Báo cáo Y học: The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea

Eur. J. Biochem. 269, 2124–2132 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.ejb.02862.x The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea Eleonora Cerasoli1, Sharon M. Kelly1, John R. Coggins1, Deborah J. Boam1, David T. Clarke2 and Nicholas C. Price1 1Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Scotland, UK; 2Synchrotron Radiation Department, CLRC Daresbury Laboratory, Warrington UK Shikimate kinase was chosen as a convenient representative example of the subclass of a/b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 M urea.Fromananalysisoftheunfoldingcurveintermsof thetwo-statemodel,thestabilityofthefoldedstatecouldbe estimated as 17 kJÆmol)1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 M. The results of spectroscopic studies indi- Despite considerable experimental and theoretical efforts over the past 30 years, the mechanism by which proteins achieve their functional three-dimensional structure repre-sents a major area of uncertainty [1,2]. The importance of an understanding of protein folding is illustrated by both biotechnological applications (for example, in the recovery of properly folded expressed proteins [3]) and by clinical consequences (where disease states are caused by protein misfolding [4]). In addition, an understanding of the principles governing protein folding would help to allow the huge amount of information from genome sequencing projects to feed through to accurate predictions of three-dimensional structure of the encoded proteins. Because of the difficulties in applying structural techniques to the acquisition of structure accompanying or following trans-lation in vivo, the usual experimental approach has been to study the refolding of denatured proteins when conditions have been changed to promote folding. Several lines of evidence indicate that this approach can give valid insights into the process of protein folding in vivo [5]. Detailed studieshaveallowedthepathwaysoffoldingofanumberof small proteins, such as barnase [6], dihydrofolate reductase [7], chymotrypsin inhibitor 2 [8], lysozyme [9] and CheY [10] to be mapped out, but a key requirement is to examine Correspondence to N. C. Price, Synchrotron Radiation Department, CLRC Daresbury Laboratory, Warrington WA4 4AD, UK. Fax: + 44 141 330 6447; Tel.: + 44 141 330 2889; E-mail: N.Price@bio.gla.ac.uk Abbreviations: SK, shikimate kinase; ANS, 8-anilino-1-naphthalene-sulfonic acid; PK, pyruvate kinase; LDH, lactate dehydrogenase; GdmCl, guanidinium chloride; SRS, synchrotron radiation source. (Received 14 November 2001, revised 6 February 2002, accepted 1 March 2002) cated that refolding occurred in at least four kinetic phases, the slowest of which (k ¼ 0.009 s)1) corresponded with the regainofshikimatebindingandofenzymeactivity.Thetwo mostrapidphaseswereassociatedwithasubstantialincrease in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. Therelevance ofthe resultsto the folding of other a/b domain proteins is discussed. Keywords: shikimate kinase; protein folding; protein unfolding; circular dichroism; fluorescence. the behaviour of protein fold families in a systematic manner. The most structurally diverse of the classes of proteins, introduced by Chothia and colleagues [11], is the a/b class, which contains nearly 100 different kinds of protein folds. One of these subclasses is the P-loop-containing nucleotide triphosphate hydrolases, the core of which forms a classical mononucleotide-binding fold found in a number of struc-turally diverse proteins such as myosin, elongation factor EF-Tu, p21ras, the NDB domain of the ABC transporters, Rec A and adenylate kinase. The structural conservation of the core within this group of proteins is illustrated by the factthatsuperimpositionoftheP-loopsresultsinrootmean square deviations in alpha C atoms of only 0.3–0.4 A [12]. The isoenzyme II of shikimate kinase (SK, EC 2.7.1.71), an enzyme which catalyses the specific phosphorylation of the 3-hydroxylgroupofshikimateusingATPasthephosphoryl donor [13,14], is a member of this subclass. This step is the fifth in the seven-step pathway leading to the synthesis of chorismate, the precursor of aromatic compounds. From the X-ray structure ofSK [15], it is clear that the ordering of thestrands23145 intheparallelbsheetplacestheenzymein the same structural family as the NMP kinases (adenylate kinase, guanylate kinase, uridylate kinase and thymidine kinase). SK has a number of experimental advantages in estab-lishing the mechanism of protein folding. It is a monomeric enzyme without disulphide bonds and, with a molecular mass of 19 kDa, it is amongst the smallest kinases so far reported. SK has a single Trp residue (Trp54)that is located in the region near the shikimate binding site [15]. Binding of shikimate leads to quenching of Trp fluorescence [16], therebyprovidingaconvenientprobefortheintegrityofthe shikimate binding site. An additional feature of SK is that the side chains of Arg11, Arg58 and Arg139 provide a Ó FEBS 2002 highly positively charged environment around the Trp side chain and the shikimate binding site [15]. The use of the iodide ion as a quencher of protein fluorescence provides an additional means of investigating the integrity of this region of the protein. In the present paper, we have undertaken a study of the unfolding and refolding of the type II SK from E. chry-santhemi, using studies of CD, fluorescence, activity and ANS fluorescence, and employing both manual mixing and rapid reaction techniques. From these studies, we have been abletoformulateanoutlinepathwayforthefoldingprocess in which at least three intermediates are involved. The results extend the less complete data available for the refolding of adenylate kinase [17] indicating that the pathway described for SK should act as a model for many other members of this subclass of a/b proteins. MATERIALS AND METHODS Enzyme purification The purification protocol was based on those used for the purification of SK II from Escherichia coli [18] and for the previous purification of the enzyme from E. chrysanthemi [19]. The latter method was adapted by reducing the salt concentration so as to prevent protein precipitation. After cell breakage, all steps were performed at 4 °C. E. coli BL21(DE3)pLysS cells (10 g) were resuspended in 10 mL of buffer (20 mM Tris/HCl, pH 7.5 containing 0.4 mM dithiothreitol plus one tablet of ÔCompleteTMÕ (Boehringer) to inhibit protease action. Cells were broken by passing them through a French pressure cell twice at 6.9 MPa and the resulting mixture was centrifuged at 100 000 g for 1 h. The supernatant was dialysed for 4 h against buffer A (20 mM Tris/HCl, pH 7.5 containing 0.4 mM dithiothreitol and 1 mM MgCl2) and loaded on to a pre-equilibrated DEAE-Sephacel anion exchange column (30 cm · 2.6 cm diameter, flow rate 50 mLÆh)1). The column was then washed with buffer A until A280 < 0.1. Elution of shiki-mate kinase was achieved using a linear gradient of 0–300 mM KCl in 600 mL buffer A with a flow rate of 50 mLÆh)1 and a fraction volume of 14 mL. Pooled fractions were dialysed against buffer A. Before adding the solution to a phenyl–Sepharose CL-4B column (4 · 2 cm), solid (NH4)2SO4 was added to 30% saturation (164 gÆL ). The solution was stirred for 20 min and then centrifuged at 20 000 g for 15 min. The supernatant was loaded onto the column pre-equilibrated in buffer B [100 mM Tris/HCl,pH 7.5containing0.4 mM dithiothreitol and 1.2 M (NH4)2SO4]. The column was washed overnight with buffer B at low flow rate (5 mLÆh ) and 10 mL fractions were collected. The enzyme was eluted using a linear gradient of 400 mL 1.2–0.0 M (NH4)2SO4 in buffer B with a flow rate of 20 mLÆh and a fraction volume of 10 mL. At the end of the gradient the column was washed with 250 mL of 100 mM Tris/HCl, pH 7.5 containing 0.4 mM dithiothreitol until residual shikimate kinase had beeneluted.Activefractionsweredialysedovernightagainst buffer A containing 10% (v/v) glycerol to concentrate the enzyme sample. After this step, the sample was loaded on to the pre-equilibrated Sephacryl S200 (superfine grade) column Refolding of shikimate kinase (Eur. J. Biochem. 269) 2125 (120 · 2.5 cm) and eluted at a flow rate of 10 mLÆh)1 in buffer C (50 mM Tris/HCl, pH 7.5 containing 0.4 mM dithiothreitol, 5 mM MgCl2 and 500 mM KCl) with a fraction volume of 4 mL. Active fractions were pooled and dialysed overnight against 50 mM Tris/HCl, pH 7.5 con-taining 0.4 mM dithiothreitol, 5 mM MgCl2 and 50% (v/v) glycerol. The purified SK was stored at )20 °C. Before use, SK was dialysed against buffer D (35 mM Tris/HCl, pH 7.6 containing 5 mM KCl, 2.5 mM MgCl2 and 0.4 mM dithiothreitol) and used within a 2-day period. Enzyme activity and CD measurements showed that the protein is stable if stored overnight at )20 °C in this buffer. The concentration of SK was determined spectrophoto-metrically using a value of 0.62 for the A280 of a 1 mgÆmL)1 solution in a cuvette of 1-cm pathlength. This value was calculated from the amino-acid composition of the enzyme [20], using the observed ratio (1.09) of absorbances in buffer and in 6 M GdmCl. This value was within 10% of that obtained using the dye-binding method [21]. The ratio A280/ A260 was greater than 1.8, confirming the absence of significant contaminant by nucleotide. Assay of enzyme activity The activity of the shikimate kinase was determined by a double coupled assay involving pyruvate kinase (PK) and lactate dehydrogenase (LDH). The production of ADP in the shikimate kinase-catalysed reaction leads to the conver-sion of NADH to NAD+, which is monitored by the decrease in A340. The assay was carried out at 25 °C in a buffer consisting of 50 mM triethanolamine hydrochloride containing 50 mM KCl and 5 mM MgCl2, titrated to pH 7.2 with KOH. Concentrations of the assay components were 1.6 mM shikimate, 5 mM ATP, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 1 Uof each of PK and LDH. Stock solutions of the substrates were stored at )20 °C after neutralization with KOH. Under these conditions, the specific activity of the enzyme was 350 lmol min)1Æmg)1. In order to measure the activity of SK in the presence of urea it was necessary to use a quenched assay because of the effects of this agent on the coupling enzymes [22]. The SK-catalysed reaction was carried out in an assay solution containing 5 mM ATP, 1.6 mM shikimate and the appropriate concentration of urea in the assay buffer. At chosen times after the start of the reaction aliquots of this solution were diluted 30-fold into a quench mixture containing the appropriate concentrations of PEP, NADH, PK and LDH. From the decrease in A340, the concentration of ADP produced in the SK-catalysed reaction at the chosen times can be determined, and hence the rate of this reaction calculated. The errors in assays of enzyme activity were less than 5% of the quoted values. Spectroscopic measurements Except where indicated, all spectroscopic measurements were made on enzyme samples in buffer D. Most CD measurements were made using a Jasco J-600 spectropolarimeter, using cells of pathlength 0.2 or 0.5 mm and protein concentrations in the range 0.1–0.5 mgÆmL)1. 2126 E. Cerasoli et al. (Eur. J. Biochem. 269) SomeCDdatawereobtainedonexperimentalstation3.1of the CLRC Daresbury Laboratory’s Synchrotron Radiation Source (SRS). This facility comprises a vacuum-UV 1 m Seya-Namioka monochromator, which provides a high flux of linearly polarized light in the wavelength range 120– 300 nm, which is converted to circularly polarized light using a photoelastic modulator [23]. The SRS CD facility was particularly useful when spectra were recorded in the presence of high concentrations of NaCl or urea which absorb strongly in the far UV. Spectra were recorded using cells of pathlength 0.1 or 0.01 mm and protein concentra-tions in the range 1–2 mgÆmL)1. Fluorescence data were obtained using a PerkinElmer LS50 spectrofluorimeter. The fluorescence of ANS was measured using excitation and emission wavelengths of 380 nm and 480 nm, respec-tively. The concentrations of solutions of ANS were checked spectrophotometrically using a value of 6.0 for the A350 of a 1-mM solution in a cuvette of 1-cm pathlength [24]. The quenching of protein fluorescence by sodium iodide (over the range of quencher concentrations from 0 to 0.2 M) was analysedbyStern–Volmerplots asdescribedpreviously [25]. Stopped flow measurements were made using an Applied Photophysics SX-17 MV apparatus using a 10 : 1 mixing ratio. The dead times for the fluorescence and CD modes have been determined as 1.7 and 8 ms, respectively [26]. As recommended by the manufacturer, the time filter applied was less than 10% of the half time of the process being studied, in order to avoid distortion of the kinetic analysis. This analysis was undertaken using the PRO/K software supplied with the instrument. The data reported represent theaveragesofthreerunseachof10shots.Unlessotherwise stated, the errors in the amplitudes and rate constants derived were less than 10% of the stated values. The concentration of enzyme during refolding was in the range 60–110 lgÆmL)1 in different experiments, with no signifi-cant variation in rate constants observed over this range. Light scattering was measured using the PerkinElmer LS50 spectrofluorimeter with excitation and emission wavelengths of 320 nm. Unfolding and refolding studies Stock solutions of Ultrapure grade urea (10 M) were made up by weight in buffer D; the actual concentrations were checked using refractive index data [27]. Unfolding and refolding of SK was performed essentially as described in our previous studies on type II dehydroqu-inase [28]. To study the extent of unfolding of SK, the enzyme was routinely incubated in buffer D in the stated concentration of denaturant for 1 h at 20 °C, before the CD,fluorescenceandactivitydatawererecorded.Refolding was routinely initiated after unfolding for 1 h in the presence of 4 M urea, by dilution with 10 vol. of buffer D, to give a residual concentration of denaturant of 0.36 M. In preliminaryexperiments,itwasshownthatunfoldingin4 M urea for periods ranging from 5 min to 3 h had no effect on either the spectroscopic properties of the unfolded enzyme, or the kinetics of refolding as monitored by changes in protein fluorescence. Where indicated ANS was included in the unfolding and refolding mixtures at a concentration of 40 lM. Ó FEBS 2002 RESULTS Unfolding of enzyme Stability of the enzyme. The loss of secondary and tertiary structure during unfolding of SK by urea were monitored by changes in far UV CD and fluorescence, respectively. On incubation of the enzyme in 4 M urea, there was essentially a complete loss of secondary structure with the ellipticity at 225 nm reduced to less than 10% of the value characteristicofnativeenzyme.Thedegreeofunfoldingwas monitored by changes in the ellipticity at 225 nm. When excited at 290 nm, the fluorescence emission maximum of SK is 346 nm, indicating that the single Trp (Trp54) is significantly exposed to the solvent, a conclusion consistent with the high value of the Stern–Volmer constant forquenching ofthefluorescenceby succinimide[16].When incubated in 4 M urea, the emission maximum shifts to 356 nm, indicating that the Trp has become completely exposed to solvent. The degree of unfolding was monitored by changes in the emission intensity at 346 nm. The unfolding data for SK (Fig. 1) could be analysed satisfactorily in terms of a two-state model [27], suggesting that no intermediate species were populated to a significant extent. From the plot of free energy change against denaturant concentration the stability of native enzyme in the absence of denaturant could be estimated as 17 ± 1 kJÆmol)1 with no significant difference in stability observed using the two measures of structural changes employed. The value of the stability is towards the lower end of those observed for a range of globular proteins [29] and is similar to the value estimated for the structurally similar enzyme adenylate kinase (19.6 kJÆmol)1) from studies of the unfolding by urea [17]. However, given the difficulties in estimating the contributions of the various non–covalent interactions to the overall stability of globular proteins [29], it is not profitable to analyse this degree of similarity in greater detail. Changes in activity in the presence of urea. Incubation with urea leads to losses in activity which run roughly in parallel with the structural changes, with 85 and 40% activity retained in the presence of 1 and 2 M urea, respectively. In the presence of 4 M urea, shikimate kinase retainsno detectableactivity(< 0.1%ofthe controlvalue). Refolding of enzyme All experiments on the refolding of shikimate kinase involved unfolding in 4 M urea for unfolding and 11-fold dilution (to 0.36 M urea) to initiate refolding. During this process, there was no significant increase in light scattering at 320 nm during refolding showing that aggregation occurred to a negligible effect. Regain of activity. The first time point at which activity can be accurately assessed was estimated to be about 80 s after the start of refolding, taking into account the time taken for appropriate dilution into the assay solution and for the double coupled assay system to achieve a constant rate. By this time 35% of the activity of the control sample (in the presence of0.36 M urea) had been regained. Over the next 15 min, a further 60% activity was regained in a first Ó FEBS 2002 Refolding of shikimate kinase (Eur. J. Biochem. 269) 2127 Fig. 2. The kinetics of regain of activity of SK after denaturation in 4 M urea. Activity values are expressed relative to a control sample incu-batedinthepresenceofthefinalconcentrationofurea,i.e.0.36 M.The dashed line shows a fit to a first order process with a rate constant of 0.007 s)1. Fig. 1. The unfolding of SK in the presence of urea. (A) Structural changes monitored by changes in ellipticity at 225 nm (triangles) and protein fluorescence at 350 nm (squares) as described in the text. The concentration of protein in each sample was 0.2 mgÆmL)1. The data shown combine the results of three separate sets of experiments for each technique, with the results of replicate determinations within 5%. (B) Data analysed according to the two-state model [27], with the regression line shown. order process with a rate constant 0.007 s)1. Thus overall 95% of the activity of the control was regained (Fig. 2). Extrapolation of the curve shows that after 15 s, the regain of activity is 10% or less. If dithiothreitol was omitted from the unfolding and refolding buffers, the extent of regain of activity was reduced to 60%, showing that some damage had occurred to either or both of the two Cys side chains (Cys13 and Cys162) in the enzyme during the unfolding/ refolding procedure. Regain of secondary structure on refolding. When the enzymewasunfoldedin4 M ureaandsubsequentlyrefolded by an 11-fold dilution using manual mixing, 75% of the recovery of ellipticity at 225 nm was complete within the dead time (20 s) of the start of recording the ellipticity. A further 15% of the signal was regained over the subsequent 500 s with a rate constant of 0.009 s)1. At the end of this period the far UV CD spectrum of the refolded enzyme was very similar to that of native enzyme (data not shown). Using stopped flow mixing to initiate refolding it was shown that the regain of ellipticity at 225 nm occurred in a number of phases. From data obtained over the first 20 s of refolding, it was shown that, within 20 ms, 15% of the total signal corresponding to the folded enzyme (i.e. the differ-ence between denatured and folded enzyme) had been regained. A further 20% of the signal was regained in a first orderprocesswitharateconstantof8 s)1;inthethirdphase a further 40% was regained with a rate constant 0.08 s)1. Finally from data over the time range 20–200 s, a fourth phase was observed accounting for an additional 10% change with a rate constant 0.008 s)1. Taken together, the four phases account for a regain of 85% of the native secondary structure (Fig. 3). Regain of tertiary structure. The regain of tertiary struc-ture was monitored by changes in protein fluorescence at 350 nm after dilutionof the denaturantfrom 4 M to 0.36 M. In the manual mixing mode, the first time point at which reliable data could be obtained was 20 s after refolding had been initiated. Within this dead time, 35% of the fluores-cence of native enzyme (in the presence of 0.36 M urea) had been regained. Over the course of 20 min, a further 55% of the fluorescence was regained in a first order process with a rate constant of 0.009 s)1 (data not shown). Thus overall 90%ofthe signal ofnative SKwas regained.Using stopped flowmixingtechniques,itwasfoundthatlessthan5%ofthe total change occurred within 5 ms and that the subsequent changes in fluorescence occurred in two phases with amplitudes 42 and 45% of the total change with first order rate constants of 0.08 and 0.009 s)1, respectively (Fig. 4A). The rate of the slower process corresponds to that observed using manual mixing techniques. Refolding in the presence of shikimate. Refolding of shikimate kinase in the presence of shikimate was carried out in order to assess the stage in the process at which the shikimate binding site is formed, using the quenching of the protein fluorescence by the ligand as the index of binding. For these experiments it was necessary to monitor the refoldingbyfluorescenceat330 nm,ratherthan350 nm.At the latter wavelength, the quenching caused by the binding of shikimate to folded enzyme was nearly equal to the 2128 E. Cerasoli et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 3. The kinetics of changes in ellipticity at 225nm during refolding of SK after denaturation in 4 M urea. The refolding was initiated by stopped flow mixing; the inset shows data in the first second of the reaction. Curves a, b and c refer to enzyme in the presence of 4 M urea, enzyme in the presence of 0.36 M urea, and enzyme during refolding, respectively. The pattern of residuals to the curve fitting is shown. enhancement of protein fluorescence which occurred on refolding, leading to a very small overall change. In a separate experiment (data not shown) the binding of shikimate to the enzyme in the presence of 0.36 M urea was shown to be very rapid. When 2 mM shikimate was added to the enzyme (0.09 mgÆmL)1), over 95% of the fluorescence change occurred within the dead time of the stopped-flow instrument (1.8 ms), implying a rate constant for the association reaction >7 · 105 M)1Æs)1. The refolding of enzyme in the absence of shikimate led to a biphasic increase in fluorescence at 330 nm (Fig. 4B); the kinetics of this process were essentially indistinguishable from those observed at 350 nm (Fig. 4A). When the refoldingwascarriedoutinthepresenceof2 mM shikimate, however, a markedly different kinetic pattern was observed (Fig. 4B). After a rapid increase in fluorescence, essentially complete within 15 s, there was a slow small decrease over the next 185 s. The rate constant for this decline (0.025 s)1) was rather higher than that of the slow increase in the absence of shikimate (0.009 s)1), which could indicate that the presence of ligand has a nucleating effect on folding of thisareaoftheenzyme[5].Thefoldingoftheprotein(which would be expected to lead to an increase in protein fluorescence) leads to the formation of a Ônative-typeÕ shikimate binding site and the consequent quenching results in the overall decrease in fluorescence in this phase of the process. The simplest interpretation of these results is that theformationofthisÔnative-typeÕsiteisonlyassociatedwith the slowest phase of the folding process. ANS as a probe during refolding. ANS has been used extensivelyasaprobefortheexistenceofÔmoltenglobuleÕor Ôcompact intermediateÕ states of proteins and their forma-tion during folding [30,31]. However, there have been Fig. 4. The kinetics of changes in protein fluorescence at during refold-ing of SK after denaturation in 4 M urea. Refolding was initiated by stopped flow mixing and the fluorescence signals have been corrected for the buffer signal. (A)Refolding in the absence ofshikimate. Curves a, b and c refer to enzyme in the presence of 4 M urea, enzyme in the presenceof0.36 M urea,and enzymeduring refolding,respectively. (B) Comparison of refolding in the absence and presence of 2 mM shiki-mate. In (A), fluorescence was monitored at 350 nm; in (B) fluores-cence was monitored at 330 nm. The pattern of residuals to the curve fitting in (A) is shown. concerns raised that the presence of ANS may in fact perturb the folding process [32]. In the case of SK, the presence of 40 lM ANS caused an 18% decrease in the activity of enzyme when assayed under the standard conditions. The presence of ANS caused less than 10% change in the Kd for shikimate using the fluorescence quenching titration. When unfolding and refolding were performed in the presence of 40 lM ANS, the regain of activity was 95% that of the control (with ANS); this activity was regained in a first order process with a rate constant 0.008 s)1. From these data, it is clear that ANS has only relatively minor effects on the catalytic site of the enzyme and its ability to refold after denaturation. During the refolding process, a characteristic pattern of changesinANSfluorescenceduringrefoldingwasobserved. When refolding was initiated by manual mixing techniques, ... - tailieumienphi.vn