Upon denaturation by 8 M urea, the maximum emission was shifted to 360 nm. The induced urea red shift was accompanied by an increase in £uorescence intensity. During the denaturation process, an initial stage of £uorescence changes takes place at low urea concentrations involving changes in both emission intensity and a gradual red shift in the wavelength of maximum emission. From 0.25 M to 1 M urea, an increase in the emission intensity with slight changes in the wavelength of the tryptophan maximum emission was observed. At urea concentrations higher than 1 M, a decrease in emission intensity and a marked red shift of the maximum emission takes place, leading to a completion at 2 M urea. Further changes in the tryptophan environment occur at urea concentrations higher than 2 M, as was evident from a new gradual increase in the emission intensity up to 8 M urea. The changes in the maximum emission wavelength and in £uorescence intensity at 360 nm as a
BBAPRO 36109 10-5-00
228
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
3.8. Quenching of tryptophan £uorescence of glucose dehydrogenase by bis-ANS
Fig. 7. Fluorescence emission spectra of B. megaterium glucose dehydrogenase under native conditions and in the presence of 8 M urea. The native protein (- - -) was in 0.1 M phosphate bu¡er (pH 7.0); the unfolded sample (9) was pre-incubated for 18 h and contained 8 M urea in addition. The £uorescence of the native enzyme at 340 nm was taken as 100%. Other conditions were as described in Section 2. The inset shows the red shift in the wavelength of the maximum £uorescence emission (b), and the relative changes in £uorescence emission intensity at 360 nm (a) as a function of urea molarity.
Bis-ANS is an aromatic compound that interact with aromatic amino acids in proteins through hydrophobic interactions [30]. To obtain further information on the environment of the tryptophan residues under native and denaturing conditions, quenching experiments were performed. In the dyefree state, native glucose dehydrogenase £uoresces with a maximum near 340 nm due to tryptophan emission (Fig. 7). Upon the addition of increasing concentrations of bis-ANS to the enzyme solution, the bis-ANS £uorescence emission (500 nm) increases with a concurrent decrease in the enzyme tryptophan emission. At 4 WM bis-ANS, quenching of enzyme £uorescence was 50% (Fig. 9). These data indicate that, in fact, tryptophan residues on the surface of the native enzyme are accessible to this quencher. When bis-ANS was added to pre-incubated enzyme solutions of increasing urea concentrations, the changes in the bis-ANS £uorescence were as described in the individual studies (Fig. 8). That the
function of urea concentration are depicted in Fig. 7 (inset). 3.7. Binding of bis-ANS to glucose dehydrogenase Anilinonaphthalene sulfonates have widely been used as sensitive reporters of apolar regions in proteins and to probe protein conformational changes [26^29]. Fig. 8 shows the £uorescence emission spectra of free and native enzyme-bound bis-ANS. On excitation at 380 nm, free bis-ANS in the aqueous bu¡er solution was non-£uorescent. The addition of the dye to the native glucose dehydrogenase in the bu¡er solution produced a marked enhancement of £uorescence intensity, indicating the binding of bisANS to the enzyme. The maximum emission of this complex was at 500 nm. The interaction of bis-ANS with glucose dehydrogenase was a¡ected by urea (Fig. 8, inset). Upon addition to pre-incubated enzyme^urea mixtures, the bis-ANS £uorescence intensity was further increased at very low urea concentrations, reaching a maximum at 0.25 M urea. Higher concentrations of denaturant were accompanied by a sharply decrease in bis-ANS £uorescence.
Fig. 8. Binding of bis-ANS to glucose dehydrogenase under native conditions and in the presence of di¡erent concentrations of urea. Fluorescence emission spectra of 3.5 WM of bis-ANS alone in 0.1 M Na-phosphate bu¡er (lower spectrum) or in enzyme solutions under native conditions and in the presence of urea. Excitation was at 380 nm. Numbers above the curves refer to the concentrations of urea at which enzyme was pre-incubated. The inset shows the relative bis-ANS £uorescence intensity at 500 nm in enzyme solutions as a function of urea concentration. The maximum of £uorescence that appeared at 0.25 M urea was taken as 100%.
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
Fig. 9. Quenching of tryptophan £uorescence of B. megaterium glucose dehydrogenase by bis-ANS. Fluorescence emission spectra of samples with glucose dehydrogenase (0.4 WM) in the presence of (1) 0, (2) 1, (3) 2, (4) 4, (5) 6, (6) 10 and (7) 20 WM bis-ANS. All the spectral measurements were carried out in 0.1 M Na-phosphate bu¡er (pH 7), using an excitation wavelength of 290 nm. The peaks near 340 nm represent tryptophan £uorescence of the enzyme, while those at 500 nm represent bis-ANS £uorescence.
bis-ANS £uorescence emission decreases at low urea concentrations in the protein titration with a concomitant increase in the tryptophan £uorescence emission is not surprising; Horowitz and Butler [31] have previously suggested that for the binding of bis-ANS is important not only having individual residues exposed, but having those residues be part of an organized hydrophobic surface. Urea disrupts hydrophobic interactions. Thus, above 0.25 M urea, bis-ANS reports the progressive loss of organized hydrophobic surfaces of glucose dehydrogenase. 4. Discussion The unfolding of proteins in denaturing compounds has been widely investigated. In the case of oligomeric proteins, inactivation, dissociation and unfolding have often been described as the structural events that occur in succession during the denaturation process. The present paper reports the activity, conformational and aggregation changes of tetrameric glucose dehydrogenase during unfolding in urea. The denaturation of this enzyme takes place in several stages. The initial changes in secondary and tertiary structure are overlapped with the formation of
229
highly aggregated species that eventually leads ¢rst to a partially unfolded, inactive monomer and then to a complete unfolded state of the molecule. It is interesting that, at very low urea concentrations (0.1^ 0.5 M), where enzyme is still completely active and tetrameric, the binding of bis-ANS is increased, reaching its maximum extent at 0.25 M urea. This increase in the bis-ANS £uorescence is accompanied by a gradual increase in emission intensity of the intrinsic £uorescence of protein; however, little changes are discernible by CD measurements. On the other hand, gel ¢ltration chromatography reveals a slight increase in Rs of the protein. These data suggest that a ¢rst stage in the urea-induced unfolding of glucose dehydrogenase is associated with a modi¢ed state of the native species to give exposed organized hydrophobic surfaces. This intermediate state retains a high degree of secondary structure. Although it is tempting to propose a molten globule like structure for this intermediate [32,33], we must take into account several experimental facts: (a) the chromatographic behavior of glucose dehydrogenase did not reveal a signi¢cant increase in its Rs at the urea concentration at which the £uorescence of enzyme-bound bis-ANS is maximal; (b) the increase of this £uorescence above the basal value in the absence of urea is not as great as expected for a molten globule intermediate; (c) the retention of full enzymatic activity at low urea concentrations. Based on the above evidence, we propose a model in which a localized region of the polypeptide chain undergoes a minor conformational change, thus exposing an organized hydrophobic surface [34]. The next stage in the unfolding of glucose dehydrogenase occurs at urea concentrations in the range of 0.75^2.0 M urea. This transition, that leads to a complete loss of activity, is concomitant with the presence of sticky species that form large aggregates. These high molecular mass forms are eluted in the exclusion volume of the HPLC gel ¢ltration column. Glutaraldehyde cross-linking reveals aggregated protein species at the top of the gel. A major conformational transition is discernible under these conditions by both CD and £uorescence, involving changes in the shape of CD spectrum, a signi¢cant decrease in the amplitude of its negative ellipticity and a gradual red shift of the maximum emission of the intrinsic protein £uorescence. Electrophoresis in a transverse
BBAPRO 36109 10-5-00
230
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
urea gradient, starting with native enzyme, shows a well de¢ned transition from the native tetrameric protein to species with decreased mobility. Concomitant with this electrophoretic transition, a less intense protein band, with a mobility corresponding to the native monomer, is observed. The latter protein band also undergoes a well de¢ned transition to slowly migrating species, but becomes more intense and sharply de¢ned at expense of the aggregated protein, which progressively decreased until it is no longer detectable. That the latter protein band represents, in the lower urea concentration side of the gel, the partially unfolded monomeric species is supported by the appearance at 2.5 M urea of a new î and in cross-linking exspecies with a Rs of 26.5 A periments, by a protein band with a molecular mass of 30 000. These results are consistent with a sharp aggregation occurring at 1.0^1.5 M urea, followed by a similarly drastic dissociation to give a monomeric protein unfolded in some extent at 2.0^2.5 M urea. Finally, a new stage takes place at urea concentrations above 2 M. In this stage, a fully unfolded state of the molecule is reached, as is evident from a further progressive decrease in the amplitude of negative ellipticity, a marked increase of the red-shifted maximum emission of its intrinsic £uorescence and a î. gradual increase in the Rs up to 49 A It is a well known fact that in the refolding and assembly of polypeptide chains of some oligomeric proteins, the observed reactivation will be a function of protein concentration due to a kinetic competition between folding and aggregation [35]. In order to dilucidate the existence of such a phenomenon in the refolding pathway of glucose dehydrogenase, the e¡ect of protein concentration on the reactivation of the enzyme was analyzed. Enzyme samples in the range of 0.001^10 WM subunit were incubated at 8 M urea, diluted and assayed for enzyme activity as described in Section 2.4. No decrease in the e¤ciency of reactivation was observed (data not shown), thus discarding the kinetic competence in the folding mechanism of glucose dehydrogenase as the rate-limiting step. On the basis of the above, a model that summarized the results is as follows: Tn3Tn0 3A3Mn0 3Mu
In this model, Tn represents native tetrameric enzyme that, at low urea concentrations, forms a fully active intermediate (TnP) with a high degree of secondary structure but with more hydrophobic surfaces exposed to the solvent. The exposed hydrophobic surfaces are still organized, since they are able to bind bis-ANS [31]. As the urea concentration is increased, there is further exposure of hydrophobic interactive area that leads to the formation of aggregated species (A). However, these newly exposed hydrophobic surfaces are not organized. Similar results have been reported previously with human placental 17 L estradiol dehydrogenase, but in this case the irreversible formation of aggregates during the refolding process leads to the inactivation of the enzyme [19]. The aggregation of glucose dehydrogenase induced by urea is fully reversible. Finally, in the region of 2.5^3 M urea, the highly aggregated species dissociate into partly unfolded monomers (MnP) that eventually are completely unfolded (Mu) accompanied by a further increase in the intrinsic maximum £uorescence emission. The reversible denaturation of glucose dehydrogenase has been taken as an example that the native conformation of an oligomeric protein is a result of its primary structure [9]. Folding studies of small monomeric proteins often are described by a simple two-state transition. In the case of large multidomain monomeric and oligomeric proteins, the folding process is further complicated by the occurrence of a heterogeneous population of intermediates that folds with di¡erent rates [36^38]. The irreversible formation of aggregates is a well documented fact in oligomeric proteins. It is now accepted that the aggregated forms are generated through wrong interactions of local structures present in folding intermediates; these interactions act as kinetic traps and result in irreversible aggregation. Recently, the occurrence of transient multimeric species during the refolding of a monomeric protein has been reported [39]. In summary, this paper describes some aspects of the denaturation behavior of tetrameric glucose dehydrogenase in urea. The results show that in the folding/assembly pathway of the enzyme, a stable population of highly aggregated species is generated. While aggregation is generally irreversible, these ag-
BBAPRO 36109 10-5-00
G. Mendoza-Herna¨ndez et al. / Biochimica et Biophysica Acta 1478 (2000) 221^231
gregated species are transient, since complete reactivation and native quaternary structure is obtained by dilution of the denaturing compound. Acknowledgements This work was supported by Research Grant IN202397 from DGAPA, UNAM. References [1] H. Jo«rnvall, H. Von Bahr-Lindstro«m, K.-D. Jany, W. Ulmer, M. Fro«schle, FEBS Lett. 165 (1984) 190^196. [2] B.D. Church, H. Halvorson, J. Bacteriol. 73 (1957) 470^476. [3] R. Doi, H. Halvorson, B. Church, J. Bacteriol. 77 (1959) 43^ 54. [4] Y. Fujita, R. Ramaley, E. Freese, J. Bacteriol. 132 (1977) 282^293. [5] M. Otani, N. Ihara, C. Umezawa, K. Sano, J. Bacteriol. 167 (1986) 148^152. [6] K. Sano, M. Otani, R. Uehara, M. Kimura, C. Umezawa, Microbiol. Immunol. 32 (1988) 877^885. [7] H.E. Pauly, G. P£eiderer, Hoppe-Seyler's Z. Physiol. Chem. 356 (1975) 1613^1623. [8] K.-D. Jany, W. Ulmer, M. Fro«schle, G. P£eiderer, FEBS Lett. 165 (1984) 6^10. [9] H.E. Pauly, G. P£eiderer, Biochemistry 16 (1977) 4599^4604. [10] E. Maurer, G. P£eiderer, Biochim. Biophys. Acta 827 (1985) 381^388. [11] E. Maurer, G. P£eiderer, Z. Nat.forsch. 42 (1987) 907^915. [12] U.K. Laemmli, Nature 227 (1970) 680^685. [13] M.M. Bradford, Anal. Biochem. 72 (1976) 248^254. [14] W.C. Deal, Biochemistry 8 (1969) 2795^2805. [15] G.K. Ackers, J. Biol. Chem. 242 (1967) 3227^3228. [16] Jaenicke, R. and Rudolph, R., in: T.E. Creighton (Ed.), Protein Structure a Practical Approach, IRL Press, Oxford, 1989, pp. 191^223.
231
[17] T.E. Creighton, J. Mol. Biol. 129 (1979) 235^264. [18] Goldenberg, D.P., in: T.E. Creighton (Ed.), Protein Structure a Practical Approach, IRL Press, Oxford, 1989, pp. 225^250. [19] G. Mendoza-Herna¨ndez, J.L. Rendo¨n, Biochim. Biophys. Acta 1297 (1996) 219^227. [20] K. Weber, M. Osborn, J. Biol. Chem. 244 (1969) 4406^4412. [21] F.J. Farris, G. Weber, C.C. Chiang, I.C. Paul, J. Am. Chem. Soc. 100 (1978) 4469^4474. [22] R. Hermann, R. Rudolph, R. Jaenicke, Nature 277 (1979) 243^245. [23] D.P. Goldenberg, T.E. Creighton, Anal. Biochem. 138 (1984) 1^18. [24] W.C. Johnson Jr., Proteins Struct. Funct. Genet. 7 (1990) 205^214. [25] J.P. Hennessey Jr., W.C. Johnson Jr., C. Bahler, H.G. Wood, Biochemistry 21 (1992) 642^646. [26] L. Stryer, J. Mol. Biol. 13 (1965) 482^495. [27] P.M. Horowitz, V. Prasad, R.F. Luduena, J. Biol. Chem. 259 (1984) 14647^14650. [28] A. Kollmann-Koch, H. Eggerer, Eur. J. Biochem. 185 (1989) 441^447. [29] J.L. Silva, C.F. Silveira, A. Correia Jr., L. Pontes, J. Mol. Biol. 223 (1992) 545^555. [30] F.Y.-H. Wu, C.-W. Wu, Biochemistry 17 (1978) 138^144. [31] P.M. Horowitz, M. Butler, J. Biol. Chem. 268 (1993) 2500^ 2504. [32] O.B. Ptitsyn, R.H. Pain, G.V. Semisotnov, E. Zerovnik, O.I. Razgulyaev, FEBS Lett. 262 (1990) 20^24. [33] C.R. Matthews, Annu. Rev. Biochem. 62 (1993) 653^683. [34] D.T. Haynie, E. Freire, Proteins Struct. Funct. Genet. 16 (1993) 115^140. [35] Garel, J.R., in: T.E. Creighton (Ed.), Protein Folding, Freeman, New York, pp. 405^454, 1992. [36] A. Mitraki, B. Fane, C. Haase-Pettingel, J. Sturtevant, J. King, Science 253 (1991) 54^58. [37] R. Jaenicke, Biochemistry 30 (1991) 3147^3161. [38] M. Nozais, J.-J. Be¨chet, M. Houadjeto, Biochemistry 31 (1992) 1210^1215. [39] F. Pecorari, P. Minard, M. Desmadril, J.M. Yon, J. Biol. Chem. 271 (1996) 5270^5276.
BBAPRO 36109 10-5-00