Báo cáo khoa học: Kinetically controlled refolding of a heat-denatured hyperthermostable protein pot

Kinetically controlled refolding of a heat-denatured hyperthermostable protein Sotirios Koutsopoulos 1 , John van der Oost 2 and Willem Norde 1,3 1 Laboratory of Physical Chemistry and Colloid Science, Wageningen University, the Netherlands 2 Laboratory of Microbiology, Wageningen University, the Netherlands 3 Department of Biomedical Engineering, University of Groningen, the Netherlands Hyperthermophilic microorganisms, which often belong to the Archaea, are able to grow optimally at 100 °C or higher [1]. After their discovery, it was nec- essary to revise our ideas about the mechanisms involved in the maintenance of protein structural integ- rity and function at elevated temperatures [2,3]. For the stabilization of proteins at high temperatures, a concerted optimization of structural features is employed. These include reduced solvent-exposed sur- face area [4], increased packing density [5–7], increased core hydrophobicity [8,9], decreased length of surface loops [6] and extended ion-pair networks [10–13]. Nat- ure uses different combinations of the same structural features to stabilize proteins that are adjusted to other environmental conditions [14]. In this work, we have investigated the unfolding ⁄ refolding process of the extracellular endo-b-1,3-glu- canase (LamA) from the hyperthermophilic micro- organism Pyrococcus furiosus that flourishes in the surroundings of low-depth undersea volcanic areas at temperatures ranging from 70 to 103 °C [15]. Proteins evolve through a balanced compromise between struc- tural rigidity, allowing for the maintenance of the native conformation at the physiological temperature of the organism, and flexibility, which is required for functionality. LamA is inactive at room temperature and shows maximum enzymatic activity at 104 °C, where ‘normal’ proteins from mesophilic organisms are already denatured [2,16]. In the simplified picture introduced 70 years ago by Anson and Mirsky [17], protein heat denaturation was described as a two-state transition between the native and the denatured state. Nowadays, the idea of one or more intermediate states is well established in many cases of heat-induced Keywords calorimetry; endo-b-1,3-glucanase; hyperthermostable enzyme; protein refolding Correspondence S. Koutsopoulos, Center for Biomedical Engineering, Massachusetts Institute of Technology, NE47-Room 307, 500 Technology Square, Cambridge, MA 02139-4307, USA Fax: +31 617 258 5239 Tel: +31 617 324 7612 E-mail: sotiris@mit.edu (Received 30 July 2007, accepted 21 September 2007) doi:10.1111/j.1742-4658.2007.06114.x The thermal denaturation of endo-b-1,3-glucanase from the hyperthermo- philic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 °C, correspond- ing to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, pre- viously denatured protein samples gave well-resolved denaturation transi- tion peaks and showed enzymatic activity after 3 and 9 months of storage, indicating slow refolding to the native conformation over time. Abbreviations DSC, differential scanning calorimetry; DNS, 3,5-dinitrosalicylic acid; DH cal , calorimetrically determined enthalpy change; DH vH , van’t Hoff enthalpy change; LamA, endo-b-1,3-glucanase; T d , denaturation temperature. FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS 5915 protein denaturation: the native state is tightly folded, the intermediate state(s) is functionally inactive and partially folded in a non-native conformation(s), and the unfolded state is characterized by significant amounts of loosely structured domains. In terms of biological functioning, the last two states are dena- tured, but only the latter resembles the random coiled conformation. A major question is whether the transi- tions between these states belong to a sequence of reversible processes that can be described thermody- namically. Apart from the profound applications of thermo- zymes in biocatalysis and biotechnology, by studying the thermal resistance properties of proteins we aim to tackle one of the most challenging problems in modern biophysics: what is the mechanism used by these pro- teins to stabilize their three-dimensional structure and sustain biological function at anthropocentrically extreme temperatures? Results Calorimetry Calorimetric studies of LamA in 0.01 m phosphate buffer showed a single denaturation transition peak. The denaturation temperature T d was dependent on pH and shifted from c. 112 °C at pH 4.0 to 109 °Cat pH 7.0 to 104 °C at pH 8.5 [18]. A typical thermogram of LamA is shown in Fig. 1A (line a). Variation of the scanning rate between 6 and 90 °CÆh )1 did not affect the T d , the shape of the endothermic peak or the enthalpy associated with the transition. This suggests that the thermal denaturation of LamA is not kineti- cally controlled [19,20]. The calorimetric criterion introduced by Privalov & Khechinashvili [21] to judge a two-state transition requires that the calorimetrically determined enthalpy change DH cal is equal to the van’t Hoff enthalpy change DH vH , which may be calculated from the dif- ferential scanning calorimetry (DSC) thermogram using the equation DH vH ¼ 4RT 2 d C p;max DH cal ð1Þ where R is the ideal gas constant, T d is the denatur- ation temperature and c p,max is the maximum heat capacity, with regard to the peak baseline, which is observed at the denaturation temperature. A two-state model implies that transient intermediate states, which should be distinguished from thermodynamically stable intermediates such as the molten globule, are not pop- ulated at the transition temperature [22]. The validity of this criterion has often been argued and therefore care should be taken when it is applied [20]. In the case of LamA, the DH cal ⁄ DH vH ratio deviates from unity, yielding a value of c. 0.5, suggesting a non-two- state transition. Furthermore, the standard functions integrated in the microcal origin dsc software (MicroCal Inc., Northampton, MA, USA) could not fit the endotherms as a two-state transition. The reversibility of the thermal transitions was tested by cooling the protein sample to room tempera- ture. Using different cooling rates between 15 and 90 °CÆh )1 , no exothermic transition suggesting protein refolding was observed (Fig. 1A, line a¢). Reheating the protein solution in the calorimeter cell, after cooling to room temperature, did not show an endo- thermic peak (Fig. 1A, line b). Heating LamA to C B A Fig. 1. Heat capacity as a function of temperature for 0.5 mgÆmL )1 LamA in 0.01 M phosphate buffer at pH 7.0. (A, B) Cooling and reheating of LamA shows no reversible denaturation peaks. (C) Transition observed when the first run is stopped just below the denaturation temperature. The scan rates tested were between 6 and 90 °CÆh )1 and, after each heating step, the sample was allowed to cool to room temperature. Kinetically controlled refolding of a denatured protein S. Koutsopoulos et al. 5916 FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS temperatures just above T d did not result in a revers- ible transition peak (Fig. 1B). In another experiment, the sample was heated up to exactly T d and was then allowed to cool to 25 °C. Subsequent reheating revealed a transition at the same T d value (Fig. 1C), which, however, was characterized by a heat exchange which was c. 50% decreased relative to that associated with the first peak. This suggests that approximately half of the LamA molecules are irreversibly denatured during the first partial transition (curve a) [20]. These are standard experiments from which we may conclude irreversible unfolding. The first experimental evidence of the reversible thermal denaturation of LamA was observed at very slow cooling rates. As mentioned previously, fast cool- ing did not result in an exothermic refolding peak. However, when very slow cooling of 0.1 °CÆh )1 was applied, a refolding transition peak was observed at the same temperature at which denaturation occurred during the first heating step (Fig. 2). Furthermore, the enthalpy released during refolding was similar to that absorbed on denaturation. Notably, this protein sam- ple, which was obtained during slow cooling, was found to be enzymatically active and, on reheating, a denaturation peak of slightly lower intensity was observed at the same temperature as before. Calorimetric tests of long-stored samples of dena- tured LamA (i.e. after 3 and 9 months of storage at ) 20 ° C) showed well-resolved transition peaks at the same temperatures as those observed during the first heating step (Fig. 3). However, the enthalpies associ- ated with these transitions were considerably lower. This indicates that the number of refolded LamA mol- ecules is smaller than that which initially gave the first strong endothermic peak and, furthermore, that refold- ing is time dependent. It is intriguing to suggest that, if the system were given more time, more denatured enzyme molecules would be natively refolded. How- ever, it was not possible to test this because, in the absence of antimicrobial agents, the denatured protein sample was not stable for longer periods. Transition phenomena similar to those shown in Figs 1–3 were also observed for LamA in solutions at pH 6.5 and pH 8.5. Enzymatic activity The specific enzymatic activity of LamA is 1547.6 unitsÆmg )1 at 90 °C. LamA samples derived from very slow cooling experiments were tested and showed recovered activity up to 83% (Fig. 4) compared with the activity of the untreated enzyme. In the fast-cooled denatured samples of LamA from a standard DSC experiment, the enzymatic activity was completely sup- pressed. However, storage of these samples for 3 and 9 months resulted in a notable increase in the enzymatic activity by 8% and 19%, respectively. The relatively long time required for the LamA molecules to show measurable activity reflects the slow kinetics of the refolding process to the native conformation. After heat incubation at 150 °C, samples of LamA did not show detectable activity, even after 6 months of storage at 4 °C. Circular dichroism The secondary structural characteristics of LamA in solution were determined using far-UV CD (Fig. 5, Fig. 2. Effect of cooling rate on the refolding of 0.5 mgÆmL )1 LamA in 0.01 M phosphate buffer at pH 7.0. Denaturation on heating (a) at a heating rate of 0.1 °CÆh )1 and the exothermic peak (b) observed on cooling with the same scanning rate. Fig. 3. LamA refolding as a function of time: (a) heat denaturation of 0.5 mgÆmL )1 native LamA in 0.01 M phosphate buffer at pH 7.0; (b) reheating the same sample 3 months later; (c) reheating the same sample 9 months later. Scan rate was 30 °CÆh )1 . S. Koutsopoulos et al. Kinetically controlled refolding of a denatured protein FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS 5917 top panel). Spectral analysis suggested that the second- ary structure of native LamA mainly consisted of b-sheets and turns, up to c. 96%. Heat-denatured LamA at 110 °C still contained 86% of b-structures, but the amount of a-helices and random coils increased to 4% and 10%, respectively. The far-UV CD spectrum of LamA after heat treatment in the CD cell unit at 110 °C resembled that obtained from the same protein sample after cooling to 25 °C. Prolonged heat incubation for 30 min at 150 °C resulted in the collapse of the secondary structure, and the polypep- tide chain of LamA appeared to be unordered (Fig. 5, top panel, curve c). Following the ellipticity of LamA in a closed cell at 220 nm as a function of temperature, we observed a transition between 105 and 110 °C (Fig. 5, top panel, inset). This transition could not be monitored further because of the instrument’s temperature limitations. At pH 7.0, the denaturation of LamA occurs at c. 109 °C, which is just below the maximum scanning tempera- ture of the instrument. Fluorescence spectroscopy The tryptophan fluorescence emission spectrum of native LamA shows a maximum at 335 nm (Fig. 5, bottom panel, curve a). Increasing the temperature of the solution resulted in a gradual decrease in the fluo- rescence intensity without a shift in the emission maxi- mum. Such a decrease in the intensity is attributed to increased tryptophan quenching as a result of thermal motion [23]. In comparison with the spectrum of native LamA at 25 °C, the spectral profile of dena- tured LamA at 110 °C (Fig. 5, bottom panel, curve b, corrected for the temperature effect on the intensity) showed slightly decreased intensity with a red shift in the emission maximum to 344 nm. This indicates a structural distortion, accompanied by partial exposure of previously confined tryptophan(s) to the solvent. After incubation for 30 min at 150 °C and cooling to 25 °C, the emission maximum shifted to 357 nm (curve c) and the intensity decreased three-fold, sug- gesting a collapsed tertiary structure [24]. Discussion The unfolding ⁄ refolding process of LamA was moni- tored using calorimetry. Analysis of the thermograms Fig. 4. Enzymatic activity of native and heat-treated LamA at differ- ent storage times. The activity was measured at 90 °C in 0.01 M phosphate buffer at pH 7.0. Fig. 5. Far-UV CD (top panel) and fluorescence emission (bottom panel) spectra of 0.25 mgÆmL )1 LamA in 0.01 M phosphate buffer at pH 7.0. Curve a, native state (recorded at 25 °C); curve b, heat- denatured at 110 °C (recorded at 110 °C; fluorescence spectrum was corrected for the tryptophan emission yield which decreases as a function of temperature); curve c, after heat incubation for 30 min at 150 °C (recorded at 25 °C). Top panel inset: thermal tran- sition of LamA monitored by the molar ellipticity at 220 nm. Kinetically controlled refolding of a denatured protein S. Koutsopoulos et al. 5918 FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS suggested that the denaturation of LamA could not be described as a two-state transition, because the calori- metric criterion, i.e. the DH cal ⁄ DH vH ratio, deviated from unity. However, the difference between DH cal and DH vH may be caused by inherent difficulties in the precise determination of the peak height and the inte- grated area of the peak, which has an unusual shape. This point needs further discussion. The heat capacity of denatured LamA, represented by the post-transition baseline, was unexpectedly low when compared with the heat capacity of the native state (pre-transition baseline). It should be noted that, in Fig. 1C, line b, the second up-scan of pre-heated LamA and, in Fig. 3 line c, the slowly refolded protein showed the same negative-like Dc p profile. Unless we assume that this is caused by an instrument artefact operating at the extreme end of its detection limit (i.e. 127 °C), such a profile is commonly attributed to aggregation of dena- tured protein molecules. Previously reported time-resolved anisotropy data did not show a significant increase in the hydrody- namic radius of the heat-denatured LamA molecule compared with the size of the native protein [25]. In addition, the fact that slow cooling resulted in an exothermic refolding transition suggests that the aggregation of LamA is not very likely (unless we assume that protein aggregation is a reversible pro- cess). In the absence of aggregation, similar denatur- ation profiles have been reported, but not discussed, for other hyperthermostable proteins [26–30]. The observed negative-like Dc p upon thermal denaturation of LamA at 109 °C may stem, in part, from the physicochemical properties of liquid water at temper- atures approaching 110 °C (H. Klump, University of Cape Town, South Africa, personal communication) [31]. Several lines of evidence support this hypothesis: (a) extrapolation of calorimetric data by Privalov [32], in his review on calorimetry in 1979, showed that the specific entropy of unfolding of several pro- teins intersects at c. 110 °C; (b) Baldwin’s hydrocar- bon model predicted that the entropy of mixing DS° of a nonpolar compound with water is negative at ambient temperatures, but approaches zero at c. 113 °C [33] (when DS° is zero, the solution shows ideal entropy of mixing and hydrophobic moieties may be readily dissolved in hydrophilic medium); (c) Shinoda [34] suggested that the increased solubility of hydrocarbons at high temperatures also depends on the enthalpy, which reflects the changes in the hydro- gen bonding interactions in water surrounding the nonpolar compound; (d) according to Ne ´ methy and Scheraga [35–37], the interaction of hydrocarbons with water at high temperatures results in changes in the local structure of the clustered water molecules adjacent to the hydrophobic surfaces (e.g. these water molecules show less hydrogen bonding and, therefore, are less hydrophilic). If the exposure of hydrophobic groups to water on denaturation does not contribute much to Dc p , perhaps other factors, such as solvation of the protein’s polar groups, become more impor- tant. Moreover, we cannot exclude the possibility that, on denaturation, the conformational changes in the pro- tein molecule are such that, from the protein core, more polar (compared to hydrophobic) amino acids are exposed to the polar solvent. The resulting struc- turally distorted, partially unfolded equilibrium inter- mediates are probably related to the kinetic folding intermediate reported by Park et al. [38]. Indeed, the surface of the native LamA molecule contains a large nonpolar fraction. A similar post-transition decrease in heat capacity, lower than that expected for a com- pletely unfolded polypeptide, was also observed in the denaturation of the recombinant human growth hor- mone [39]. Therein, it was suggested that the protein retained residual structure and, hence, was not fully hydrated after thermal denaturation. Whatever the case may be in the LamA system, these conjectures suggest that a negative Dc p on pro- tein denaturation and unfolding may be possible. It is also possible that the observed transition profile rests on an eluding component or mechanism that has not been considered so far. A more detailed characterization of the state of the protein at temperatures beyond 110 °C was not possi- ble because of limitations in the existing instrumenta- tion, which is not designed to operate at such biologically extreme temperatures. We were able to show that the state of denatured LamA was signifi- cantly different from that of the native protein with regard to secondary and tertiary structural elements (Fig. 5). However, as the transition could not be completely monitored by CD or fluorescence spec- troscopy, we could not unambiguously determine whether the post-transitional state of the protein rep- resents unfolding, or if it is just an intermediate which unfolds completely only upon heating to even higher temperatures. To answer the question about the state of LamA at temperatures above the denaturation point (i.e. at 109 °C), we used a calorimeter with a scanning tem- perature efficiency up to 200 °C (MC-DSC 4100, Cal- orimetry Sciences Corporation, Lindon, UT, USA). This experiment revealed that, following the main transition peak at 109 °C, another small exothermic peak appeared at 144 °C (Fig. 6), which suggests that S. Koutsopoulos et al. Kinetically controlled refolding of a denatured protein FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS 5919 the first peak does not represent complete unfolding, and that specific protein domains remain folded up to the temperature of the second transition. This implies that refolding proceeds through an intermediate state. Notably, this partially folded non-native state of LamA, after thermal denaturation at 109 °C, may refold to the native conformation either by ultra-slow cooling immediately after denaturation or by storing it for prolonged times. Slow cooling resulted in an exothermic refolding peak, indicating protein refold- ing. This was confirmed not only by enzymatic activ- ity tests but also by calorimetric studies: (a) on reheating the refolded protein, a DSC denaturation profile similar to that observed in the first heating step was found; and (b) heating a previously dena- tured LamA sample gave, after long storage, well- resolved denaturation peaks with partial recovery of the enthalpy exchange. In this work, we have shown that the temperature- induced transition of LamA from the native to the denatured state can be reversed if sufficient time is given for the system to equilibrate. Irreversible dena- turation is commonly observed on heating of pro- teins. However, the effect of time is rarely considered, even though theoretical studies have predicted that, in the absence of aggregation, reversible transitions are possible when slow relaxation is involved [40]. When the system relaxes more slowly than the time window of the measurement, i.e. the duration of the experi- ment, we ‘see’ the process as irreversible, but, given sufficient time, it may well be restored to the original state. This is the case for the refolding of LamA. In the article by Kaushik et al. [41], the unfold- ing ⁄ refolding kinetics were investigated, and it was shown that a predenatured hyperthermophilic pepti- dase from P. furiosus could refold completely after 36 h of incubation at 32 °C. Refolding required a few days on incubation at lower temperatures. These experiments resemble those presented here, where we showed reversible transition in long-stored frozen LamA samples. It is interesting to speculate that this behaviour may also be found in other heat-denatured proteins: refolding to the native conformation may be possible if sufficient time is given to the system. By contrast with the above-mentioned study, we were able to observe by calorimetry an exothermic refolding peak on very slow cooling of denatured LamA. It is not clear yet whether the denatured, par- tially folded state of LamA is kinetically trapped as a result of slow relaxation refolding kinetics, or whether this state is a thermodynamically stable form trapped in a local energy minimum of the energy distribution funnel. We can speculate that one of the reasons for the slow refolding process may be the relatively slow cis ⁄ trans isomerization of one or more of the 18 prolines of the protein [42]. This process requires hundreds of seconds to be completed [43]. LamA con- tains only one cysteine, and therefore post-transitional improper intramolecular disulfide bond formation is not possible, which would result in irreversible pro- tein denaturation. Chemical modification of amino acids on protein denaturation is possible in denatur- ation processes occurring at such high temperatures. However, temperature-induced deamidation of gluta- mines and asparagines could not be detected by mass spectroscopy, because this chemical reaction did not lead to significant changes in the protein mass. It is conceivable that this possibility is not very likely because LamA was able to refold to the active pro- tein conformation: extensive chemical changes on deamidation would irreversibly prevent correct protein folding. In conclusion, the calorimetric analysis showed that the transition of LamA from the native state to a par- tially unfolded intermediate was reversible if conditions were selected to give the system sufficient time. There is a strong biological argument supporting the conclu- sions presented here: extracellular LamA is exposed to temperature changes occurring in the microorganism’s environment (e.g. volcanic underwater milieu). During an environmental temperature change, such a system relaxes to the initial state very slowly. During this per- iod, denatured LamA molecules may recover their native conformation and biological activity. Fig. 6. Heat capacity as a function of temperature up to 200 °Cof 0.7 mgÆmL )1 LamA in 0.01 M phosphate buffer at pH 7.0 (scan rate was 30 °CÆh )1 ). Kinetically controlled refolding of a denatured protein S. Koutsopoulos et al. 5920 FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Purification of LamA The gene encoding LamA (GenBank accession no.AF013169) was expressed in Escherichia coli (strain BL 21 DE3) and cloned into pGEF+ under control of the T7 pro- moter [16]. Further purification was achieved by gel filtration (Superdex 200, Amersham Pharmacia, Uppsala, Sweden). The purity of the enzyme was tested using high-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectroscopy (PerSeptive Bio- systems Voyager DE-RP mass spectrometer, Framingham, MA, using sinapinic acid crystallized on a gold-coated well plate; spectra were calibrated with protein standards). LamA is a single domain globular-ellipsoid protein with a molecular mass of 30 085 Da. Its isoelectric point is at pH 4.4, as deter- mined by isoelectric focusing. Pure LamA was stored at ) 20 °C in 0.01 m phosphate buffer at pH 7.0, without anti- microbial agents, which might affect the protein’s physico- chemical characteristics. Differential scanning calorimetry Calorimetric studies were carried out in a VP-DSC calori- meter (MicroCal Inc.). Very small heat exchanges of LamA were recorded between 20 and 130 °C using, as reference, the buffer solution. All samples were degassed under vac- uum for 15 min prior to loading the cells, which were main- tained under a pressure of 2.5 bar to avoid boiling of the sample. The concentration of LamA was 0.5 mgÆmL )1 ; experiments were also performed at different concentrations between 0.1 and 2 mgÆmL )1 with the same results normal- ized per mass of the enzyme. Unless stated otherwise, the temperature was increased at a rate of 30 °CÆh )1 and, after reaching the maximum desired value, the sample was allowed to cool to room temperature. Heating rates between 6 and 90 °CÆh )1 were also used. A very slow cool- ing rate of 0.1 °CÆh )1 (i.e. 0.002 °CÆmin )1 ) was also tested. The normalized excess heat capacity functions were obtained after baseline subtraction and data processing using the formulation of Privalov [32]. Enzymatic activity tests The enzymatic activity of LamA before and after heat treat- ment was measured using the colorimetric reagent 3,5-dini- trosalicylic acid (DNS) [44]. This method is based on the spectrophotometric determination of the hydrolysed ends of oligosaccharides resulting from degradation of the substrate (i.e. laminarin). For the assay, the hyperthermostable enzyme and the substrate in 0.01 m phosphate buffer at pH 6.5 were incubated for 10 min at 90 °C. The enzymatic reaction was stopped by rapidly cooling the sample at room temperature. After the addition of DNS, the sample was incubated at 100 °C for 5 min and diluted (1 : 5, v ⁄ v) in water. The sample was then allowed to cool to room tem- perature and the absorbance was measured at 595 nm. Circular dichroism measurements CD spectroscopy was used to investigate the secondary structure of LamA before and after heat treatment. Far- UV (190–260 nm) CD spectra of 0.25 mgÆmL )1 LamA in quartz cuvettes (path length, 0.1 cm) were recorded in a J-715 spectrophotometer (JASCO, Tokyo, Japan). The scan rate was 100 nmÆmin )1 , with a resolution of 0.2 nm and response time of 0.25 s. Spectra were recorded in a closed metal-caged quartz cuvette under pressure to prevent the evaporation of water. The CD spectra of LamA after heat incubation at 150 °C were collected on samples which had been previously heated and then cooled to room tempera- ture (heat incubation for 30 min at 150 °C was performed in a temperature-controlled oil bath using thick-walled glass tubes with a lid capable of withstanding the vapour pres- sure of water). After subtraction of blank spectra, data analysis was performed by fitting the spectra to reference spectra using contin software [45,46]. Fluorescence spectroscopy Fluorescence emission spectra of 0.025 mgÆmL )1 LamA in quartz cuvettes (path length, 1 cm) were recorded in the range 300–400 nm in a Varian Cary Eclipse spectrophotometer (Palo Alto, CA). Spectra of denatured LamA at 110 °C were recorded in a closed cuvette under pressure to prevent solvent evaporation. Fluorescence spectra of LamA after heat incu- bation at 150 °C were collected on samples that had been cooled to room temperature. Excitation was set at 300 nm to excite only the tryptophans. The excitation and emission slit widths were 5.0 and 2.5 nm, respectively. All spectra were corrected for the background emission peak of water. Acknowledgements The authors gratefully acknowledge discussions with Horst Klump (University of Cape Town, South Africa) on the shape of the denaturation profile of the protein from calorimetric data. This research was supported by an Individual Marie Curie Fellowship of the Euro- pean Community programme ‘Improving Human Research Potential and the Socio-Economic Knowl- edge Base’ to S.K. References 1 Brown JR & Doolittle WF (1997) Archaea and the pro- karyote-to-eukaryote transition. Microbiol Mol Biol Rev 61, 456–502. S. Koutsopoulos et al. 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Kinetically controlled refolding of a denatured protein FEBS Journal 274 (2007) 5915–5923 ª 2007 The Authors Journal compilation ª 2007 FEBS 5923 . years ago by Anson and Mirsky [17], protein heat denaturation was described as a two-state transition between the native and the denatured state. Nowadays,. LamA refolding as a function of time: (a) heat denaturation of 0.5 mgÆmL )1 native LamA in 0.01 M phosphate buffer at pH 7.0; (b) reheating the same sample