Reversible and irreversible unfolding of multi-domain proteins

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Biochimica et Biophysica Acta 1774 (2007) 1591 – 1603 www.elsevier.com/locate/bbapap

Reversible and irreversible unfolding of multi-domain proteins K.H. Strucksberg, T. Rosenkranz, J. Fitter ⁎ Forschungszentrum Jülich, IBI-2, Biologische Strukturforschung, D-52425 Jülich, Germany Received 23 May 2007; received in revised form 6 September 2007; accepted 10 September 2007 Available online 25 September 2007

Abstract In contrast to single-domain proteins unfolding of larger multi-domain proteins is often irreversible. In a comparative case study on three different multi-domain proteins (phosphoglycerate kinase: PGK and two homologous α-amylases: TAKA and BLA) we investigated properties of unfolded states and their ability to fold back into the native state. For this purpose guanidine hydrochloride, alkaline pH, and thermal unfolded states were characterized. Structural alterations upon unfolding and refolding transitions were monitored using fluorescence and CD spectroscopy. Static and dynamic light scattering was employed to follow aggregation processes. Furthermore, proper refolding was also investigated by enzyme activity measurements. While for PGK at least partial reversible unfolding transitions were observed in most cases, we found reversible unfolding for TAKA in the case of alkaline pH and GndHCl induced unfolding. BLA exhibits reversible unfolding only under conditions with high concentrations of protecting osmolytes (glycerol), indicating that aggregation of the unfolded state is the main obstacle to achieve proper refolding for this protein. Structural properties, such as number and size of domains, secondary structure contents and compositions within domains, and domain topology were analyzed and considered in the interpretation of differences in refolding behavior of the investigated proteins. © 2007 Elsevier B.V. All rights reserved. Keywords: alpha-amylase; Aggregation; Unfolded states; Protecting osmolyte; Hydrodynamic radius

1. Introduction By far the most detailed investigations of protein folding processes have been performed with small globular proteins (100–200 amino acids) which are consisting of a single domain. In vitro most of these proteins exhibit a reversible unfolding transition. The application of equilibrium thermodynamics, for which the reversibility of the transitions is a prerequisite, provided a wealth of valuable insides on mechanisms of protein folding and stability [1–3]. However, the majority of proteins in the cell belong to the category of larger multi- domain proteins which typically unfold irreversible under most in vitro conditions. One of the major problems in this respect is related to Abbreviations: DLS, dynamic light scattering; SLS, static light scattering; CD, circular dichroism; PGK, phosphoglycerate kinase from Bakers yeast; BLA, Bac. licheniformis α-amylase; TAKA, Aspergillus oryzae α-amylase; DTT, dithiothreitol; Mops, 3-(N-morpholino)propanesulfonic acid; GndHCl, guanidine hydrochloride; EDTA, ethylenediamine tetraacid; RMSD, Root mean square deviation ⁎ Corresponding author. Tel.: +49 2461 612036; fax: +49 2461 612020. E-mail address: [email protected] (J. Fitter). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.09.005

aggregation of non native states which often inherently reduces the efficiency of the (re-)folding process. In vivo at least two factors play an important role to achieve reasonable folding yields of larger multi-domain proteins which are generally more prone for aggregation. First, the cytosol is a highly crowded solution (300–400 g/L of proteins and other macromolecules in Escherichia coli). Although macromolecular crowding increases the effective concentration of proteins and thereby enhances aggregation of non-native states, it is assumed that crowding (or more precisely the exclude volume effect) provides a nonspecific force for molecular compaction which principally promotes the collapse of polypeptide chains and stabilizes the native state [4,5]. Second, in vivo special folding helper proteins, namely molecular chaperones, serve to prevent protein aggregation and misfolding [6,7]. Depending on environmental conditions of the cell a substantial fraction of in particular larger multi-domain proteins (∼ 30%) use the assistance of various chaperones during the folding process [8]. Nevertheless, even in cells protein folding can be problematic. In the case of heterologous over-expression of recombinant proteins in bacteria or in other hosts proteins quite often fail to

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fold into their native states due to heavy aggregation (the inclusion body problem). In this context various strategies have been developed, first, to solubilize the inclusion bodies and in subsequent steps to transfer the proteins into conditions which provide high folding yields. In particular the latter step was studied in detail by employing co-solvent assisted folding (sugars, osmolytes), artificial chaperone systems (detergent, cyclodextrins), and in vitro chaperonin systems (GroEL/GroES) (see for example [9]). Many of the above mentioned principles and strategies are also relevant for the reversibility of induced unfolding transitions. In this work, we studied reversibility of unfolding transition for three different multi-domain proteins. Besides conventional unfolding treatments, such as employing elevated temperatures and GndHCl, we applied alkaline pH to induce unfolding transitions. The latter treatment was chosen, because it often produces unfolding transitions without apparent aggregation [10,11]. One enzyme, the phosphoglycerate kinase (PGK) from bakers yeast (44.3 kDa), consists of two domains, and for a multi-domain protein, yields some remarkable properties with respect to the reversibility of unfolding. It was demonstrated that for some conditions the thermal unfolding [12] as well as the GndHCl induced unfolding [13,14] transition was reversible, while for other conditions the thermal unfolding was irreversible [15,16]. For most other multi-domain proteins, in particular in the case of thermal unfolding, the transitions are irreversible at least for moderate and higher protein concentrations. This holds also for two α-amylases, namely BLA from Bacillus licheniformis (58.3 kDa) and TAKA from Aspergillus oryzae (52.5 kDa), for which unfolding transitions under various conditions were analyzed in the past [17–21]. Although both enzymes show an irreversible thermal unfolding transition, only BLA exhibits pronounced aggregation upon thermal unfolding, while for TAKA aggregation was not observed [22]. As known from many previous studies it is obvious that besides some other factors (e.g., incorrect disulfide bond formation, absence of co-factors) aggregation is one main obstacle to achieve proper (re-)folding. In contrast to PGK, for the two homologous α-amylases (as well as for many other α-amylases) only very few studies were able to demonstrate reversible unfolding transitions [23,24]. Therefore, the question arose, whether we would observe reversible unfolding for the α-amylases under similar conditions as where PGK (but also other multi-domain proteins) exhibits reversible unfolding transitions. In order to archive a more extended knowledge about possible reversible unfolding transitions we performed a systematic case study on the three multi- domain proteins. In a comparative analysis we investigated structural (and in some cases functional) alterations of the enzymes during unfolding and refolding transitions. Accompanying measurements using static as well as dynamic light scattering were performed in order to monitor the state of aggregation [25]. We employed various complementary techniques (CD- and fluorescence spectroscopy, enzyme activity assays, and DLS) to obtain results which state with high confidence whether experimental observations indicate refolding or not. For example, intrinsic tryptophan fluorescence measurements are often strongly biased by protein aggregation [22] and therefore need to be verified by

complementary techniques. One major goal was to analyze under which conditions unfolding transitions of the three enzymes are reversible and which properties enable reversibility. In particular the role of protein aggregation and the possible impact of structural properties of the proteins, such as domain topology, on the ability to obtain reversible unfolding transitions are discussed in this work. 2. Materials and methods 2.1. Enzymes α-Amylase from Bacillus licheniformis (BLA; purchased from Sigma) and from A. oryzae (TAKA; from Sigma) was obtained as lyophilized powder. Phosphoglycerate kinase from Bakers Yeast (PGK; from Sigma) was obtained in ammonium sulfate solution. Powders were dissolved in buffer and all enzymes were purified and transferred into desired buffers by the use of a desalting column (PD-10, Sephadex G-25, Amersham Biosciences). If not stated otherwise a 30mM Mops, 50 mM NaCl, 2 mM EDTA, pH 7.4 buffer was used for all proteins. In the case of CD-spectroscopy this buffer was used with only 10 mM Mops. Alkaline induced unfolding was performed in a Glycine/NaOH buffer. Protein concentrations were determined by weight measurements of lyophilized protein and by measuring the absorption at 280 nm (PGK: ε280: 20,340 M− 1 cm− 1; TAKA: ε280: 113,560 M− 1 cm− 1; BLA: ε280: 136,410 M− 1 cm− 1).

2.2. Enzyme activity measurements The enzyme activity of PGK was measured spectroscopically at 25 °C as described by Bücher [26]. The activity of both α-amylases was measured using the starch-iodine method after Bird and Hopkins [27]. For TAKA activity measurements were performed at 25 °C while for BLA 35 °C was used. For all enzymes the enzymatic activity was measured first in native buffer at their respective reference temperature (assigned to 100% activity). The enzymatic activity of the unfolded and refolded states is given in percentages of the native state (reference) activity.

2.3. Fluorescence spectroscopy Fluorescence emission spectra were recorded with protein solutions (protein concentration: 0.05–0.1 mg/mL) in quartz cuvettes (104F-QS, Hellma, Muehlheim, Germany) using a RF-1501 fluorospectromer (Shimadzu, Duisburg, Germany) or a QuantaMaster spectrofluorometer (QM-7) from Photon Technology International (Lawrenceville, NJ, USA). In contrast to PGK, having only two tryptophan residues, both α-amylases exhibit much more tryptophan residues (TAKA: 12, BLA: 17) per molecule. With excitation wavelengths of 280 nm for both α-amylases and of 295 nm for PGK we obtained emission spectra which are dominated by tryptophan emission (for details see Fig. 2). All emission spectra (recorded between 300 and 450 nm) were corrected for background intensities as measured with pure buffer solutions. Unfolding transitions were analyzed by determining the wavelength of the emission intensity maximum (λmax) as a function of environmental conditions (temperature, pH, GndHCl).

2.4. CD spectroscopy CD spectra in the far UV-region (200–280 nm) were recorded on a Jasco J810 under constant nitrogen flow. The spectra were recorded in a 2-mm or in a 1mm cell, averaged over three scans, and finally corrected for the buffer signal. Cell cuvette thickness and protein concentration were chosen in a way that the maximum high-tension voltage of the photomultiplier was not exceeding 600 V at the lowest wavelength (200–210 nm). Mean residue ellipticity (MRE) values were calculated by using

½h ¼

hd Mw 10d nd Cd l

ð1Þ

K.H. Strucksberg et al. / Biochimica et Biophysica Acta 1774 (2007) 1591–1603 where θ is the measured ellipticity in mdeg, Mw is the molecular mass of the protein in Da, n is the number of amino acid residues, C is the protein concentration in mg/mL, and l is the path length in cm. Typically we used protein concentrations between 0.1 and 0.8 mg/mL. Thermal unfolding transitions were monitored by taking the CD signal at 222 nm as a function of temperature.

2.5. Static and dynamic light scattering The detection and characterization of aggregates was performed by employing light scattering techniques. With both fluorescence spectrometers we measured the elastic scattering at 500 nm. For this purpose the same experimental setup was used as for fluorescence studies. This approach was helpful to check whether pronounced aggregation of proteins occurred during the unfolding transition. Dynamic laser light scattering was employed to determine the size distribution of proteins for folded and unfolded states. We used a DynaPro dynamic light scattering system from ProteinSolutions (Lakewood, NJ, USA) with a 3-mm path length 45 μL quartz cuvette. Samples with protein concentrations between 0.7 and 1.5 mg/mL were filtered through 0.2 μm Anotop filters before measured in the DLS. Data of 10–20 acquisitions were averaged and analyzed with the Dynamics V6 software. Using this software autocorrelation functions were calculated and a regularization fit was performed in order to obtain the size distribution (histogram). Depending on the number of peaks (each peak represents a distinct population of particles with a defined size) we were able to distinguish between monomodal and multimodal distributions. Based on this analysis, hydrodynamic radii of monomeric proteins and the state of aggregation (polydispersity, modality) were determined.

2.6. Unfolding and refolding transitions All thermal unfolding and refolding transitions were performed with heating (cooling) rates of 1 °C per minute. For refolding studies with GndHCl and alkaline pH unfolded samples, we prepared stock solutions for unfolded states (i.e. at 6 M GndHCl or at pH 12, respectively) with high protein concentrations (∼ 10 mg/mL). The proteins were incubated under these unfolding conditions for 12 h, which was sufficient to reach equilibrium. Thereafter fractions of the stock solution were diluted into corresponding refolding buffers. The end concentration of protein in refolding buffers was between 0.05 and 0.1 mg/mL, if not stated otherwise. In order to check the samples for putative refolding, we measured under refolding conditions (also in the case of thermal unfolding/ refolding transitions) after various exposure times ranging from 5 min to 2 days. For refolding studies with TAKA we always used refolding buffers with 1 mM DTT. In some cases a mixture of 2 mM reduced glutathione and 0.2 mM oxidized glutathione was used in the refolding buffer [23]. Furthermore, refolding of α-amylases was performed with and without addition of calcium ions (5–10 mM CaCl2). Experimental data points as measured during unfolding transitions were fitted with a sigmoidal Boltzmann function using OriginPro 7.5 (OriginLab Corp., Northhampton, MA, USA).

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creases the melting temperature for thermal unfolding drastically. We analyzed both α-amylases in EDTA buffer (calcium depleted samples) because in this case thermal unfolding transitions can occur at lower temperatures. Lower melting temperatures help to avoid some problems (deamidation, or other chemical modifications) which are supposed to occur at more elevated temperatures (N80 °C) and are also thought to prevent reversibility of unfolding [30,31]. The results of structural characterization as obtained for native and for the three unfolded states are shown in Fig. 2. With respect to the fluorescence data we observe a characteristic red shift of unfolded states as compared to the native state. The shifts are more or less identical for alkaline and GndHCl induced unfolding with λmax values characteristic for solvent accessible tryptophan residues (see Table 1). In contrast, thermal unfolded states exhibit less pronounced red shifts for all enzymes. As we know from earlier studies smaller red shifts of the emission spectra are mainly caused by aggregation rather than indicating a less distinct unfolding [22]. For both α-amylases a pronounced tyrosine fluorescence emission (λmax = 305 nm) is visible in the case of GndHCl unfolded states which is caused by an expanded unfolded state and a reduction of Förster resonance energy transfer from tyrosines to tryptophans. For alkaline unfolded states we do not observe tyrosine emission, because the tyrosine residues are deprotonated which gives rise to a strong reduction in fluorescence (for more details see legend of Fig. 2). The CD data indicate that all proteins are more or less fully unfolded at the given unfolding conditions (see also Table 1). With the exception of thermal unfolded PGK and BLA, all other unfolded states do not exhibit pronounced aggregation. Therefore we were able to determine hydrodynamic radii which clearly reveal rather expanded structures for all unfolded states (Table 1). None of the proteins exhibit any enzymatic activity for thermal, GndHCl and alkaline induced unfolded states (0% activity in the limits of error).

3. Results 3.1. Native and unfolded states The three proteins under investigation in this study belong to the category of medium sized multi-domain proteins with a number of 415–483 amino acid residues per molecule (Fig. 1). Only the TAKA structure is inherently stabilized by four disulfide bonds, while BLA has no cysteins and PGK has only one. Both α-amylases are stabilized by calcium ions (BLA has three binding sites and TAKA has two). As known from some earlier studies these calcium ions are not essential to maintain the native structural integrity, at least at room temperature [28,29]. However, for thermostability (and also for stability in general) the calcium ions play an important role, since bound calcium in-

Fig. 1. Schematic representation of protein structures for the enzymes studied in this work (PDB code for BLA: 1BLI, for TAKA: 6TAA, and for PGK: 3PGK). The color code (green, orange, cyan) represents the domain structure as given with the published structures [51–53], while calcium ions are shown in red, disulfide bonds in magneta, and tryptophan residues in yellow. Structure presentations were produced using PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System (2002), DeLano Scientific, San Carlos, CA, USA).

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Fig. 2. Fluorescence emission spectra (a–c) and CD spectra (d–f) are shown for the native and for three different unfolded states. Fluorescence emissions spectra (dominated by tryptophan residues) exhibit a more or less pronounced red shift for all unfolded states. While in PGK (2 tryptophans, 7 tyrosines) only the tryptophan residues were excited (λexc. 295 nm), in samples with BLA (17 tryptophans, 31 tyrosines) and with TAKA (12 tryptophans, 35 tyrosines) both intrinsic fluorophores were excited (λexc. 280 nm). As a result we observe distinct tyrosine fluorescence emission for some unfolded states of BLA and TAKA. CD spectra of the unfolded states emphasize that these states have lost nearly completely their secondary structure with respect to the native state.

3.2. Temperature induced unfolding in native and almost native buffers It is already known from various earlier studies, that mild denaturating conditions (e.g., 0.5–0.9 M GndHCl) can lower the

thermal melting temperature and also increase the transition temperature of cold denaturation (which often is experimentally inaccessible because it occurs below the freezing point of water) [32]. Such a behavior is also observable for PGK as shown in Fig. 3. Based on fluorescence and CD data the temperature

Table 1 Structural properties of folded and unfolded states Native

BLA TAKA PGK

6 M GndHCl

λmax [nm]

RH [nm]

Residual CD signal [%]

λmax/shift [nm]

RH [nm]

Residual CD signal [%]

334 328 328

3.2 3.2 3.0

100 100 100

348/14 346/18 349/21

5.9 5.0 5.6

0 0 0

λmax/shift [nm]

RH [nm]

Residual CD signal [%]

λmax/shift [nm]

RH [nm]

Residual CD signal [%]

340/6 339/11 342/14

n.d. 6.1 n.d

0 b5 0

348/14 351/23 348/20

5.1 5.0 6.2

0 b5 b15

Heat unfolded a

BLA TAKA PGK

Alkaline unfolded (pH 12)

a The heat unfolded states were achieved by keeping protein solutions 10 °C above the corresponding thermal transitions temperatures (T1/2 for BLA: 52 °C, for TAKA: 58 °C, and for PGK: 62 °C, see also [21]). In the case of heat unfolded states for BLA and for PGK a determination of the monomeric hydrodynamic radius RH was not possible due to pronounced aggregation (for details see next section).

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induced unfolding transitions are only visible for samples with 0.7 M GndHCl buffers between one and 55 °C. (Fig. 3a, b). In contrast, under native buffer conditions no thermal induced unfolding occurs in the given temperature range. Moreover, both temperature induced transitions are fully reversible under mild denaturating conditions. A rather similar result was already observed for PGK earlier [12]. Accompanying DLS data (Fig. 3c) nicely show how for both, heat and cold unfolded states, PGK is structurally expanded upon unfolding. Earlier DLS studies reveal similar results for the cold unfolded state [33]. Interestingly, the reversibility of thermal unfolding transitions with 0.7 M GndHCl buffers is observed for rather high protein concentrations. DLS measurements were performed with protein concentration of about 1 mg/mL and we obtained more than 95% of all proteins in the monomeric state, indicating that aggregation is negligible. For native buffer conditions the thermal unfolding transition takes places at much higher temperatures (T1/2 ∼62 °C), is clearly irreversible (Fig. 3d), and is characterized by a pronounced aggregation of the unfolded state (data not shown). In order to check whether a similar reversibility can be achieved for α-amylases under mild denaturating conditions, we employed several rather low GndHCl concentrations (0.1– 0.8 M) and monitored thermal unfolding. The result is shown in Fig. 4 for both α-amylases. For mild denaturating conditions the thermal unfolding transitions are shifted to lower temperatures with respect to the native buffer, but the transitions last irreversible. For the native buffer BLA starts to exhibit distinct aggregation when the proteins enter the unfolded state. The aggregation remains unchanged upon cooling back the samples down to room temperature. In contrast, TAKA stays without aggregation upon unfolding, even under refolding conditions (when cooled down). With respect to the native buffer, low GndHCl concentrations even increase the tendency of aggregation for both α-amylases. To concluded, we can state that for both α-amylases low GndHCl conditions do not provide a reversible thermal unfolding transition as observed for PGK. This result was also supported by enzyme activity measurements. PGK exhibits 85–95% enzymatic activity at room temperature conditions after heating and cooling treatments in 0.7 M GndHCl buffer. For the α-amylases and for PGK in native buffer the apparent refolded states do not show any enzymatic activity. 3.3. GndHCl and alkaline induced unfolding transitions In contrast to thermal unfolding, GndHCl unfolded samples in most cases do not exhibit strong aggregation. The unfolding transition upon the increase of GndHCl concentration is shown for all three enzymes in Fig. 5a, c, e. The data show that at around 1.5 M GndHCl all enzymes are already fully unfolded. At 6 M GndHCl all samples are not only fully unfolded, but also reside perfectly dissolved without any tendency of aggregation (best solubility of unfolded states as observed with DLS). In subsequent refolding studies, fractions of 6 M GndHCl solution were diluted into respective GndHCl concentrations (solid symbols in Fig. 5a, c, e). For refolding conditions with GndHCl concentrations below the transition point, the fluorescence data reveal emission spectra which are completely shifted back. At

Fig. 3. The results of thermal unfolding transitions as measured with PGK are shown in this figure. It is demonstrated that for samples with 0.7 M GndHCl heat and cold induced unfolding is fully reversible (heating: open symbols; cooling: full symbols). This result is proven by three independent techniques: (a) tryptophan fluorescence; (b) CD spectroscopy; (c) dynamic light scattering. In contrast, samples in native buffers (i.e. without GndHCl) exhibit higher melting temperatures and absolutely no reversibility for the unfolding transitions (d).

least for BLA (see Fig. 5a, g) and for PGK the recovery of the native state after unfolding appears to be nearly 100%, while for TAKA we obtain around 60% recovery. However, accompanying measurements of sample aggregation upon refolding reveal drastic difference between the three samples (Fig. 5h). In the range from 6 M to 2 M GndHCl all samples are more or less

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Fig. 4. Thermal unfolding transitions as measured for BLA (a) and for TAKA (b). The open symbols represent measurements during heating and the corresponding unfolding transitions have been fitted with a sigmoidal Boltzmann function (solid and dotted lines). The following transition temperatures were obtained from these fits: for BLA in native buffer T1/2: 52 °C, in 0.5 M GndHCl T1/2: 45 °C; for TAKA in native buffer T1/2: 59 °C, in 0.2 M GndHCl T1/2: 55 °C. The solid symbols represent data points as measured during cooling (i.e. refolding conditions).

free of aggregates. Below 2 M GndHCl only PGK stays without any tendency for aggregation, while for TAKA the scattering increases by a factor of three (which still is rather low) and for BLA the elastic scattering increases drastically by a factor of more than 250. In particular BLA exhibits a strong aggregation when the unfolded state is transferred into native state conditions (i.e. GndHCl concentration below 1.5 M). Although, the fluorescence emission spectrum of BLA for the refolded stated perfectly superpose with the native state (see Fig. 5g), the corresponding CD data for BLA reveal another result (Fig. 5b). The CD data clearly demonstrate that GndHCl unfolded BLA which is transferred back into native buffer conditions stays with a fully unfolded state, in contrast to what is indicated by the fluorescence data. Most probably the fluorescence data are biased by strong aggregation effects. The apparent blue shift with respect to the unfolded state is not related to protein compaction during a potential refolding process, but is caused by the burial of tryptophan residues due to a conglomeration of protein monomers in the aggregation process [22]. For PGK and for TAKA fluorescence and CD data (Fig. 5d, f) reveal coinciding results. For the enzymatic activity of refolded TAKA and PGK we obtained 38% and 80% respectively, while BLA exhibits no enzymatic activity under refolding condition. Our data indicate that almost fully (for PGK) and partially (for TAKA) refolded states are obtained from GndHCl unfolded proteins. In the case of BLA a strong aggregation seems to prevent a proper refolding.

As already shown in the previous section, buffers with alkaline pH can produce fully unfolded states (Fig. 2). The unfolding transition as a function of pH is shown for the three enzymes in Fig. 6a–c. Similar to the results as obtained from GndHCl induced unfolding we observe that TAKA is least stable as compared to PGK and BLA. TAKA exhibits an unfolding transition around pH 9.5, while PGK and BLA unfold above pH 11. Only TAKA and PGK show a partial reversibility of unfolding which was also observed in CD data (Fig. 6d, e). In contrast, BLA unfolds absolutely irreversible, similar to what we observed for thermal and GndHCl induced unfolding. The elastic scattering, as shown in Fig. 6f, stays for all enzymes at rather low levels during the potential refolding process. These data are not indicative for the fact, that aggregation hinders a refolding process. However, the use of DLS allows a more detailed insight. Fig. 6g shows the size distribution for native BLA at pH 8.5. In this state the distribution is dominated by the monomer peak around 3.2 nm and a rather small peak width (i.e. small polydispersity). This kind of distribution is characteristic for the native state, and similar results were obtained for the other proteins in the native state. For BLA transferred from pH 12 to pH 8.5 (Fig. 6h) we observe a rather different distribution. The main peak is much broader and is peaked around 17 nm, which is indicative for the fact that the enzyme is no longer monomeric but starts to form micro-aggregates. Because these aggregates are not much bigger than monomers (in the case of heavy aggregation the particles easily reach a size of 1000– 10,000 nm) the elastic scattering is not significantly increased. However, even small aggregates can be considerable obstacles in the refolding process. For PGK and TAKA corresponding DLS data show size distributions of the refolded enzyme which are much more similar to that of the native state. A rather dominant narrow monomer peak with a mean value of RH between 3.5 and 4.5 indicates that aggregation is almost negligible and that the structures are at least partially refolded. Again enzymatic activity measurements support, at least qualitatively results from fluorescence and CD measurements. While BLA exhibits no activity under refolding conditions, both, TAKA and PGK, show an enzymatic activity of about 20% for the refolded states. 3.4. Unfolding transitions in highly concentrated protecting osmolyte solutions It is obvious from the previous sections, that refolding was often hindered by distinct aggregation. One strategy to overcome this problem is to use protecting co-solvents which are either employed in buffers during the whole unfolding/refolding transitions or simply added to refolding buffers. A well-understood co-solvent in this respect is glycerol [34], which was already employed in several studies aiming to enhance refolding yields [35,36]. We employed 7 M glycerol in the refolding buffer for enzymes unfolded with 6 M GndHCl. As shown in Fig. 7 we obtain for BLA an unfolding transition which is highly reversible. Both, fluorescence (Fig. 7a) and CD (Fig. 7b) data clearly indicate a high refolding yield. Furthermore, the refolded sate of BLA exhibits no visible aggregation (data not

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Fig. 5. Unfolding and refolding transitions as induced by GndHCl. (a, c, e) Fluorescence emission peak wavelengths are given as a function of GndHCl concentration for unfolding (open symbols) and for refolding (closed symbols) transitions. (b, d, f) The corresponding CD spectra are shown for the unfolded and refolded states. (g) The fluorescence emission spectra for BLA indicate a reversible unfolding process, which is in contradiction to result from CD spectroscopy, see panel b. (h) As a result from elastic scattering measurements the apparent aggregation is shown for different GndHCl concentrations. For these measurements fraction from 6 M GndHCl stock solutions were diluted to obtain proteins solutions with lower denaturant concentrations. The large scattering of BLA seems to induce a blue shift of the corresponding fluorescence spectrum (g).

shown). A subsequent removal of glycerol from the refolding buffer as performed with a desalting column, yields BLA which remains almost unchanged (i.e. still similar to the native state, see Fig. 7a). Even thermal unfolding transitions performed in glycerol are characterized by a high extent of reversibility. In contrast to thermal unfolding in native buffers (see Fig. 4) BLA

exhibits a reversibility of about 70% (Fig. 7c, d). The effect of 7 M glycerol is also reflected in a much higher transition temperature (76 °C instead of 52 °C in native buffers). For PGK the reversibility in glycerol is even higher (∼ 80%) as compared to BLA (Fig. 7e, f). Nearly identical reversibility of the unfolding transition in glycerol for both enzymes was observed

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Fig. 6. Unfolding and refolding transitions as induced by alkaline pH. (a–c) Open symbols represent measurements starting from pH 8 with increasing pH values up to pH 13, while solid symbols represent measurements where samples in pH 12 buffer were transferred into buffers with lower pH values. (d, e) Corresponding CD spectra are shown for unfolded and refolded states. (f) For samples which were transferred from pH 12 to lower pH values the corresponding elastic scattering data are shown here. The elastic scattering intensity of all samples directly dissolved in native buffers (pH 8.0) was around a value of 1600. (g, h) Using DLS we analyzed the measured correlation function by applying a regularization fit and the obtained size distributions are shown in this figure. Figure (g) exhibits a characteristic pattern for a sample where 99.8% of the total sample mass corresponds to protein in monomeric state (i.e. without any aggregation) with RH = 3.2 nm. In contrast, figure (h) exhibits a much broader distribution peaked around 17 nm. This contribution represents 97.3% of the total protein mass and indicates that the enzymes are conglomerated to small aggregates.

in the CD data. In contrast to PGK and BLA, TAKA shows no reversibility during thermal unfolding in glycerol and a reversibility of GndHCl unfolding transition which is not altered in the presence of 7 M glycerol (i.e. similar to the results shown in Figs. 4b and 5c). The enzymatic activity of refolded BLA

from 6 M GndHCl in glycerol was about 80%, which nicely coincides with the CD data (see Fig. 7b). For thermal unfolded states in glycerol we observed a pronounced scan rate dependence of the reversibility. Only cooling rates of less than 1–2 degree per minute reveal reversibility of enzymatic

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Fig. 7. Unfolding and refolding transitions as observed in native buffers with 7 M glycerol. (a, b) Fluorescence and CD data of GndHCl unfolded BLA and of samples refolded in 7 M glycerol. Both techniques indicate that glycerol at high concentrations in the refolding buffer provides a pronounced refolding yield (∼ 80%). Thermal unfolding of BLA (c, d) and of PGK (e, f) was analyzed by fluorescence spectroscopy. The open symbols represent data points measured during heating, while solid symbols were obtained during cooling. For both samples, 7 M glycerol in native buffers permits a distinct reversibility of the unfolding transitions. In all refolding studies presented in this figure the final protein concentration was 0.1 mg/mL.

activities in the order of 70% for BLA and PGK. TAKA shows no activity after thermal unfolding in glycerol. We employed glycerol and two further osmolytes (ethylene glycol and sorbitol) also at different concentrations between one and seven molar in the refolding buffer (data not shown). With respect to reversibility of the unfolding transition, sorbitol in all cases exhibits rather similar results as compared to glycerol. For both osmolytes a concentration of 5–7 M in the refolding buffer yields the highest extent of refolding. For ethylene glycol the degree of refolding was smaller even at a concentration of 7 M. 4. Discussion We analyzed for three different multi-domain proteins unfolding transitions employing in total five different conditions to induce unfolding. The results concerning the reversibility of the particular unfolding transitions are summarized in Table 2. The data reveal that at least one unfolding condition for

reversible unfolding transition was found for every protein. Nevertheless, we observed significant differences between the proteins with respect to their ability to refold. PGK exhibits reversible unfolding transitions at four out of five conditions, BLA unfolds reversibly only at one condition, and TAKA, as an in-between, seems to share some similarities with BLA and some with PGK. In the following we will discuss our results with respect of two major subjects, aggregation phenomena and structural properties of the proteins including domain topology. From the presented results it became evident that non-aggregated unfolded states are crucial for a proper refolding. In the case of thermal unfolding we are dealing already with aggregation for the unfolded state which follows the scheme: A

A

NY U Y R :

ð2Þ

Here we observe a soluble native state (N), an aggregated unfolded state (U), and an aggregated potential refolded state

1600

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(R).1 For GndHCl and alkaline pH induced unfolding only the refolded states exhibit aggregation: A

N YU Y R :

ð3Þ

GndHCl as well as Urea are destabilizing osmolytes which do not only destabilize the native state, but also protect proteins in the unfolded state from aggregation [37–39]. Therefore, thermal unfolded states in most cases are much more prone for aggregation than GndHCl or Urea unfolded states (see Figs. 4 and 5). Since the buffer with high concentrations of GndHCl is a good solvent for unfolded states, we observe that the thermal unfolded states which are highly aggregated can easily be dissolved in 6 M GndHCl buffers. However, a successful refolding from GndHCl unfolded states can only take place by a reduction of GndHCl concentration which shifts the equilibrium to the native state, but at the same time decreases protecting effect against aggregation of unfolded states. For PGK and for TAKA the tendency of aggregation of GndHCl unfolded states near the transitions concentrations M1/2 (see Fig. 5) seems to be sufficient low, to ensure at least partial refolding. This is not the case for BLA, where we observe a strong aggregation (Fig. 5d) which hinders a proper refolding. To circumvent this dilemma, we employed refolding studies with protecting osmolytes (glycerol). The co-solvent in refolding buffers suppresses efficiently aggregation and promotes significant refolding yields for BLA (Fig. 7). Glycerol was also successfully employed for reversible thermal unfolding transition of BLA and PGK where at least a partial refolding is observed in 7 M glycerol which is absolutely absent without glycerol (Figs. 3c and 4). In the case of BLA we also used increased protein concentration for thermal unfolding in 7 M glycerol. Up to 0.2 mg/mL we still observed reversibility of thermal unfolding, while at 0.5 mg/mL the unfolding was no longer reversible and aggregation occurred. This clearly demonstrates that even high concentrations of glycerol are limited in their ability to hinder protein aggregation of unfolded states. However, higher refolding yields are generally achieved at much lower protein concentrations [23,36]. For PGK we observed reversible thermal unfolding under mild denaturation conditions (0.7 M GndHCl). This rather unique property of PGK, we do not observed for the α-amylases, and to our knowledge, this property was also not observed for any other multi-domain protein. In the past various unfolding and folding studies have been performed with PGK [12,33,40,41], where reversibility of the unfolding process was achieved with 0.7 M GndHCl. However, without GndHCl PGK behaves similar to BLA (and most probable to many other multi- domain proteins), displaying strong aggregation as well as irreversibility of the unfolding transition upon thermal unfolding, which could be surmounted efficiently by the use of glycerol. Another aspect of glycerol is given by the fact, that glycerol in some cases

1 The shown scheme displays a sequence of subsequent steps. In the case of successful refolding R would be identical with N, and we would obtain a scheme which is often displayed with a double arrow for a reversible forth and back reaction.

Table 2 Conditions of reversible and irreversible unfolding a Heat induced Heat induced Heat induced GndHCl Alkaline (native buffer) (0.7 M GndHCl) (7 M glycerol) induced induced PGK − TAKA − BLA −

+ − −

+ − +

+ + −

+ + −

a

For the respective unfolding treatments the transitions are marked with (+) for (at least partial) reversible and with (−) for irreversible processes.

stabilizes the enzymes significantly, which leads to much higher thermal transition temperatures [42]. For calcium depleted BLA we observe a T1/2 which is 24 °C above the transition temperature of that without glycerol. It is known from previous studies [29] that several α-amylases have a large potential for additional stabilization upon calcium binding (for BLA we observe ΔT1/2 = 50 °C upon calcium binding). With respect to calcium binding stabilization, glycerol exhibits at least half of the stabilization potential. In contrast, for PGK and for TAKA (data not shown) the increase of T1/2 in glycerol is only marginal as compared to buffers without glycerol. PGK and TAKA revealed a higher tendency for irreversible unfolding upon heat treatment as compared to GndHCl and alkaline induced unfolding. Under native buffer conditions for all proteins irreversible thermal induced unfolding was observed (see Table 2). The higher tendency for irreversible transitions induced by elevated temperatures might be explained by an interesting observation Gruebele and co-workers described recently [43]. The study reports a generic property of folded or partly unfolded proteins which start to form β-sheet like structures upon heating. These structures lead to a higher probability of forming random interdomain or intermolecular H-bonds which increase the chance of conformational scrambling or protein aggregation and thereby irreversibility of the unfolding transition. Besides cases where aggregation is the crucial process that controls the ability of refolding, in particular TAKA shows unfolding transitions which are irreversible but not accompanied by aggregation. While for GndHCl and for alkaline induced unfolding at least partial reversibility is observed (see also [23]), we do not observe any reversibility upon thermal unfolding. The thermal unfolded state was characterized by predominant monomeric protein structures, for which aggregation was not observed and the determination of the hydrodynamic radius of the unfolded state was possible (see Table 1). Therefore, the addition of glycerol was not helpful to overcome the irreversibility, since aggregation was not the obstacle for refolding. TAKA is the only protein in our study exhibiting disulfide bonds. Therefore we used DTT as well as a mixture of oxidized and reduced glutathione in the refolding buffer for this enzyme. However, in no case we observed reversibility of thermal unfolding. Therefore, it seems that some unknown thermal induced modifications in TAKA occur which hinder refolding. For the α-amylases studied here calcium ions are integral parts of the native structure. To our surprise the presence of calcium ions was not crucial for proper refolding of α-amylases. Neither in thermal unfolding studies, nor in GndHCl or in alkaline induced unfolding, the presence of calcium ions had a positive effect on refolding yields. Although

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calcium can be utilize to promote refolding of non-aggregated unfolded states [24], calcium ions themselves seems not to be essential to enable the (re-)folding process. On the contrary, even small amounts of calcium can enhance the aggregation (e.g., for concentration of more than 10 mM CaCl2 we observed aggregation of TAKA in the refolding buffer), indicating a specific role of the metal in the aggregation process (see also [44]). The summary given in Table 2 reveals similarities and dissimilarities between PGK and both α-amylases and between BLA and TAKA. In principle we can state the following: (1) with respect to GndHCl and alkaline pH induced unfolding PGK and TAKA exhibit similarities, while BLA behaves differently. (2) In the case of thermal unfolding the results are more divers. Both α-amylases are similar with respect to thermal unfolding at low concentrations of GndHCl (irreversible unfolding) and dissimilar at high concentrations of glycerol where BLA behaves like PGK (reversible unfolding). An attempt to understand these results in more detail is to take into account structural properties of the proteins. Fig. 1 allows the inspection of the most obvious structural features. As expected both α-amylases share a pronounced structure homology. They consist of three domains with the highest structural homology for the central α/β-barrel structure in domain A (RMSDvaluesof corresponding Cα-positions between 1 and 2 Å; Duy and Fitter, unpublished results). In both proteins domain C includes a so-called greek-key motif which is formed by antiparallel β-strands. The structural homology of domain C between TAKA and BLA is already less pronounced as compared to domain A. Domain B differs considerably in size and in content of secondary structure elements (both much smaller for TAKA) between both α-amylases. In addition, domain B consists of an insertion of 59 and 100 residues (for TAKA and BLA, respectively) connected at both termini to the sequence of the much bigger domain A. PGK is composed of only two α/β-domains, the N-terminal domain (185 residues) and the slightly larger C-terminal domain (229 residues). The impact of various aspects concerning domain topology on folding of multidomain proteins has been described in numerous studies. It was demonstrated that sequence diversity, domain interactions, and properties of domain connectivity of neighbouring domains in multi-domain proteins can have a profound influence on proper folding and thereby on the reversibility of unfolding [45–47]. However a take-over of these results to other multi-domain proteins is not easy and straightforward. Moreover, even the definition of a domain is not rigorous. In most cases a classification is used which defines a domain as a compact region of the native protein structure within which elements interact more extensively than with elements outside of it. Another, for our purpose of a more helpful definition, defines a domain as an independent folding unit. This more rigorous definition holds also for PGK, for which domain folding was investigated in several studies [13,48,49]. For GndHCl induced unfolding, it was shown that both isolated domains of PGK are able to refold completely, but exhibit differences in folding pathways and in refolding kinetics as compared to the case where the domains are embedded within the full protein structure. Therefore, at least for GndHCl unfolded states, there is

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no evidence that domain interactions in PGK considerably hinder refolding or reduce the refolding efficiency. As already mentioned above, one important issue in multi-domain protein folding is how domains avoid misfolding with their neighbours [46]. Besides other aspects, the sequence homology and structural properties of the linker region between two adjacent domains are supposed to play an important role. In both respects PGK seems to be a favourable candidate to ensure proper refolding, since PGK exhibits unfolding reversibility for four out of five unfolding conditions. Both domains in PGK exhibit a low sequence homology which is favourable to avoid aggregation propensity in multi-domain proteins [45]. In addition, the linker region appears rather flexible and thereby unwanted topological constrain on the domains which can lead to misfolding intermediates is reduced [47]. The question is: what is similar and what is different in the folding of TAKA and BLA with respect to PGK and what is the difference between both α-amlyases? The following differences between the α-amylases and PGK may contribute to the differences in the refolding ability. (1) The α-amylases consist of three domains instead of two domains for PGK. (2) Although the total size of the α-amylases is not much larger than that of PGK, the central domain A in BLA and TAKA is significantly larger (∼300 residues) as compared to the N- or C-domain (each ∼200 residues) of PGK. (3) For PGK the structure of both domains is dominated by α-helices. In the case of α-amylase α-helices predominated in the structure of domain A, while in domain C only β-sheets occur. However, TAKA is also able to refold from GndHCl and alkaline unfolded states like PGK, but different to BLA which does not show reversibility under these conditions. So, why do we observe rather different refolding abilities for the homologous α-amylases? A comparison of both α-amylase structures reveals two main features: (1) in contrast to BLA, the domain B in TAKA is much smaller (59 residues instead of 100 residues for BLA) and is consisting of only very few and small secondary structure elements. The small and very flexible domain B might have a minor influence on the overall folding process and therefore may ensure more efficient folding of TAKA. In this respect TAKA seems to behave more like a two domain protein, such as PGK. (2) TAKA (32% α-helix; 17% β-sheet) is characterized by a larger α-helix content and a smaller β-sheet content as compared to BLA (26% α-helix; 24% β-sheet). For comparison PGK consists of 35% α-helix and of 12% β-sheet. It is known from various studies, that more extended β-sheet structures in proteins are correlated with a stronger tendency of protein aggregation (see for example [50]). For PGK it was shown that increased temperatures during the unfolding transition leads to an extended β-sheet structure which could funnel the protein into aggregates [43]. A larger β-sheet content in the native structure found for BLA as compared to TAKA might cause the stronger tendency of aggregation during unfolding and thereby more often leads to irreversible unfolding. With respect to aggregation and to the irreversibility of unfolding, a comparison with other homologous α-amylases revealed that most of them (from Bacillus amyloliquefaciens (BAA), from Bacillus subtilis (BSUA), or from pig pancreas (PPA)) are similar to BLA [21]. There is only one exception, the psychrophilic α-amylase from alteromonas haloplanctis (AHA), which exhibits almost fully reversible unfolding

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transitions for heat and GndHCl induced unfolding [19]. Interestingly, AHA shares those structural features with TAKA which we assume to be important to ensure proper refolding. AHA is characterized by a rather small β-sheet content (31%α-helix, 13% β-sheet) and by a small size of domain B (59 residues) with only few secondary structure elements. In contrast PPA (28% αhelix, 23% β-sheet, domain B consisting of 67 residues with many secondary structure elements, mainly β-strands) shows very pronounced aggregation upon unfolding, similar to BLA. It seems that the structural homology between TAKA and AHA on one hand, and structural homology between BLA and PPA on the other hand support our interpretations. Nevertheless, we cannot claim that the discussed structural properties are the only relevant features for reversible unfolding transitions. We have investigated in a comparative study a two-domain protein and two homologous three-domain proteins with respect to reversibility of unfolding transitions. We observed clear differences in the ability to refold from unfolded states between the investigated proteins. PGK was able to refold at least partially from most unfolded states, while the α-amylases exhibit a reduced ability to refold. The analysis of structural properties (number and size of the domains, domain topology, β-sheet content) of the respective proteins was elucidative to understand at least tentatively the different behavior in the refolding abilities. In order to pinpoint the relevant aspects of multi-domain protein folding in more detail, it would be important also to study isolated domains from the α-amylases. To our knowledge, folding studies of isolated domains from αamylases have not been reported yet. Furthermore, mutants of full-length proteins with modifications of the inter-domain linker regions would help to understand the role of domain interactions.

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Acknowledgements

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G. Büldt is acknowledged for the generous support from his institute. We are indebted to Eva Reimer for support with enzymatic activity measurements.

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