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Human glycine N-methyltransferase is unfolded by urea through a compact monomer state
ABB Archives of Biochemistry and Biophysics 420 (2003) 153–160 www.elsevier.com/locate/yabbi
Human glycine N-methyltransferase is unfolded by urea through a compact monomer stateq Zigmund Luka and Conrad Wagner* Department of Biochemistry, Medical Center, Vanderbilt University, School of Medicine, 620 Light Hall, Nashville, TN 37232-0146, USA Received 24 July 2003, and in revised form 4 September 2003
Abstract Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0–3.5 M urea with enzyme inactivation. The equal to that of globular proteins with the same molecular compact monomers were characterized by Stokes radius (Rs ) of 40.7 A mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully , exposed tryptophan residues, and disrupted secondary structure in 8 M urea. unfolded state with Rs of 50 A Ó 2003 Elsevier Inc. All rights reserved. Keywords: Glycine N-methyltransferase; Unfolding; Dissociation; Inactivation; Stability; Conformation
Glycine N-methyltransferase (GNMT) is an abundant mammalian enzyme that transfers the methyl group from S-adenosylmethionine (AdoMet)1 to glycine producing sarcosine and S-adenosylhomocysteine (AdoHcy) [1,2]. Sarcosine has no known metabolic function and is reconverted to glycine by a mitochondrial enzyme system. Therefore, the biological role of GNMT was proposed to maintain the ratio of AdoMet/ AdoHcy by converting excess AdoMet to AdoHcy without the synthesis of a metabolically active product. GNMT is a key enzyme in the control of the methylation capacity of the cell. GNMT is a tetrameric protein consisting of four 32kDa subunits [3]. The amino acid sequence of GNMT q This work was supported by Grants DK15289 and DK54859 from the US Public Health Service and from the Office of Research and Development, Medical Research Service, Department of Veteran Affairs. * Corresponding author. Fax: 1-615-343-0407. E-mail address: [email protected] (C. Wagner). 1 Abbreviations used: GNMT, glycine N-methyltransferase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; Ve , elution volume of proteins determined by position of maximum of elution peak; SEC, size-exclusion chromatography; Rs , Stokes radius.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.09.009
from rat, mouse, rabbit, pig, and human has been derived from the nucleotide sequence of cDNAs [4–7]. Identity among protein sequences is 89–93% [4]. The crystal structure of recombinant rat GNMT apoprotein and its complex with AdoMet and with AdoHcy has been obtained by X-ray analysis [8–10]. Recently, the crystal structure of recombinant human GNMT was solved (Pakhomova et al. unpublished results), which appeared to be very similar to the crystal structure of rat enzyme. In the crystal, the tetrameric structure of GNMT is formed by multiple hydrogen bonds between b-sheets as well as by ion-pair interactions between charged residues of different subunits [8,9]. Native rat GNMT is isolated with bound 5-methyltetrahydrofolate pentaglutamate (5-CH3 THFG5 ) [11]. We showed that GNMT is inhibited by 5-CH3 THFG5 when one molecule of the inhibitor is bound to the purified tetramer [12]. Activity of the enzyme is also regulated in vivo by a variety of factors like methionine and vitamin A [13,14]. Another possible mode of regulation of GNMT activity includes association/dissociation of the tetramer. It was shown that fluorescein-labeled GNMT could be transported into isolated rat liver nuclei as a monomer [15]. While the physiological
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relevance of the latter observation is unclear, it is a further reason to study the conformational stability of GNMT by biophysical methods. We examined GNMT unfolding as the first step in the biophysical characterization of GNMT. The following questions were addressed: (1) How stable is the tetrameric structure of GNMT? (2) What is the mechanism of dissociation and unfolding of GNMT by a chemical denaturant? (3) What conditions are required for refolding of the tetramer from unfolded monomers? To answer these questions, we analyzed the urea unfolding of human GNMT by enzyme activity, size-exclusion chromatography (SEC), fluorescence spectroscopy, and circular dichroism.
Materials and methods Protein preparation The human GNMT cDNA was cloned and expressed in the pET-17b expression vector as reported earlier [16]. Briefly, the GNMT cDNA was synthesized by RT-PCR from the human poly(A) RNA (Clontech, Palo Alto, CA). Cloned cDNA was introduced into a pET-17b expression vector. Expression was done at 20 °C for 17– 18 h with 1 mM IPTG. The GNMT protein was isolated and purified by ammonium sulfate precipitation from the crude extract and ion-exchange chromatography on DE-52 column. In the final step, the protein was passed through a Sephacryl S-200 size-exclusion column. The final purity of GNMT samples was at least 95–97%. GNMT activity This was assayed using the charcoal adsorption method described earlier [12]. In 100 ll of reaction mixture, the concentration of the reagents was: 50 mM Tris–HCl buffer, pH 8.0, 150 mM NaCl, 200 nM [methyl-3 H]AdoMet (3–5 103 dpm), 20.0 mM glycine, 5 mM DTT, and 0.3–1.5 lg enzyme. After incubation at 25 °C for 15 min, the reaction was stopped by 50 ll of 10% trichloroacetic acid and 250 ll of a suspension of acid-treated charcoal was added to adsorb the unreacted labeled AdoMet. The suspension was incubated in ice for 15 min. After centrifugation in a microcentrifuge, the radioactivity in the supernatant containing the labeled sarcosine was counted in a Tri-Carb Liquid Scintillation Analyzer (Packard Instrument, Meriden, CT). Enzyme activity in urea solution was measured essentially as above with some modifications. First, two master mixes were prepared. Mix I contained buffer, substrates, and other necessary additives for activity assay, and the desired concentration of urea. Enzyme unfolding was done in a separate Mix II containing buffer solution and
the same concentration of urea in which activity was measured later. The volume ratio of Mix II/Mix I was kept constant (1:1) and high in order to minimize any effect of protein dilution on the dissociation parameters. Fluorescence spectroscopy showed that GNMT in various urea solutions reached an equilibrium between folded and unfolded protein in 4 M urea in 15 min and in 8 M urea in about 5 min. Therefore, the protein in all urea unfolding experiments was incubated at ambient temperature for 1 h before measurement. After incubation for 1 h, both solutions were mixed and the reaction was assayed at 25 °C for 15 min. No degradation of proteins after the unfolding experiments was found by SDS–electrophoresis. The working solutions containing urea were prepared by dilution from a 10 M urea stock solution. The 10 M urea solution was prepared fresh for each experiment to minimize carbamylation of the GNMT by cyanate formed by urea hydrolysis [17]. The urea solutions were filtered through a 0.2 lm filter and its concentration was determined from refractive index [18]. Size-exclusion chromatography € KTA purifier System Experiments were done on the A (Amersham–Pharmacia Biotech., Piscataway, NJ) using a Superose-12 column in Column Buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 14 mM bmercaptoethanol at a flow rate of 0.5 ml/min at 22 °C. Some SEC experiments were done with Column Buffer at pH 7.5. The sample volume was 100 ll. The elution of the proteins was monitored by absorbance at 280 nm. The column was calibrated with cytochrome c, lysozyme, chymotrypsin, carbonic anhydrase, ovalbumin, BSA, alcohol dehydrogenase, potato b-amylase, and aldolase without added urea and also in Column Buffer containing 8 M urea. The void volume Vo was determined with blue dextran and the total solvent-accessible volume Vt was determined with acetone. The GNMT samples were incubated in urea solution for 1 h before applying to the column. To determine whether AdoMet affected the quaternary structure of GNMT, a smaller column (1 15 cm, volume 15 ml) with Sephacryl S-200 resin was used. The elution of GNMT was done in the Column Buffer without and with 100 lM AdoMet. Fluorescence spectroscopy Protein fluorescence spectra were recorded on a Perkin–Elmer 650-40 Fluorescence Spectrophotometer (Perkin–Elmer, Norwalk, CT). Fluorescence emission spectra were recorded from 300 to 400 nm with slits of 5 nm and a scanning speed of 2 nm/s in a 10 mm cuvette at 22 °C. Simultaneous intrinsic tryptophan and tyrosine fluorescence was measured with excitation at 285 nm and tryptophan fluorescence was recorded by using excita-
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tion at 300 nm. The protein concentrations were 0.05– 0.13 mg/ml with absorbance values not higher than 0.1 at the excitation wavelength to avoid the inner filter effect. Circular dichroism Spectra were recorded on a Jasco-720 spectropolarimeter. The spectral data of the samples were scanned in the range 250–190 nm four times and averaged and smoothed by the instrument software. The protein concentration of the samples was 0.13 mg/ml with an optical path length of 1.0 or 10.0 mm. The scan speed was 10 nm/min, at a sensitivity of 20 mdeg, resolution of 1 nm, and a time constant of 2 s. The spectropolarimeter was calibrated with D ())-pantolactone according to the instrument manual. Protein assay The concentration of protein samples was determined by the BCA method [19] using bovine serum albumin as a standard. Protein purity was determined by SDS– electrophoresis and by N-terminal sequencing. Gels were documented by NucleoVision 760 Imaging Workstation (NucleoTech, San Mateo, CA).
Results Enzyme activity It was found that GNMT lost enzyme activity in increasing concentrations of urea and became completely inactive at 4.0 M urea (Fig. 1). The sigmoidal shape of the inactivation curve indicates that a two-state transition from active to inactive state of the enzyme is most likely. Unfolding of GNMT tetramers may be a multistep process that includes tetramer dissociation. Loss of enzyme activity may occur at a different step. To find out whether loss of enzyme activity is a result of tetramer dissociation, the inactivation curve was carried out at a different concentration of the enzyme. As shown in Fig. 1, the midpoint of the transition of loss of enzyme activity depends on the concentration of the enzyme. This was found to be 1.5 M for 3.0 lg/ml and 2.3 M urea for 15.0 lg/ml. The dependence of the transition midpoint upon protein concentration indicates that inactivation of GNMT coincides with dissociation of the enzyme tetramer. Quaternary structure To investigate the change of quaternary and tertiary structure of GNMT upon urea unfolding, size-exclusion chromatography studies were performed. Using the methods described by Uversky [20] and Gualfetti et al.
Fig. 1. Dependence of GNMT activity on urea concentration. Open circles, GNMT concentration 3 lg/ml; closed circles, 15 lg/ml.
[21] we determined that the calibration curve of our Superose-12 column in 50 mM Tris–HCl, 150 mM NaCl, pH 8.0, and 14 mM b-mercaptoethanol is described by: 1000=Ve ¼ ð0:690 0:028ÞRs þ 51:3ð 1:29Þ:
ð1Þ
The same held true for the unfolded state of proteins in urea solutions. We used this dependence to determine the Stokes radii of GNMT both in the absence and presence of urea. In the absence of urea, GNMT was eluted from the Superose-12 column in one peak with an elution volume of 12.60 (Figs. 2 and 3). The Stokes radius of the native GNMT tetramer calculated by Eq. (1) was found to be . Similar values were obtained under other con40.7 A ditions (20 and 100 mM Tris, pH 7.5, 0.1 M citrate, pH 5.6). The presence of one of the substrates, AdoMet, did not change the quaternary structure, since no change of elution profile of GNMT was found in Column Buffer containing 100 lM AdoMet (data not shown). In urea solutions, the elution profiles of GNMT consisted of one or two peaks depending on the urea concentration (Fig. 2). At urea concentrations higher than 1.0 M in addition to the peak of native tetramer, a second peak appeared with an elution volume of 14.00 ml in 2.0 M urea. As the concentration of urea was increased, the native tetramer progressively dissociated to a smaller molecular mass species and the elution volume of both peaks gradually decreased. In 4 M urea, all native tetramers dissociated and only the molecular species with the lower molecular mass remained. To analyze the elution profile of GNMT, the elution volume of both peaks was plotted against urea concentration. The results are presented in Fig. 3. It was found
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transition was seen in which the elution volume of the second peak sharply decreased suggesting a complete unfolding of GNMT. Finally, in 8 M urea GNMT was eluted as a single peak with an elution volume of . 11.63 ml. These correspond to Rs of 50.3 A We assumed that the changes of the elution profile in 0–4 M urea reflect the first step of GNMT unfolding, namely dissociation of the tetramer to smaller subunits and gradual increasing of the Stokes radii of both molecular species. In 5.0–7.0 M urea, the smaller GNMT subunits undergo full unfolding. To find out what type of subunits, dimers or monomers, are eluted as lower molecular mass species, we analyzed the SEC data by comparison of the experimental Stokes radii with predicted ones. From analysis of the literature, two empirical equations for dependence of Stokes radius on molecular mass of globular proteins were used [20]. The dependence for Stokes radius on MW for native globular proteins was found to be logðRs Þ ¼ 0:254 þ 0:369 logMW:
ð2Þ
For globular proteins that are fully unfolded in 8 M urea, the dependence was found to be logðRs Þ ¼ 0:657 þ 0:524 logMW:
Fig. 2. The elution profiles of GNMT in different concentrations of urea. All buffers contained 50 mM Tris, pH 8.0, 150 mM NaCl, and 14 mM b-mercaptoethanol. Urea concentrations in mol/L are indicated on the curves. Concentration of the protein was 0.15 mg/ml.
ð3Þ
The values of experimental Rs and predicted Rs by these equations of different molecular species of GNMTs are presented in Table 1. For the native GNMT tetramer, the value predicted by Eq. (2) and the experimental value of Rs are very close. Using this approach, we could estimate the Stokes radii of the urea-dissociated subunits of GNMT. Assuming that extrapolation of the dependence of elution volume of these subunits on urea concentration to zero molarity is linear, we estimated the elution volume of subunits in the ‘‘native’’ state to be 14.40 ml. By Eq. (1) . This this gives the experimental Stokes radius of 26.3 A value is nearly equal to the Rs value of GNMT predicted by Eq. (3) for globular GNMT monomers (32.6 kDa), . This means that human which was found to be 25.8 A GNMT is dissociated by urea directly to monomers. Moreover, the monomers of GNMT in the urea concentration range 1.0–5.0 M are in a conformation similar to a globular protein since the Stokes radius is close to that predicted for native globular protein with the same Table 1 The Stokes radii of human GNMT
Fig. 3. Dependence of the elution volume of different forms of GNMT on urea concentration. Squares denote tetramers; circles denote monomers.
that the changes in elution volume of native protein and the molecular species with higher Ve are linear up to 5 M urea concentration. This change for native GNMT and smaller molecular mass species is 0.15 and 0.23 ml/M, respectively. In the 5–7 M urea concentration, another
Conditions
Rs , exp.
Rs , pred. (MW)
Native 4 M urea–0 Ma 8 M urea
40.7 26.3a 50.3
43.0 (130 kDa) 25.8 (32.6 kDa) 51.0 (32.6 kDa)
Rs exp., values obtained by SEC; Rs pred., values calculated by Eqs. (2) and (3) for native GNMT tetramers, native GNMT monomers and fully unfolded monomers. a Rs exp. obtained for GNMT monomers extrapolated to zero molar urea concentration.
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molecular mass. The Stokes radius of GNMT monomers in 8 M urea calculated by Eq. (3) was found , which corresponds to the experimental to be 51.0 A ). This values of Rs of the urea unfolded GNMT ð50:3 A means that GNMT monomers are in a fully unfolded state in 8 M urea solution. Reversibility For every protein, the conditions of refolding are different in terms of buffer composition, temperature, and kinetics [22]. We found that about 95% recovery of native tetramer could be achieved by stepwise dilution. Refolding of GNMT from 8 M urea was done through the following steps: (1) 8–5.5 M (monomer refolding); (2) 5.5–4.0 M; (3) 4.0–2.0 M (tetramer association); and (4) 2.0–1.0 M urea at 22 °C in the buffer used in the unfolding experiment. At each step, the protein was incubated for 1 h. At the final step, the protein in 1 M urea was passed through a Superose-12 column in Column Buffer. The critical point was the incubation time at the midpoint of association of compact monomers. When a sample of GNMT from step 3 in 2 M urea after 15 min incubation was analyzed by SEC, it was found that only about 60% of protein had formed tetramers. Tetramer formation was complete after 1 h incubation. Re-equilibration of the GNMT tetramer–monomers equilibrium during elution Size-exclusion chromatographic studies of multimeric protein dissociation rise a question as to what extent the data obtained are modified by re-equilibration of the tetramer to monomer transition during the time needed for the chromatographic procedure. In the approximately 30 min needed to elute the compact monomers, one could expect that tetramers will dissociate and some of the monomers that are formed will reassociate to tetramers. This introduces some uncertainty in the estimate of the relative amounts of tetramer and monomer formed under different urea concentrations and will depend on the relative rates of dissociation and association. To study how re-equilibration of GNMT changes the elution profile, we carried out SEC after different times of incubation in urea solution before applying to the column. By comparing zero time incubation with time needed for equilibration, we could estimate how much effect re-equilibration could have on the data. A urea concentration of 2.5 M is the midpoint of the tetramer–monomer transition (Fig. 3). Therefore, we subjected GNMT to SEC in 2.5 M urea after different times of pre-incubation in this buffer before applying to the column. The data obtained are presented in Fig. 4. When the protein sample was applied to the column immediately after mixing with urea, the elution profile consisted of mainly of a tetramer peak with an elution
Fig. 4. Re-equilibration of GNMT tetramer–monomer dissociation on the SEC column. The GNMT was mixed with 2.5 M urea and incubated for different times before chromatography. (A) The elution profiles of GNMT in 2.5 M urea after different pre-incubation times. Curves 1, 2, 3, 4, and 5 correspond to 1, 15, 30, 60, and 120 min of preincubation. Concentration of GNMT was 0.15 mg/ml. (B) Ratio of the height of monomer to tetramer peaks at times shown in (A).
volume of 12.35 ml and a minor monomer peak at a higher elution volume. As the pre-incubation time increased, the ratio of the monomer to tetramer peak increased and stabilized at a maximal value after about one hour of pre-incubation. The elution volume of both peaks did not change except at ‘‘zero time’’ when the monomer peak has lower Ve value. In this case, the appearance of monomers is entirely a result of dissociation on the column. Assuming that immediately after mixing GNMT with urea all the protein is in the form of tetramer, we can conclude that appearance of the monomer is entirely a result of dissociation during elution. If so, then the ratio of the area of monomer to tetramer in the elution profile is a quantitative measure of reequilibration. As shown in Fig. 4 at zero time, the reequilibration resulted in 16% of monomer. Therefore, the change in the relative amounts of tetramer and monomer cannot be more than 16% in error due to reequilibration during SEC.
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Fluorescence spectroscopy The conformational changes in GNMT during dissociation and unfolding were studied by fluorescence spectroscopy. The excitation maximum of human GNMT was found to be 285 nm. This is due to excitation of both tyrosine and tryptophan residues. When intrinsic emission spectra were recorded with excitation at 285 nm, it was characterized by a maximum at 339– 340 nm and a width of about 58–60 nm (Fig. 5). When intrinsic fluorescence was excited at 300 nm, both the intensity and the shape of emission spectra were changed due to the excitation of only tryptophan residues. The large number of fluorophores and possible difference in microenvironment make use of fluorescence spectroscopy for the monitoring of GNMT unfolding more difficult compared to simple cases of the proteins with only one or a few tryptophan and tyrosine residues. When emission spectra of GNMT in urea solutions were recorded with excitation at 285 nm, they showed a decrease in fluorescence intensity and a red shift of the emission maxima compared to the native state in the absence of urea (Fig. 5). When excitation at 300 nm was used, a more complex dependence of emission spectra on urea concentration was found. In this case, some tryptophan residue(s) were found that did not change the maximum of emission at 334 nm while other tryptophan residues shifted their emission maxima to 356 nm. We found that the fraction of denatured GNMT is best characterized by fluorescence intensity at 334 nm when fluorescence was excited at 300 nm. To monitor the general microenvironment of tryptophan and tyrosine residues, the best parameter is a change of maxima of emission spectra with excitation at 285 nm. These data are presented in Fig. 6. The fluorescence intensity at 334 nm was sharply decreased in the 1.5–5.0 M urea concentration range having a sigmoidal shape similar to
Fig. 6. Variation of GNMT fluorescence at different concentrations of urea. (A) Urea dependence of the intensity of fluorescence emission at 334 nm (excitation at 300 nm). The intensity of fluorescence emission was measured at a protein concentration of 0.013 mg/ml (filled squares) and 0.13 mg/ml (open circles). (B) Urea dependence of the maximum of the emission spectra (excitation 285 nm). Protein concentration was 0.13 mg/ml.
that of activity. Fig. 6A shows that the denaturation is also dependent on protein concentration. If the first transition is a dissociation of GNMT tetramer dissociation to monomers N4 () 4D
Fig. 5. GNMT fluorescence emission spectra under native conditions (1,3) and in 8 M urea (2,4). The spectra were recorded at 285 nm (excitation), solid line and 300 nm (excitation) dashed line.
ð4Þ
an equilibrium shift toward denatured monomers should be observed upon decrease of the total protein concentration according to the Law of Mass Action. This was confirmed by unfolding GNMT at different protein concentrations. At a protein concentration of 0.013 mg/ml, the midpoint of the dissociation of tetramer to monomers was significantly lower (2.75 M urea) compared to a protein concentration of 0.13 mg/ml (3.4 M). In contrast to fluorescence intensity, the emission maximum was not changed in the 0–3.5 M urea concentration range, i.e., the region where the fluorescence intensity undergoes a transition (Fig. 6B). An increase of maximum was seen only in urea concentrations higher than 3.5 M. The emission maximum of GNMT in 8 m
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urea corresponds to that of tryptophan in water and was found to be 355–356 nm. Circular dichroism To characterize the change in secondary structure, the unfolding of GNMT was monitored by circular dichroism. The CD spectrum of GNMT under native conditions is a typical protein spectrum with a-helices and b-sheet (Fig. 7A). As the urea concentration increased, the CD spectra changed to that of a protein with less secondary structure When ellipticity at 222 nm was plotted against urea concentration, two clearly different transition regions were identified (Fig. 7B). The first transition occurs in the 1.0–4.0 M urea concentration and the second tran-
Fig. 7. Analysis of GNMT urea unfolding by circular dichroism. (A) CD spectra of GNMT under native conditions (20 mM Na-phosphate, pH 8.0), solid line; 4 M urea; dashed line and 8 M urea; dotted line. The CD spectrum of GNMT under native conditions was recorded in phosphate buffer to get better resolution. GNMT solutions in 4 and 8 M urea contained 50 mM Tris–HCl, pH 8.0, 150 mM NaCl to be comparable with Column Buffer used for SEC. The CD spectra in these buffers were identical to that recorded in phosphate buffer but the signal was less stable. (B) Urea dependence of ellipticity of GNMT at 222 nm in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl. The protein concentration in all solutions was 0.13 mg/ml.
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sition was found in 4.0–7.0 M urea. The molar ellipticity at 224 nm of GNMT in 4 M urea was about 50% of that of native protein. In 8 M urea concentration, the content of secondary structure was minimal as shown by the low value of molar ellipticity.
Discussion Three possible models of GNMT unfolding were considered. The first model included dissociation of the native tetramer to dimers with dissociation to monomers in a second step with unfolding of monomers in the last step. The second model was based on consecutive tetramer dissociation to compact monomers with unfolding of the latter in the last step. Another possible mechanism was direct dissociation of native tetramer to unfolded monomers without any intermediate states. The data presented here indicate that the unfolding of the tetrameric structure of the GNMT is a two-step process. The first step is a dissociation of native tetramer to compact monomers in 1–4.0 M urea. At higher concentrations of urea, the monomers with compact structure undergo an unfolding transition, which leads to a completely unfolded state in 8 M urea. The scheme of this process is presented in Fig. 8. GNMT dissociation coincides with loss of enzyme activity. This implies that the only active structure of GNMT is the native tetramer, not the monomers. This is direct confirmation of a structural feature of the GNMT tetramer where each monomer interacts with two other subunits as shown by the crystal structure [8,9]. The protein concentration dependence of tetramer/monomer equilibrium raises a question as to whether the concentration of GNMT in the cell is sufficient to maintain the stable active tetrameric conformation of enzyme. It is known that in rat liver tissue the content of GNMT is about 1–3% [2–4]. Simple calculations give the
Fig. 8. Proposed scheme of GNMT unfolding in urea solutions.
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concentration of GNMT as about 1 lM (0.13 mg/ml). Our experiments showed that at this concentration under native conditions GNMT exists as an active tetramer. The strongest evidence for the proposed mechanism of GNMT unfolding was obtained by SEC. This method is extensively used for the study of protein unfolding [20–24] and its validity for such investigations is well justified. By this method it was shown that native , GNMT is a tetramer with a Stokes radius of 40.7 A which is close to the predicted Rs of 43.0 A for a 130 kDa globular protein. Dissociated monomers of GNMTs have a compact conformation in urea concentrations of 1.0–5.0 M. This conclusion is based on coincidence of the experimental value of the Stokes radius and that predicted for a globular protein with a molecular mass equal to GNMT monomers (Table 1). The compact monomers of GNMT are characterized by an unchanged maximum of fluorescence emission spectra up to 3.5 M urea (Fig. 6B). This indicates that the tryptophan residues are still in a hydrophobic microenvironment in the monomer form. The monomers of GNMT still contain about 50% of the secondary structure as that of the native tetramer. Spectroscopic data showed also that while being in a compact structure, the monomers of GNMT are partially disordered. First, about 50% of secondary structure is disordered as it was shown by CD analysis. Second, the intensity of fluorescence emission at 334 nm in 4.0–5.0 M urea concentration reached a minimal value (Fig. 6A). This indicates that flexibility of tryptophan residues increased significantly. Despite disruption of some fraction of secondary structure and long-range interactions in the protein globule, the compact conformation of monomer is surprisingly stable. Its final unfolding step began only at urea concentrations higher than 5 M. The second step of GNMT urea unfolding is the transition from the compact structure of monomers to a fully unfolded state. The SEC data showed that this process might be considered as molten globule as only one molecular species was found in solution with a gradually increased Rs in increasing urea concentrations. Missense mutations in human glycine N-methyltransferase have been shown to be one cause of persistent isolated hypermethioninaemia [25,26]. The exact reasons for the loss of GNMT activity in these cases are
now under investigation and the stability of the human GNMT mutants is part of this study.
References [1] J.E. Heady, S.J. Kerr, J. Biol. Chem. 248 (1973) 69–72. [2] M. Balaghi, D.W. Horne, C. Wagner, Biochem. J. 291 (1993) 145– 149. [3] H. Ogawa, T. Gomi, F. Takusagawa, M. Fujioka, Int. J. Biochem. Cell Biol. 30 (1998) 13–26. [4] H. Ogawa, T. Gomi, M. Fujioka, Comp. Biochem. Physiol. B 106 (1993) 601–611. [5] Y.M. Chen, J.Y. Shiu, S.J. Tzeng, L.S. Shih, Y.J. Chen, W.Y. Lui, P.H. Chen, Int. J. Cancer 75 (1998) 787–793. [6] K. Aida, M. Tawata, M. Negishi, T. Onaya, Horm. Metab. Res. 29 (1997) 646–929. [7] H. Ogawa, T. Gomi, T. Horii, H. Ogawa, M. Fujioka, Biochem. Biophys. Res. Commun. 124 (1984) 44–50. [8] Z. Fu, Y. Hu, K. Konishi, Y. Takata, H. Ogawa, T. Gomi, M. Fujioka, F. Takusagawa, Biochemistry 35 (1996) 11985–11993. [9] R. Pattanayek, M.E. Newcomer, C. Wagner, Protein Sci. 7 (1998) 1326–1331. [10] F. Takusagawa, H. Ogawa, M. Fujioka, in: X. Cheng, R.M. Blumental (Eds.), S-Adenosylmethionine-dependent Methyltransferases: Structure and Functions, World Scientific Publishing, Singapore, 1999, pp. 93–122. [11] C. Wagner, W.T. Briggs, R.J. Cook, Biochem. Biophys. Res. Commun. 127 (1985) 746–752. [12] E.J. Yeo, W.T. Briggs, C. Wagner, J. Biol. Chem. 274 (1999) 37559–37564. [13] M.J. Rowling, M.H. McMullen, D.C. Chipman, K.L. Schalinske, J. Nutr. 132 (2002) 2545–2550. [14] M.J. Rowling, M.H. McMullen, K.L. Schalinske, J. Nutr. 132 (2002) 365–369. [15] N.I. Krupenko, C. Wagner, J. Biol. Chem. 272 (1997) 27140– 27146. [16] Z. Luka, C. Wagner, Prot. Expr. Purif. 28 (2003) 280–286. [17] J. Lippincott, I. Apostol, Anal. Biochem. 267 (1999) 57–64. [18] C.N. Pace, Methods Enzymol. 131 (1986) 200–266. [19] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76–85. [20] V.N. Uversky, Biochemistry 32 (1993) 13228–13298. [21] P.J. Gualfetti, M. Iwakura, J.C. Lee, H. Kihara, O. Bilsel, J.A. Zitzewitz, C.R. Matthews, Biochemistry 38 (1999) 13367–13378. [22] R. Jaenicke, R. Rudolph, Methods Enzymol. 131 (1986) 218–250. [23] I.V. Baskakov, D.W. Bolen, Biochemistry 37 (1998) 18010–18017. [24] A.M. Al-Obeidi, A. Light, J. Biol. Chem. 263 (1988) 8642–8645. [25] P. Augoustides-Savvopoulou, Z. Luka, S.P. Stabler, R.H. Allen, K. Patsiaoura, C. Wagner, S.H. Mudd, J. Inherit. Metab. Dis. (1993) in press. [26] Z. Luka, R. Cerone, J. Phillips, S.H. Mudd, C. Wagner, Hum. Genet. 110 (2002) 68–74.
Human glycine N-methyltransferase is unfolded by urea through a compact monomer stateq Zigmund Luka and Conrad Wagner* Department of Biochemistry, Medical Center, Vanderbilt University, School of Medicine, 620 Light Hall, Nashville, TN 37232-0146, USA Received 24 July 2003, and in revised form 4 September 2003
Abstract Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0–3.5 M urea with enzyme inactivation. The equal to that of globular proteins with the same molecular compact monomers were characterized by Stokes radius (Rs ) of 40.7 A mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully , exposed tryptophan residues, and disrupted secondary structure in 8 M urea. unfolded state with Rs of 50 A Ó 2003 Elsevier Inc. All rights reserved. Keywords: Glycine N-methyltransferase; Unfolding; Dissociation; Inactivation; Stability; Conformation
Glycine N-methyltransferase (GNMT) is an abundant mammalian enzyme that transfers the methyl group from S-adenosylmethionine (AdoMet)1 to glycine producing sarcosine and S-adenosylhomocysteine (AdoHcy) [1,2]. Sarcosine has no known metabolic function and is reconverted to glycine by a mitochondrial enzyme system. Therefore, the biological role of GNMT was proposed to maintain the ratio of AdoMet/ AdoHcy by converting excess AdoMet to AdoHcy without the synthesis of a metabolically active product. GNMT is a key enzyme in the control of the methylation capacity of the cell. GNMT is a tetrameric protein consisting of four 32kDa subunits [3]. The amino acid sequence of GNMT q This work was supported by Grants DK15289 and DK54859 from the US Public Health Service and from the Office of Research and Development, Medical Research Service, Department of Veteran Affairs. * Corresponding author. Fax: 1-615-343-0407. E-mail address: [email protected] (C. Wagner). 1 Abbreviations used: GNMT, glycine N-methyltransferase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; Ve , elution volume of proteins determined by position of maximum of elution peak; SEC, size-exclusion chromatography; Rs , Stokes radius.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.09.009
from rat, mouse, rabbit, pig, and human has been derived from the nucleotide sequence of cDNAs [4–7]. Identity among protein sequences is 89–93% [4]. The crystal structure of recombinant rat GNMT apoprotein and its complex with AdoMet and with AdoHcy has been obtained by X-ray analysis [8–10]. Recently, the crystal structure of recombinant human GNMT was solved (Pakhomova et al. unpublished results), which appeared to be very similar to the crystal structure of rat enzyme. In the crystal, the tetrameric structure of GNMT is formed by multiple hydrogen bonds between b-sheets as well as by ion-pair interactions between charged residues of different subunits [8,9]. Native rat GNMT is isolated with bound 5-methyltetrahydrofolate pentaglutamate (5-CH3 THFG5 ) [11]. We showed that GNMT is inhibited by 5-CH3 THFG5 when one molecule of the inhibitor is bound to the purified tetramer [12]. Activity of the enzyme is also regulated in vivo by a variety of factors like methionine and vitamin A [13,14]. Another possible mode of regulation of GNMT activity includes association/dissociation of the tetramer. It was shown that fluorescein-labeled GNMT could be transported into isolated rat liver nuclei as a monomer [15]. While the physiological
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relevance of the latter observation is unclear, it is a further reason to study the conformational stability of GNMT by biophysical methods. We examined GNMT unfolding as the first step in the biophysical characterization of GNMT. The following questions were addressed: (1) How stable is the tetrameric structure of GNMT? (2) What is the mechanism of dissociation and unfolding of GNMT by a chemical denaturant? (3) What conditions are required for refolding of the tetramer from unfolded monomers? To answer these questions, we analyzed the urea unfolding of human GNMT by enzyme activity, size-exclusion chromatography (SEC), fluorescence spectroscopy, and circular dichroism.
Materials and methods Protein preparation The human GNMT cDNA was cloned and expressed in the pET-17b expression vector as reported earlier [16]. Briefly, the GNMT cDNA was synthesized by RT-PCR from the human poly(A) RNA (Clontech, Palo Alto, CA). Cloned cDNA was introduced into a pET-17b expression vector. Expression was done at 20 °C for 17– 18 h with 1 mM IPTG. The GNMT protein was isolated and purified by ammonium sulfate precipitation from the crude extract and ion-exchange chromatography on DE-52 column. In the final step, the protein was passed through a Sephacryl S-200 size-exclusion column. The final purity of GNMT samples was at least 95–97%. GNMT activity This was assayed using the charcoal adsorption method described earlier [12]. In 100 ll of reaction mixture, the concentration of the reagents was: 50 mM Tris–HCl buffer, pH 8.0, 150 mM NaCl, 200 nM [methyl-3 H]AdoMet (3–5 103 dpm), 20.0 mM glycine, 5 mM DTT, and 0.3–1.5 lg enzyme. After incubation at 25 °C for 15 min, the reaction was stopped by 50 ll of 10% trichloroacetic acid and 250 ll of a suspension of acid-treated charcoal was added to adsorb the unreacted labeled AdoMet. The suspension was incubated in ice for 15 min. After centrifugation in a microcentrifuge, the radioactivity in the supernatant containing the labeled sarcosine was counted in a Tri-Carb Liquid Scintillation Analyzer (Packard Instrument, Meriden, CT). Enzyme activity in urea solution was measured essentially as above with some modifications. First, two master mixes were prepared. Mix I contained buffer, substrates, and other necessary additives for activity assay, and the desired concentration of urea. Enzyme unfolding was done in a separate Mix II containing buffer solution and
the same concentration of urea in which activity was measured later. The volume ratio of Mix II/Mix I was kept constant (1:1) and high in order to minimize any effect of protein dilution on the dissociation parameters. Fluorescence spectroscopy showed that GNMT in various urea solutions reached an equilibrium between folded and unfolded protein in 4 M urea in 15 min and in 8 M urea in about 5 min. Therefore, the protein in all urea unfolding experiments was incubated at ambient temperature for 1 h before measurement. After incubation for 1 h, both solutions were mixed and the reaction was assayed at 25 °C for 15 min. No degradation of proteins after the unfolding experiments was found by SDS–electrophoresis. The working solutions containing urea were prepared by dilution from a 10 M urea stock solution. The 10 M urea solution was prepared fresh for each experiment to minimize carbamylation of the GNMT by cyanate formed by urea hydrolysis [17]. The urea solutions were filtered through a 0.2 lm filter and its concentration was determined from refractive index [18]. Size-exclusion chromatography € KTA purifier System Experiments were done on the A (Amersham–Pharmacia Biotech., Piscataway, NJ) using a Superose-12 column in Column Buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 14 mM bmercaptoethanol at a flow rate of 0.5 ml/min at 22 °C. Some SEC experiments were done with Column Buffer at pH 7.5. The sample volume was 100 ll. The elution of the proteins was monitored by absorbance at 280 nm. The column was calibrated with cytochrome c, lysozyme, chymotrypsin, carbonic anhydrase, ovalbumin, BSA, alcohol dehydrogenase, potato b-amylase, and aldolase without added urea and also in Column Buffer containing 8 M urea. The void volume Vo was determined with blue dextran and the total solvent-accessible volume Vt was determined with acetone. The GNMT samples were incubated in urea solution for 1 h before applying to the column. To determine whether AdoMet affected the quaternary structure of GNMT, a smaller column (1 15 cm, volume 15 ml) with Sephacryl S-200 resin was used. The elution of GNMT was done in the Column Buffer without and with 100 lM AdoMet. Fluorescence spectroscopy Protein fluorescence spectra were recorded on a Perkin–Elmer 650-40 Fluorescence Spectrophotometer (Perkin–Elmer, Norwalk, CT). Fluorescence emission spectra were recorded from 300 to 400 nm with slits of 5 nm and a scanning speed of 2 nm/s in a 10 mm cuvette at 22 °C. Simultaneous intrinsic tryptophan and tyrosine fluorescence was measured with excitation at 285 nm and tryptophan fluorescence was recorded by using excita-
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tion at 300 nm. The protein concentrations were 0.05– 0.13 mg/ml with absorbance values not higher than 0.1 at the excitation wavelength to avoid the inner filter effect. Circular dichroism Spectra were recorded on a Jasco-720 spectropolarimeter. The spectral data of the samples were scanned in the range 250–190 nm four times and averaged and smoothed by the instrument software. The protein concentration of the samples was 0.13 mg/ml with an optical path length of 1.0 or 10.0 mm. The scan speed was 10 nm/min, at a sensitivity of 20 mdeg, resolution of 1 nm, and a time constant of 2 s. The spectropolarimeter was calibrated with D ())-pantolactone according to the instrument manual. Protein assay The concentration of protein samples was determined by the BCA method [19] using bovine serum albumin as a standard. Protein purity was determined by SDS– electrophoresis and by N-terminal sequencing. Gels were documented by NucleoVision 760 Imaging Workstation (NucleoTech, San Mateo, CA).
Results Enzyme activity It was found that GNMT lost enzyme activity in increasing concentrations of urea and became completely inactive at 4.0 M urea (Fig. 1). The sigmoidal shape of the inactivation curve indicates that a two-state transition from active to inactive state of the enzyme is most likely. Unfolding of GNMT tetramers may be a multistep process that includes tetramer dissociation. Loss of enzyme activity may occur at a different step. To find out whether loss of enzyme activity is a result of tetramer dissociation, the inactivation curve was carried out at a different concentration of the enzyme. As shown in Fig. 1, the midpoint of the transition of loss of enzyme activity depends on the concentration of the enzyme. This was found to be 1.5 M for 3.0 lg/ml and 2.3 M urea for 15.0 lg/ml. The dependence of the transition midpoint upon protein concentration indicates that inactivation of GNMT coincides with dissociation of the enzyme tetramer. Quaternary structure To investigate the change of quaternary and tertiary structure of GNMT upon urea unfolding, size-exclusion chromatography studies were performed. Using the methods described by Uversky [20] and Gualfetti et al.
Fig. 1. Dependence of GNMT activity on urea concentration. Open circles, GNMT concentration 3 lg/ml; closed circles, 15 lg/ml.
[21] we determined that the calibration curve of our Superose-12 column in 50 mM Tris–HCl, 150 mM NaCl, pH 8.0, and 14 mM b-mercaptoethanol is described by: 1000=Ve ¼ ð0:690 0:028ÞRs þ 51:3ð 1:29Þ:
ð1Þ
The same held true for the unfolded state of proteins in urea solutions. We used this dependence to determine the Stokes radii of GNMT both in the absence and presence of urea. In the absence of urea, GNMT was eluted from the Superose-12 column in one peak with an elution volume of 12.60 (Figs. 2 and 3). The Stokes radius of the native GNMT tetramer calculated by Eq. (1) was found to be . Similar values were obtained under other con40.7 A ditions (20 and 100 mM Tris, pH 7.5, 0.1 M citrate, pH 5.6). The presence of one of the substrates, AdoMet, did not change the quaternary structure, since no change of elution profile of GNMT was found in Column Buffer containing 100 lM AdoMet (data not shown). In urea solutions, the elution profiles of GNMT consisted of one or two peaks depending on the urea concentration (Fig. 2). At urea concentrations higher than 1.0 M in addition to the peak of native tetramer, a second peak appeared with an elution volume of 14.00 ml in 2.0 M urea. As the concentration of urea was increased, the native tetramer progressively dissociated to a smaller molecular mass species and the elution volume of both peaks gradually decreased. In 4 M urea, all native tetramers dissociated and only the molecular species with the lower molecular mass remained. To analyze the elution profile of GNMT, the elution volume of both peaks was plotted against urea concentration. The results are presented in Fig. 3. It was found
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transition was seen in which the elution volume of the second peak sharply decreased suggesting a complete unfolding of GNMT. Finally, in 8 M urea GNMT was eluted as a single peak with an elution volume of . 11.63 ml. These correspond to Rs of 50.3 A We assumed that the changes of the elution profile in 0–4 M urea reflect the first step of GNMT unfolding, namely dissociation of the tetramer to smaller subunits and gradual increasing of the Stokes radii of both molecular species. In 5.0–7.0 M urea, the smaller GNMT subunits undergo full unfolding. To find out what type of subunits, dimers or monomers, are eluted as lower molecular mass species, we analyzed the SEC data by comparison of the experimental Stokes radii with predicted ones. From analysis of the literature, two empirical equations for dependence of Stokes radius on molecular mass of globular proteins were used [20]. The dependence for Stokes radius on MW for native globular proteins was found to be logðRs Þ ¼ 0:254 þ 0:369 logMW:
ð2Þ
For globular proteins that are fully unfolded in 8 M urea, the dependence was found to be logðRs Þ ¼ 0:657 þ 0:524 logMW:
Fig. 2. The elution profiles of GNMT in different concentrations of urea. All buffers contained 50 mM Tris, pH 8.0, 150 mM NaCl, and 14 mM b-mercaptoethanol. Urea concentrations in mol/L are indicated on the curves. Concentration of the protein was 0.15 mg/ml.
ð3Þ
The values of experimental Rs and predicted Rs by these equations of different molecular species of GNMTs are presented in Table 1. For the native GNMT tetramer, the value predicted by Eq. (2) and the experimental value of Rs are very close. Using this approach, we could estimate the Stokes radii of the urea-dissociated subunits of GNMT. Assuming that extrapolation of the dependence of elution volume of these subunits on urea concentration to zero molarity is linear, we estimated the elution volume of subunits in the ‘‘native’’ state to be 14.40 ml. By Eq. (1) . This this gives the experimental Stokes radius of 26.3 A value is nearly equal to the Rs value of GNMT predicted by Eq. (3) for globular GNMT monomers (32.6 kDa), . This means that human which was found to be 25.8 A GNMT is dissociated by urea directly to monomers. Moreover, the monomers of GNMT in the urea concentration range 1.0–5.0 M are in a conformation similar to a globular protein since the Stokes radius is close to that predicted for native globular protein with the same Table 1 The Stokes radii of human GNMT
Fig. 3. Dependence of the elution volume of different forms of GNMT on urea concentration. Squares denote tetramers; circles denote monomers.
that the changes in elution volume of native protein and the molecular species with higher Ve are linear up to 5 M urea concentration. This change for native GNMT and smaller molecular mass species is 0.15 and 0.23 ml/M, respectively. In the 5–7 M urea concentration, another
Conditions
Rs , exp.
Rs , pred. (MW)
Native 4 M urea–0 Ma 8 M urea
40.7 26.3a 50.3
43.0 (130 kDa) 25.8 (32.6 kDa) 51.0 (32.6 kDa)
Rs exp., values obtained by SEC; Rs pred., values calculated by Eqs. (2) and (3) for native GNMT tetramers, native GNMT monomers and fully unfolded monomers. a Rs exp. obtained for GNMT monomers extrapolated to zero molar urea concentration.
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molecular mass. The Stokes radius of GNMT monomers in 8 M urea calculated by Eq. (3) was found , which corresponds to the experimental to be 51.0 A ). This values of Rs of the urea unfolded GNMT ð50:3 A means that GNMT monomers are in a fully unfolded state in 8 M urea solution. Reversibility For every protein, the conditions of refolding are different in terms of buffer composition, temperature, and kinetics [22]. We found that about 95% recovery of native tetramer could be achieved by stepwise dilution. Refolding of GNMT from 8 M urea was done through the following steps: (1) 8–5.5 M (monomer refolding); (2) 5.5–4.0 M; (3) 4.0–2.0 M (tetramer association); and (4) 2.0–1.0 M urea at 22 °C in the buffer used in the unfolding experiment. At each step, the protein was incubated for 1 h. At the final step, the protein in 1 M urea was passed through a Superose-12 column in Column Buffer. The critical point was the incubation time at the midpoint of association of compact monomers. When a sample of GNMT from step 3 in 2 M urea after 15 min incubation was analyzed by SEC, it was found that only about 60% of protein had formed tetramers. Tetramer formation was complete after 1 h incubation. Re-equilibration of the GNMT tetramer–monomers equilibrium during elution Size-exclusion chromatographic studies of multimeric protein dissociation rise a question as to what extent the data obtained are modified by re-equilibration of the tetramer to monomer transition during the time needed for the chromatographic procedure. In the approximately 30 min needed to elute the compact monomers, one could expect that tetramers will dissociate and some of the monomers that are formed will reassociate to tetramers. This introduces some uncertainty in the estimate of the relative amounts of tetramer and monomer formed under different urea concentrations and will depend on the relative rates of dissociation and association. To study how re-equilibration of GNMT changes the elution profile, we carried out SEC after different times of incubation in urea solution before applying to the column. By comparing zero time incubation with time needed for equilibration, we could estimate how much effect re-equilibration could have on the data. A urea concentration of 2.5 M is the midpoint of the tetramer–monomer transition (Fig. 3). Therefore, we subjected GNMT to SEC in 2.5 M urea after different times of pre-incubation in this buffer before applying to the column. The data obtained are presented in Fig. 4. When the protein sample was applied to the column immediately after mixing with urea, the elution profile consisted of mainly of a tetramer peak with an elution
Fig. 4. Re-equilibration of GNMT tetramer–monomer dissociation on the SEC column. The GNMT was mixed with 2.5 M urea and incubated for different times before chromatography. (A) The elution profiles of GNMT in 2.5 M urea after different pre-incubation times. Curves 1, 2, 3, 4, and 5 correspond to 1, 15, 30, 60, and 120 min of preincubation. Concentration of GNMT was 0.15 mg/ml. (B) Ratio of the height of monomer to tetramer peaks at times shown in (A).
volume of 12.35 ml and a minor monomer peak at a higher elution volume. As the pre-incubation time increased, the ratio of the monomer to tetramer peak increased and stabilized at a maximal value after about one hour of pre-incubation. The elution volume of both peaks did not change except at ‘‘zero time’’ when the monomer peak has lower Ve value. In this case, the appearance of monomers is entirely a result of dissociation on the column. Assuming that immediately after mixing GNMT with urea all the protein is in the form of tetramer, we can conclude that appearance of the monomer is entirely a result of dissociation during elution. If so, then the ratio of the area of monomer to tetramer in the elution profile is a quantitative measure of reequilibration. As shown in Fig. 4 at zero time, the reequilibration resulted in 16% of monomer. Therefore, the change in the relative amounts of tetramer and monomer cannot be more than 16% in error due to reequilibration during SEC.
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Fluorescence spectroscopy The conformational changes in GNMT during dissociation and unfolding were studied by fluorescence spectroscopy. The excitation maximum of human GNMT was found to be 285 nm. This is due to excitation of both tyrosine and tryptophan residues. When intrinsic emission spectra were recorded with excitation at 285 nm, it was characterized by a maximum at 339– 340 nm and a width of about 58–60 nm (Fig. 5). When intrinsic fluorescence was excited at 300 nm, both the intensity and the shape of emission spectra were changed due to the excitation of only tryptophan residues. The large number of fluorophores and possible difference in microenvironment make use of fluorescence spectroscopy for the monitoring of GNMT unfolding more difficult compared to simple cases of the proteins with only one or a few tryptophan and tyrosine residues. When emission spectra of GNMT in urea solutions were recorded with excitation at 285 nm, they showed a decrease in fluorescence intensity and a red shift of the emission maxima compared to the native state in the absence of urea (Fig. 5). When excitation at 300 nm was used, a more complex dependence of emission spectra on urea concentration was found. In this case, some tryptophan residue(s) were found that did not change the maximum of emission at 334 nm while other tryptophan residues shifted their emission maxima to 356 nm. We found that the fraction of denatured GNMT is best characterized by fluorescence intensity at 334 nm when fluorescence was excited at 300 nm. To monitor the general microenvironment of tryptophan and tyrosine residues, the best parameter is a change of maxima of emission spectra with excitation at 285 nm. These data are presented in Fig. 6. The fluorescence intensity at 334 nm was sharply decreased in the 1.5–5.0 M urea concentration range having a sigmoidal shape similar to
Fig. 6. Variation of GNMT fluorescence at different concentrations of urea. (A) Urea dependence of the intensity of fluorescence emission at 334 nm (excitation at 300 nm). The intensity of fluorescence emission was measured at a protein concentration of 0.013 mg/ml (filled squares) and 0.13 mg/ml (open circles). (B) Urea dependence of the maximum of the emission spectra (excitation 285 nm). Protein concentration was 0.13 mg/ml.
that of activity. Fig. 6A shows that the denaturation is also dependent on protein concentration. If the first transition is a dissociation of GNMT tetramer dissociation to monomers N4 () 4D
Fig. 5. GNMT fluorescence emission spectra under native conditions (1,3) and in 8 M urea (2,4). The spectra were recorded at 285 nm (excitation), solid line and 300 nm (excitation) dashed line.
ð4Þ
an equilibrium shift toward denatured monomers should be observed upon decrease of the total protein concentration according to the Law of Mass Action. This was confirmed by unfolding GNMT at different protein concentrations. At a protein concentration of 0.013 mg/ml, the midpoint of the dissociation of tetramer to monomers was significantly lower (2.75 M urea) compared to a protein concentration of 0.13 mg/ml (3.4 M). In contrast to fluorescence intensity, the emission maximum was not changed in the 0–3.5 M urea concentration range, i.e., the region where the fluorescence intensity undergoes a transition (Fig. 6B). An increase of maximum was seen only in urea concentrations higher than 3.5 M. The emission maximum of GNMT in 8 m
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urea corresponds to that of tryptophan in water and was found to be 355–356 nm. Circular dichroism To characterize the change in secondary structure, the unfolding of GNMT was monitored by circular dichroism. The CD spectrum of GNMT under native conditions is a typical protein spectrum with a-helices and b-sheet (Fig. 7A). As the urea concentration increased, the CD spectra changed to that of a protein with less secondary structure When ellipticity at 222 nm was plotted against urea concentration, two clearly different transition regions were identified (Fig. 7B). The first transition occurs in the 1.0–4.0 M urea concentration and the second tran-
Fig. 7. Analysis of GNMT urea unfolding by circular dichroism. (A) CD spectra of GNMT under native conditions (20 mM Na-phosphate, pH 8.0), solid line; 4 M urea; dashed line and 8 M urea; dotted line. The CD spectrum of GNMT under native conditions was recorded in phosphate buffer to get better resolution. GNMT solutions in 4 and 8 M urea contained 50 mM Tris–HCl, pH 8.0, 150 mM NaCl to be comparable with Column Buffer used for SEC. The CD spectra in these buffers were identical to that recorded in phosphate buffer but the signal was less stable. (B) Urea dependence of ellipticity of GNMT at 222 nm in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl. The protein concentration in all solutions was 0.13 mg/ml.
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sition was found in 4.0–7.0 M urea. The molar ellipticity at 224 nm of GNMT in 4 M urea was about 50% of that of native protein. In 8 M urea concentration, the content of secondary structure was minimal as shown by the low value of molar ellipticity.
Discussion Three possible models of GNMT unfolding were considered. The first model included dissociation of the native tetramer to dimers with dissociation to monomers in a second step with unfolding of monomers in the last step. The second model was based on consecutive tetramer dissociation to compact monomers with unfolding of the latter in the last step. Another possible mechanism was direct dissociation of native tetramer to unfolded monomers without any intermediate states. The data presented here indicate that the unfolding of the tetrameric structure of the GNMT is a two-step process. The first step is a dissociation of native tetramer to compact monomers in 1–4.0 M urea. At higher concentrations of urea, the monomers with compact structure undergo an unfolding transition, which leads to a completely unfolded state in 8 M urea. The scheme of this process is presented in Fig. 8. GNMT dissociation coincides with loss of enzyme activity. This implies that the only active structure of GNMT is the native tetramer, not the monomers. This is direct confirmation of a structural feature of the GNMT tetramer where each monomer interacts with two other subunits as shown by the crystal structure [8,9]. The protein concentration dependence of tetramer/monomer equilibrium raises a question as to whether the concentration of GNMT in the cell is sufficient to maintain the stable active tetrameric conformation of enzyme. It is known that in rat liver tissue the content of GNMT is about 1–3% [2–4]. Simple calculations give the
Fig. 8. Proposed scheme of GNMT unfolding in urea solutions.
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concentration of GNMT as about 1 lM (0.13 mg/ml). Our experiments showed that at this concentration under native conditions GNMT exists as an active tetramer. The strongest evidence for the proposed mechanism of GNMT unfolding was obtained by SEC. This method is extensively used for the study of protein unfolding [20–24] and its validity for such investigations is well justified. By this method it was shown that native , GNMT is a tetramer with a Stokes radius of 40.7 A which is close to the predicted Rs of 43.0 A for a 130 kDa globular protein. Dissociated monomers of GNMTs have a compact conformation in urea concentrations of 1.0–5.0 M. This conclusion is based on coincidence of the experimental value of the Stokes radius and that predicted for a globular protein with a molecular mass equal to GNMT monomers (Table 1). The compact monomers of GNMT are characterized by an unchanged maximum of fluorescence emission spectra up to 3.5 M urea (Fig. 6B). This indicates that the tryptophan residues are still in a hydrophobic microenvironment in the monomer form. The monomers of GNMT still contain about 50% of the secondary structure as that of the native tetramer. Spectroscopic data showed also that while being in a compact structure, the monomers of GNMT are partially disordered. First, about 50% of secondary structure is disordered as it was shown by CD analysis. Second, the intensity of fluorescence emission at 334 nm in 4.0–5.0 M urea concentration reached a minimal value (Fig. 6A). This indicates that flexibility of tryptophan residues increased significantly. Despite disruption of some fraction of secondary structure and long-range interactions in the protein globule, the compact conformation of monomer is surprisingly stable. Its final unfolding step began only at urea concentrations higher than 5 M. The second step of GNMT urea unfolding is the transition from the compact structure of monomers to a fully unfolded state. The SEC data showed that this process might be considered as molten globule as only one molecular species was found in solution with a gradually increased Rs in increasing urea concentrations. Missense mutations in human glycine N-methyltransferase have been shown to be one cause of persistent isolated hypermethioninaemia [25,26]. The exact reasons for the loss of GNMT activity in these cases are
now under investigation and the stability of the human GNMT mutants is part of this study.
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