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Originally published In Press as doi:10.1074/jbc.M207011200 on May 7, 2003

J. Biol. Chem., Vol. 278, Issue 30, 27945-27955, July 25, 2003
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Circular Permutation of 5-Aminolevulinate Synthase

EFFECT ON FOLDING, CONFORMATIONAL STABILITY, AND STRUCTURE*

Anton V. Cheltsov {ddagger} §, Wayne C. Guida ¶ || ** and Gloria C. Ferreira {ddagger} || {ddagger}{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, College of Medicine, the Department of Interdisciplinary Oncology and ||H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 and the **Department of Chemistry, Eckerd College, St. Petersburg, Florida 33711

Received for publication, July 12, 2002 , and in revised form, May 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The first and regulatory step of heme biosynthesis in mammals begins with the pyridoxal 5'-phosphate-dependent condensation reaction catalyzed by 5-aminolevulinate synthase. The enzyme functions as a homodimer with the two active sites at the dimer interface. Previous studies demonstrated that circular permutation of 5-aminolevulinate synthase does not prevent folding of the polypeptide chain into a structure amenable to binding of the pyridoxal 5'-phosphate cofactor and assembly of the two subunits into a functional enzyme. However, while maintaining a wild type-like three-dimensional structure, active, circularly permuted 5-aminolevulinate synthase variants possess different topologies. To assess whether the aminolevulinate synthase overall structure can be reached through alternative or multiple folding pathways, we investigated the guanidine hydrochloride-induced unfolding, conformational stability, and structure of active, circularly permuted variants in relation to those of the wild type enzyme using fluorescence, circular dichroism, activity, and size exclusion chromatography. Aminolevulinate synthase and circularly permuted variants folded reversibly; the equilibrium unfolding/refolding profiles were biphasic and, in all but one case, protein concentration-independent, indicating a unimolecular process with the presence of at least one stable intermediate. The formation of this intermediate was preceded by the disruption of the dimeric interface or dissociation of the dimer without significant change in the secondary structural content of the subunits. In contrast to the similar stabilities associated with the dimeric interface, the energy for the unfolding of the intermediate as well as the overall conformational stabilities varied among aminolevulinate synthase and variants. The unfolding of one functional permuted variant was protein concentration-dependent and had a potentially different folding mechanism. We propose that the order of the ALAS secondary structure elements does not determine the ability of the polypeptide chain to fold but does affect its folding mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, heme biosynthesis is initiated with the condensation of glycine with succinyl-CoA to form 5-aminolevulinic acid (ALA),1 CoA, and CO2 (1, 2). 5-Aminolevulinate synthase (ALAS) (EC 2.3.1.37 [EC] ), the enzyme responsible for this catalytic reaction, has two distinct mammalian isoforms: the housekeeping ALAS, expressed in all cell types; and the erythroid-specific ALAS, only expressed in developing erythrocytes. The two human ALAS-encoding genes were assigned to chromosome 3 (3) and the X chromosome (4) for the housekeeping and erythroid-specific enzymes, respectively. Erythroid, ALAS-produced heme accounts for 90% of the total heme synthesized in humans (5, 6), and mutations in human erythroid ALAS lead to X-linked sideroblastic anemia (7–10), which is distinguished by inadequate heme synthesis and an overaccumulation of iron in erythroblast mitochondria (7).

ALAS is a pyridoxal 5'-phosphate (PLP)-dependent homodimer with 56-kDa subunits and the active site located at the dimer interface (11). The steady-state kinetics of the ALAS-catalyzed reaction follows an ordered Bi-Bi mechanism, in which glycine binds first, succinyl-CoA second, and ALA is released last (12). Along the catalytic pathway the PLP forms several intermediates (13). In the absence of substrates or products, PLP is covalently bound via a Schiff base to the {epsilon}-amino group of Lys-313 of murine erythroid ALAS, forming an internal aldimine (14). Elucidation of the roles of specific ALAS amino acids in substrate recognition and binding and catalysis has been accomplished mainly through mutagenesis and structural homology modeling (13, 15–18). Specifically in murine erythroid ALAS, Lys-313 appears to play a dual role, i.e. holding the PLP cofactor in the active site as an internal aldimine (14) and catalysis (13), and Arg-439 is crucial in the binding of glycine substrate (18).

Previously, we demonstrated that circular permutation of ALAS, which changed the primary sequence without altering the amino acid composition, led to equally active ALAS variants (19). In fact, we concluded that the natural continuity of the ALAS polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the PLP cofactor, or assembly of the two subunits into a functional enzyme. Moreover, at the primary structure level, the order of the two identified functional elements (i.e. the catalytic and the glycine-binding domains) did not affect the functioning of the enzyme. However, despite the similarity of the predicted overall tertiary structures, ALAS and its circularly permuted variants displayed distinct arrangements of the secondary structure elements (19).

These observations underscore the importance of elucidating the folding mechanism(s) of ALAS and circularly permuted variants to understand its(their) role in attaining a stable dimeric interface, conducive to PLP anchoring and ALAS catalysis. In this study, we analyze and compare the folding mechanism, conformational stability, and predicted structures of the circularly permuted ALAS variants in relation to wild type enzyme. We report that the differences in the folding processes of ALAS and active, circularly permuted ALAS variants are translated in distinct overall conformational stabilities, unique cofactor environments, and identical stabilities associated with the protein domains defining the dimeric interface.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The following reagents were from Sigma: DEAE-Sephacel, {beta}-mercaptoethanol, Gdm-HCl, p-dimethylaminobenzaldehyde, acetylacetone, Tween 20, PLP, bovine serum albumin, {alpha}-ketoglutarate dehydrogenase, {alpha}-ketoglutarate, NAD+, thiamin pyrophosphate, succinyl-CoA, HEPES-free acid, aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, the bicinchoninic acid protein concentration determination kit, and the gel filtration molecular weight markers kit (cytochrome c, carbonic anhydrase, bovine serum albumin, alcohol dehydrogenase, and {beta}-amylase). Glycerol, mono and dibasic potassium phosphate, sodium acetate, perchloric acid, acetic acid, and disodium EDTA dihydrate were provided from Fisher. Ultrogel AcA-44 was obtained from IBF Biotechnics. SDS-PAGE reagents were supplied by Bio-Rad. Vent DNA polymerase, BamHI, SalI, and T4 DNA ligase were purchased from New England Biolabs. The Superdex 200 gel filtration resin was purchased from Amersham Biosciences. DNA oligonucleotides were synthesized by Cybersyn Inc. The Escherichia coli strain LC24 (20) was obtained from the ATCC.

Nomenclature Used for the Different Proteins—L25, Q69, N404, and N408 represent circularly permuted ALAS variants with the N-terminal amino acids corresponding to leucine 25, glutamine 69, asparagine 404, and asparagine 408 of wild type murine erythroid ALAS, respectively (19). 2X-ALAS is a monomeric protein consisting of two wild type ALAS subunits covalently linked through the N terminus of one subunit to the C terminus of the other subunit.

pAC1 Plasmid—The pAC1 plasmid, constructed as described previously (19), contains the cDNA coding for 2X-ALAS, which corresponds to the sequence of two tandem ALAS cDNAs linked through an MfeI site.

Purification of Wild Type ALAS, 2X-ALAS, and Selected Circularly Permuted ALAS Variants—Recombinant wild type ALAS, 2X-ALAS, and selected circularly permuted variants were purified from E. coli overproducing cells containing the different ALAS-encoding cDNAs under the control of the alkaline phosphatase (pho A) promoter (19, 21). E. coli strain BL21(DE3) cells were used to overexpress 2X-ALAS, whereas wild type ALAS and ALAS circularly permuted variants L25, Q69, N404, and N408 variants were overproduced and purified as described previously (19, 21). The purification of 2X-ALAS began with the resuspension of the harvested cells harboring 2X-ALAS in buffer A (20 mM potassium phosphate, pH 7.5, containing 10% glycerol, 1 mM EDTA, 20 µM PLP, 5 mM mercaptoethanol, and the following protease inhibitors: 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml phenylmethylsulfonyl fluoride). Upon cell lysis, the cell debris was removed by centrifugation, as described previously (21), and the supernatant was brought to 25% ammonium sulfate. After stirring for 10 min at 4 °C, the solution was centrifuged at 27,000 x g for 30 min at 4 °C. The pellet was discarded, and the supernatant was further fractionated with ammonium sulfate to a final concentration of 35% and centrifuged as above. The chromatographic steps using Ultragel AcA and DEAE-Sephacel columns were as described previously (21) with the following modifications: the DEAE-Sephacel resin was washed with buffer A until A280 was lower than 0.1, and 2X-ALAS was eluted with buffer A containing 60 mM KCl. 2X-ALAS-containing fractions were pooled and concentrated in an Amicon 8050 stirred cell with an YM30 membrane. The purified and concentrated enzyme was stored under liquid nitrogen until use.

Molecular Mass Determination of 2X-ALAS by Gel Filtration Chromatography—The native molecular mass of 2X-ALAS was determined by gel filtration chromatography on Superdex 200 column (1.0 x 50 cm). The Superdex 200 gel filtration column, connected to a PerkinElmer Life Sciences high pressure liquid chromatography system, was equilibrated with 20 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol, 1 mM EDTA, 20 µM PLP, and 5 mM mercaptoethanol. The flow rate was set at 1.0 ml/min. The purified ALAS (10 µM), 2X-ALAS (10 µM), and gel filtration molecular weight markers were prepared in the same buffer and applied onto the column under the same conditions. The molecular mass of 2X-ALAS was calculated from the calibration curve generated using the molecular weight markers.

Refolding of Unfolded Wild Type ALAS, 2X-ALAS, and ALAS Circularly Permuted Variants followed by Recovery of Enzymatic Activity— Protein unfolding was achieved by incubating any of the proteins, typically at 5 µM, in buffer B (20 mM potassium phosphate, pH 7.5, 2% Tween 20, 100 µM PLP, 10% glycerol, and 5 mM mercaptoethanol) containing 5.0 M Gdm-HCl for 1 h at room temperature (~25 °C). Refolding was initiated by diluting the unfolded protein in buffer B, to which the 100 µM PLP was freshly added, to 0.01–0.2 µM. Thus, the Gdm-HCl dilution was 25–500-fold. After incubation at 25 °C for 2 h, the recovery of enzymatic activity was monitored by assaying aliquots of the refolded proteins according to the method described previously (22). The yield of reactivation was expressed relative to the activity of a native control sample (i.e. in the absence of Gdm-HCl) maintained under identical conditions.

ALAS Unfolding Monitored by Gel Filtration Chromatography—Analysis of the size of ALAS during unfolding experiments was performed at 4 °C using the same high pressure liquid chromatography system and gel filtration column as for the determination of the molecular mass of 2X-ALAS. For each run, the gel filtration column was equilibrated with buffer B containing the appropriate guanidinium chloride (Gdm-HCl) concentration. The flow rate was 1.0 ml/min, and the detection was determined from the protein intrinsic protein fluorescence (see below). As a control for each run, 2X-ALAS was used as a molecular weight marker.

Fluorescence and CD Spectroscopies—Fluorescence spectra were collected on a Shimadzu RF-5301 PC spectrofluorophotometer. Intrinsic protein fluorescence was monitored with excitation and emission wavelengths at 280.6 and 334.2 nm, respectively, whereas protein-bound PLP cofactor fluorescence was followed with excitation and emission wavelengths set at 434 and 515 nm, respectively. CD spectra (210–240 nm) were obtained on a Jasco model 710 spectropolarimeter with a cylindrical cell of 0.1-cm path length and a total volume of 300 µl. The observed rotation degrees ({theta}obs) were converted to molar ellipticity. All spectra were obtained at 25 °C and corrected for buffer contribution.

Equilibrium Unfolding and Refolding Studies—Stock solutions of either native or 5.0 M Gdm-HCl-denatured proteins were diluted to 0.1 µM in buffer B containing the indicated final concentration of Gdm-HCl and incubated for 2 h at 25 °C. Both unfolding and refolding experiments were done in 0.0–5.0 M Gdm-HCl range, because5.0 M Gdm-HCl was sufficient to unfold completely wild type ALAS and its circularly permuted variants (see "Results"). Folding transitions were examined upon determination of catalytic activity (see below), oligomeric size, CD, intrinsic tryptophan fluorescence, and protein-bound PLP fluorescence properties. In experiments where the PLP cofactor fluorescence was used as the spectroscopic probe to monitor the unfolding transitions, PLP was excluded from the buffer composition. All measurements were done in triplicate.

Enzymatic Assay—ALAS activity was measured according to the method of Lien and Beattie (23) with subtle modifications. Briefly, the 2-h equilibrium unfolded samples were incubated in 25.0 mM Tris, pH 7.4, containing 41.7 mM glycine, 0.14 mM succinyl-CoA, 2.1 mM MgCl2, 2.1 mM EDTA, and 0.017 mM PLP for 1 h at 30 °C. The samples were then placed on ice, and freshly prepared 10% acetylacetone in 1.0 M sodium acetate, pH 4.7, was added to a final concentration of 0.33% acetylacetone and 0.3 M sodium acetate. An incubation for 10 min at 80 °C was followed by the addition, at 25 °C, of modified Ehrlich's reagent (24) to a final concentration of 0.05 M. The colored salt of the pyrrole formed with Ehrlich's reagent was quantitated by reading the absorbance at 552 nm using a Biotek µQuant plate reader.

Analysis of the Equilibrium Unfolding Profile Using a Three-state Model—Before data analysis, the raw data were converted into fraction unfolded (F.U.) according to Equation 1,

(Eq. 1)
where xi is the system parameter being monitored during the unfolding reaction and xnative and xunfolded are the parameter values corresponding to the native and completely unfolded states of a protein, respectively.

A unimolecular, three-state unfolding process of a dimeric protein is described as follows: N {leftrightarrow} I {leftrightarrow} U where N, I, and U are native, intermediate, and unfolded states, respectively (25, 26). As shown in Equation 2, if y represents the experimental variable being used to follow the transition, and yN, yI, and yU are the values of y for N, I, and U, respectively (27), then

(Eq. 2)
where fI and fU are fractions of I and U species, respectively, and can be expressed through equilibrium constants as shown in Equations 3 and 4,

(Eq. 3)

(Eq. 4)

When F.U. is used as the experimental variable, then yN and yU in Equation 2 are set to 0.0 and 1.0, respectively.

The equilibrium constants can be related to the free energy and denaturant concentration using the linear dependence of the unfolding free energy upon denaturant concentration (28) as shown in Equations 5 and 6,

(Eq. 5)

(Eq. 6)
By substituting Equations 6, 5, 4, and 3 into Equation 2, an equation relating y to denaturant concentration can be obtained. , , mNI, mIU, and yI parameters were obtained by fitting experimental data to the derived equation, using the program DataFit version 7.0 (Oakdale Engineering Inc.).

Protein-bound PLP Fluorescence Quenching Studies—Accessibility of the PLP cofactor in the wild type ALAS and variants was assessed by monitoring the PLP fluorescence upon the addition of external quenchers. Cs+, I–, and acrylamide, which are cationic, anionic, and neutral, respectively, were among the quenchers tested. The quenching reactions were performed at 25 °C with 2.0 µM protein samples in 20 mM potassium phosphate, pH 7.5, containing 10% glycerol. While studying quenching by Cs+ and I–, the ionic strength was maintained constant by adding 0.5 M KCl to the same buffer. The fluorescence data were analyzed by fitting to one of the two forms (Equations 7 or 8) of the Stern-Volmer equation (29), where KSV is the dynamic quenching constant, V is the static quenching constant, and Q is the quencher, using the program DataFit version 7.0 (Oakdale Engineering Inc.).

(Eq. 7)

(Eq. 8)

Modeling of Wild Type ALAS and Circularly Permuted Variants and PLP Cofactor Docking—Comparative protein modeling of the three-dimensional structures of wild type ALAS and its circularly permuted variants (i.e. L25, Q69, and N408) was performed using the amino acid sequence and coordinates for E. coli 8-amino-7-oxononanoate synthase (AONS) (30, 31) (Protein Data Bank accession code 1BS0 [PDB] ) as template. AONS and ALAS share 30.2% sequence identity and 31.3% sequence similarity. According to Chothia and Lesk (32), the 30% sequence identity threshold guarantees three-dimensional similarity, and thus, AONS appeared to be an acceptable template for homology modeling of ALAS and its variants. The AONS and the target amino acid sequences were aligned using the ClustalW algorithm (33). Then the initial three-dimensional structures for wild type ALAS and its circularly permuted variants were obtained using the comparative protein structure modeling method of "satisfaction of spatial restraints" as implemented in Modeler 4.0 (34). The obtained protein models were further optimized using MacroModel 7.2 (35) purchased from Schrödinger, Inc. (www.schrodinger.com). The models were also validated using PRO-CHECK (36). The AMBER* (37) force field with all atom S-LP treatment was used for all structure calculations, and the energy optimization of the models was done in several steps, by gradually removing energy constrains from 500.0 kJ/mol·Å2 to 0.0 kJ/mol·Å2. A distance dependent dielectric "constant" further attenuated by a factor of 4 was used for the AMBER* electrostatic treatment. Following optimization, the PLP co-factor was linked to the Schiff base lysine of the wild type ALAS and variants (i.e. L25, Q69, and N408). In order to find the best cofactor conformation for each structure, a truncated model of the protein was employed. A 15-Å radius was used to define a shell of atoms around this lysine. The entire residue was incorporated into the shell if any atom in the residue was at least within 15 Å from any atom in this lysine residue. The resulting polypeptide chains were extended out to the {alpha}-carbon in both directions. All of these residues were "frozen" at their starting positions so that only their electrostatic and van der Waals energies were included in the subsequent calculations, whereas the lysine side chain and the PLP cofactor were allowed to move freely. The explicit torsion angles were allowed to vary as well. The cofactor conformations with energies within a 25-kJ energy window were found by applying a multiple-conformation search using Macromodel 7.2. The Low-Mode method was employed for the conformational search (38). The TNCG minimizer was used for energy minimization. The global minima were used to interpret the docking results. The molecular surfaces of the structures were computed using Swiss PDB Viewer (39). The secondary structures of the ALAS and circularly permuted variants were predicted using the DSSP program (40). Protein topology schematics (TOPS) of the predicted three-dimensional structures were generated using the TOPS program (41, 42).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Unfolding/Refolding Transitions of Wild Type ALAS and Circularly Permuted Variants, Defining Experimental Conditions for Reversibility—To assess the impact of circular permutation on the thermodynamic stability and folding mechanism of ALAS, the Gdm-HCl-induced equilibrium unfolding/refolding transitions of ALAS circularly permuted variants, at pH 7.5 and 25 °C, were compared with those of wild type ALAS and 2X-ALAS. The latter protein corresponds to two wild type ALASs in tandem. Although the structural content and molecular mass of 2X-ALAS, as verified by CD and gel filtration chromatography, respectively, are identical to those of the wild type enzyme, the subunit molecular mass is twice that of wild type ALAS. These features confirm the monomeric state of 2X-ALAS and make it an important control in the folding studies. We rationalized that if monomerization were an unfolding stage detectable by any of the spectroscopic probes used, the comparison of the unfolding profiles for ALAS and 2X-ALAS would be instrumental in assigning this transition. The equilibrium unfolding/refolding transitions were monitored by far-UV CD at 222 nm, which probes the secondary structure content, and by intrinsic fluorescence, which measures tertiary structure formation. Unfolding was accompanied by a red shift of the fluorescence emission (from 334 to 360 nm), indicating change of tryptophan residues from the hydrophobic interior of the protein to a more polar environment. Under the defined experimental conditions, the Gdm-HCl concentration of 5.0 M was sufficient to unfold completely both wild type ALAS and circularly permuted variants (Fig. 1A illustrates the Gdm-HCl-induced unfolding of ALAS). All transitions were reversible, as illustrated for one circularly permuted variant (Q69) in Fig. 1B; however, complete recovery of enzymatic activity was not achieved (see below).



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  		FIG. 1.
The reversibility of Gdm-HCl-induced unfolding/refolding equilibrium transitions of wild type ALAS and circularly permuted variants. Unfolding (•) and refolding ({circ}) transitions of 0.1 µM wild type ALAS (A) and Q69 variant (B), at pH 7.5 and 25 °C, were followed by fluorescence ({lambda}ex = 280.6 nm; {lambda}em = 334.2 nm) Note the definition of the unfolding base line in A; concentrations equal or greater than 5.0 M led to complete unfolding of the protein (see "Experimental Procedures" for details). Gu-HCl, Gdm-HCl.

 

Experimental conditions were carefully scrutinized, given that during our initial attempts at studying ALAS unfolding, protein aggregation was observed at 1.0–2.0 M Gdm-HCl. Apparent protein aggregation was manifested as discontinuities at the probable transitions in the refolding profiles (data not shown). The aggregation might have resulted from the exposure, during unfolding, of protein hydrophobic surfaces that are interdomain contacts in the folded protein (43–45). To circumvent this problem and ensure reversibility of the Gdm-HCl-induced folding/refolding process, a non-denaturing detergent (i.e. 2% Tween 20) and PLP were required to be included in the reactions, and the protein concentration could not be higher than 0.1 µM. (The presence of a detergent can stabilize exposed hydrophobic surfaces and prevent protein aggregation during unfolding (44, 45).) Moreover, 2% Tween 20 had no effect on the enzymatic activity of ALAS or the variants (data not shown). PLP was also found to be crucial in protein stability; in fact, attempts to obtain apoALAS by dialysis of the cofactor under the designed reaction conditions led to destabilization of the protein in solution and its precipitation.

Recovery of enzymatic activity was also used to monitor the extent of refolding, because ALAS activity reflected the integrity of the active site. Whereas ALAS and all of the variants exhibited reversibility in their unfolding/refolding profiles as monitored by intrinsic protein fluorescence, the degree of catalytic activity recovery upon refolding varied among the different proteins. For example, the recovery of active enzyme was 48 and 30% for ALAS and 2X-ALAS, respectively, whereas the catalytic activity recoveries reached 68 and 86% for the L25 and N404 variants, respectively.

Gdm-HCl-dependent Unfolding Transitions of Wild Type ALAS and Circularly Permuted Variants as Monitored by Intrinsic Tryptophan Fluorescence and CD—The equilibrium Gdm-HCl-induced unfolding transitions of wild type ALAS and its circularly permuted variants were monitored by intrinsic fluorescence and far-UV CD (Fig. 2). For all the proteins, the unfolding profiles were biphasic (Fig. 2A), indicating the presence of at least one stable folding intermediate. No additional unfolding intermediates were apparent, because the normalized transitions were completely independent of the spectroscopic probe used (i.e. fluorescence and CD at 222 nm). Fig. 2B illustrates the similarity between the unfolding profiles of wild type ALAS as followed by two independent spectroscopic probes. It should be stressed that although more experimental points over 5.0 M Gdm-HCl were collected for wild type ALAS (i.e. 3) than the circularly permuted variants, the reversibility of the unfolding/refolding transitions was verified for all proteins, and no changes in intrinsic tryptophan fluorescence or CD were observed at Gdm-HCl concentrations equal to or higher than 5.0 M. Thus, we believe the data fitting and their interpretation have not been compromised.



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  		FIG. 2.
Gdm-HCl-induced unfolding of wild type ALAS and its circularly permuted variants. A, unfolding profiles of 0.1 µM wild type ALAS (•), L25 ({triangleup}), and N404 (+) variants monitored by following intrinsic tryptophan fluorescence. B, unfolding of 0.1 µM wild type ALAS monitored by intrinsic fluorescence (•) and far-UV CD ({circ}) spectroscopies; C, protein concentration independence of ALAS unfolding in the 0.01–0.1 µM range: 0.01 (•), 0.05 ({circ}), and 0.1 µM ({blacktriangledown}). Each data point represents the average of 3 independent experiments, and the error bars are plotted over and partially obscured by the data points. The experimental data were fitted to a three-state model for a unimolecular unfolding process as described under "Experimental Procedures." Gu-HCl, Gdm-HCl.

 

Despite the common biphasic nature of the unfolding profiles of ALAS and circularly permuted variants, there are significant differences among them. Specifically, the transition to which we assigned the formation of a stable folding intermediate occurred at 2.0 M Gdm-HCl for the wild type ALAS, whereas denaturant concentrations of 0.9, 0.8, 2.5, and 3.0 M were necessary to reach equivalent transitions in the unfolding of Q69, N408, L25 and N404 variants, respectively (Fig. 2A). Curiously, a similar transition was observed in the unfolding of the monomeric 2X-ALAS (i.e. at 2.0 M Gdm-HCl), suggesting that the detected folding intermediate did not correspond to dissociated monomers but instead to an intermediate whose apparent structural content is common to both ALAS and 2X-ALAS. Of relevance, the wild type ALAS folding intermediate, obtained in the presence of 2.0 M Gdm-HCl, retained about 30% of the native state structural content, as revealed by far-UV CD spectroscopy (data not shown).

The equilibrium unfolding profile of wild type ALAS was independent of protein concentration within 0.01–0.1 µM (Fig. 2C), and thus, as one would expect, the derived values from the unfolding curves for the thermodynamic parameters at different protein concentrations were identical (see below). Like ALAS, the unfolding processes of L25, Q69, and N408 variants were independent of protein concentration within the 0.01–0.1 µM range (data not shown). The superimposition of the unfolding curves for wild type ALAS, L25, Q69, and N408 variants indicate that their Gdm-HCl-induced unfolding reactions correspond to unimolecular processes. Actually, the unfolding curves are best described by a three-state model for a unimolecular process. In contrast, the unfolding of the N404 variant and 2X-ALAS varied with protein concentration in the 0.01–0.1 µM range (data not shown), and thus their data fitting to an unimolecular, three-state process was used as an approximation of their Gdm-HCl-induced folding model.

Examination of the Gdm-HCl transitions for the different proteins, applying the three-state model, yielded the thermodynamic stabilities of the native proteins and their folding intermediates (Table I). The free energy of the N {leftrightarrow} I transition () for all of the circularly permuted variants was similar to that of the wild type ALAS. This transition was assigned to the disruption of the dimeric interface with formation of a stable folding intermediate. In contrast, the free energy of the I {leftrightarrow} U transition (), assigned to correspond to the conversion of the folding intermediate into a completely unfolded protein, differed significantly among the circularly permuted variants (Table I). The similarity of the free energies for the first transition among the different proteins suggests that the dimeric interface of the circularly permuted variants is similar to that of wild type ALAS. This seems reasonable as the ALAS active site is located at the subunit interface (11), and thus functional circularly permuted variants might not tolerate drastic variabilities at the subunit interface. The different ( energies among ALAS and permuted variants indicate that the circular permutation of ALAS affected mainly the folding of the core of the individual subunits.


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  		TABLE I
The thermodynamic stability parameters of wild type ALAS, 2X-ALAS, and circularly permuted variants for the Gdm-HCl-induced unfolding

The thermodynamic stability parameters were determined from fitting the unfolding profile data to a three-state model for a unimolecular process as described under "Experimental Procedures." Parameter errors are expressed as standard deviation of their values obtained from independent fitting of three data sets.

 

With the exception of the Q69 variant, values for all of the proteins were smaller than the values of , suggesting that the folding intermediate is energetically closer to the native state than to the fully unfolded state (Table I). Although the overall stabilities of the Q69 and the N408 variants were lower than wild type ALAS, the L25 variant overall stability was substantially higher (Table I). Finally, the N404 variant and 2X-ALAS native states were significantly stabilized by increased protein concentration (data not shown). Taken together, these results indicate that the stability of native ALAS and functional variants appears to be attributed primarily to the stabilization of the dimeric interface (Table I).

Gdm-HCl-dependent Unfolding Transitions of Wild Type ALAS and Circularly Permuted Variants as Monitored by Enzyme Activity and PLP Cofactor Fluorescence—Given that ALAS is a PLP-dependent homodimeric protein, the possibility that certain transitions (e.g. dimer dissociation) might not be detectable using the conventional spectroscopic probes (i.e. intrinsic tryptophan fluorescence and CD) could not be ruled out. Therefore, we also followed the unfolding of wild type ALAS and variants by determining the enzyme activity and protein-bound PLP fluorescence at different denaturant concentrations. Enzyme activity is a sensitive probe to examine induced enzyme conformational changes, as it makes possible the detection of small protein structural changes that are translated in alterations, even if slight, of the active site. The fluorescent PLP cofactor was used as an alternate probe, given that changes of protein-bound PLP fluorescence reflect perturbations introduced in the PLP environment during unfolding, which probably correspond to structural changes at the dimeric interface.

Although exhibiting biphasicity, the unfolding profiles of ALAS and variants monitored by PLP fluorescence were distinct from those obtained when tryptophan fluorescence and far-UV CD were used as spectroscopic probes (Fig. 3, A–D). For the wild type ALAS and N404 variant, the first, and more accentuated, unfolding transition occurred at a significantly lower Gdm-HCl concentration than when the unfolding processes were monitored by far-UV CD or tryptophan fluorescence spectroscopies (Fig. 3, A and D), although the transition was barely detectable in the unfolding of the L25 variant (Fig. 3B). Strikingly, the loss of enzymatic activity of unfolded, wild type ALAS and N404 variant at different Gdm-HCl concentrations correlated well with the folding processes as monitored by PLP fluorescence (Fig. 4, A and D). For ALAS and N404, the unfolding transition, as monitored by the protein-bound PLP fluorescence, occurred at a lower denaturant concentration than when unfolding was followed by intrinsic fluorescence or far-UV CD (Fig. 3, A and D). This suggests that the transition detected by the protein-bound PLP, and coincident with the abolishment of enzymatic activity, corresponds to the disruption of the integrity of the active site and possibly the dissociation of the dimer into inactive monomers (Fig. 4, A and D). The determination of the oligomeric state of the species present at Gdm-HCl concentrations capable of promoting these unfolding transitions corroborated this assignment of monomerization (see below). Finally, the dissociation of the ALAS dimer into subunits caused insignificant unfolding of the individual subunits (see below).



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  		FIG. 3.
Gdm-HCl-induced unfolding of wild type ALAS and its circularly permuted variants as monitored by PLP-bound fluorescence (•) and intrinsic fluorescence ({circ}) spectroscopies. A, wild type ALAS (0.1 µM); B, L25 (0.1 µM); C, Q69 (0.1 µM); D, N404 (0.1 µM). Each data point represents the average of 3 independent experiments, and the error bars are plotted over and partially obscured by the data points. The experimental data were fitted to a three-state model for a unimolecular unfolding process as described under "Experimental Procedures." Gu-HCl, Gdm-HCl.

 


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  		FIG. 4.
Determination by activity, intrinsic tryptophan fluorescence, and PLP cofactor fluorescence of the unfolding equilibrium transitions of ALAS and circularly permuted variants. A, wild type ALAS (0.1 µM); B, L25 (0.1 µM); C, Q69 (0.1 µM); D, N404 (0.1 µM). Each data point represents the average of 3 independent experiments, and the error bars are plotted over and partially obscured by the data points. The enzyme activity expressed as fraction unfolded was determined relative to the native enzyme under the same conditions. The enzyme activity data were fitted to a two-state state model for a unimolecular unfolding process, whereas the intrinsic tryptophan and PLP cofactor fluorescence data were fitted to a three-state model. The symbols represent the following: •, activity; {circ}, intrinsic tryptophan fluorescence; and {blacktriangleup}, PLP cofactor fluorescence. Gu-HCl, Gdm-HCl.

 

It is worth noting that for all of the studied proteins over 90% loss of enzymatic activity occurred at about 0.4 M Gdm-HCl (Fig. 4, A–D); however, at this denaturant concentration no sharp transitions were observed in unfolding processes of L25 and Q69 when monitored using protein-bound PLP fluorescence (Fig. 3, B and C). These two observations indicate that although 0.4 M Gdm-HCl was sufficient to perturb the dimeric interface of the L25 and Q69 variants, probably by disrupting specific inter-subunit interactions necessary for ALAS catalysis, this denaturant concentration was insufficient to promote their monomerization. Collectively, these results suggest the following: 1) dimeric interface stability required for enzymatic activity is similar among wild type ALAS and circularly permuted variants, because a similar denaturant concentration causes total inactivation of the different proteins; 2) abolishment of ALAS activity can occur prior to complete monomerization, as verified with the L25 and Q69 variants; and 3) the dissociation of the ALAS and variant dimers is not accompanied by substantial unfolding of the subunits, as monomers retain a considerable degree of their secondary and tertiary structures.

Oligomeric State of Species Detected at the Unfolding Transitions of Wild Type ALAS and Circularly Permuted Variants—To verify whether the first event in the unfolding process of the wild type ALAS and circularly permuted variants corresponds to dimer dissociation, the oligomeric state of species generated during Gdm-HCl-induced unfolding of ALAS and variants was determined in relation to those of 2X-ALAS treated under the same experimental conditions. Under non-denaturing conditions, ALAS and 2X-ALAS exhibit similar elution profiles in gel filtration chromatography, as expected for the dimeric ALAS with a subunit mass of 56 kDa and the monomeric 2X-ALAS with a mass of 112 kDa (Fig. 5A). However, in the presence of 0.6, 2.0, or 5.0 M Gdm-HCl, the elution profiles for ALAS and 2X-ALAS differed, with the ALAS-derived species having a delayed peak, and thus an estimated smaller size than that of the 2X-ALAS-derived unfolded species (Fig. 5B); this confirms the occurrence of the ALAS dimer dissociation at the above denaturant concentrations. N404 and N408 variants and their derived unfolded species displayed similar retention times to those of ALAS treated under similar conditions (data not shown), suggesting that, for ALAS, N404 and N408 variants, monomerization of the dimer is an early event in the unfolding of both ALAS and circularly permuted variants. In contrast, 0.6 M Gdm-HCl did not cause complete dissociation of the L25 and Q69 dimers, as revealed by the elution profiles of L25 and Q69 variants, viz. those of their derived unfolded species, which exhibited a mixed population of dimer and monomers (data not shown). This finding is consistent with the fluorescence results described above. Finally, far-UV CD indicated that the dissociated subunits retained more than 90% of the structural content of the native protein (Fig. 5B). The fact that the dissociation of the ALAS subunits during Gdm-HCl-induced unfolding does not involve dramatic changes in their conformation is consistent with the non-detection of this event by intrinsic tryptophan fluorescence and far-UV CD spectroscopies.



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  		FIG. 5.
A, determination of the oligomeric state of unfolding intermediates of ALAS. ALAS (•;11.2 µg) and 2X-ALAS ({circ};11.2 µg) (either native or unfolded in either 0.6 or 2.2 M Gdm-HCl) were independently applied to a Superdex 200 filtration column and eluted with the corresponding Gdm-HCl concentration-containing buffer. a.u., arbitrary units. B, far-UV CD spectra of native wild type ALAS (–) and its folding intermediate at 0.6 M Gdm-HCl (- -). For both cases, the protein concentration was 0.1 µM. GuHCl, Gdm-HCl.

 

Fluorescence Quenching Studies of Protein-bound PLP—To investigate the effects of circular permutation on the local environment of the active site and accessibility of the PLP cofactor in ALAS and circularly permuted proteins, the fluorescence quenching properties of the protein-bound PLP were tested using three differently charged quenchers: Cs+, acrylamide, and I. For all proteins, the quenching of the protein-bound PLP varied linearly with increasing concentrations of I– (Fig. 6A). The KSV values for the N404 and N408 variants are significantly lower than those for the wild type ALAS and L25 and Q69 variants, indicating that the PLP cofactor is not as easily accessible to the iodide quencher in the N404 and N408 variants as it is in the wild type ALAS and other circularly permuted variants (Fig. 6A and Table II). With exception of N404 and N408 variants, acrylamide produced no significant quenching of the PLP cofactor (Table II). The upward-curving behavior in the Stern-Volmer plot for the PLP fluorescence quenching data of the N404 variant (Fig. 6B) suggests an exposed active site, to which the bulky acrylamide quencher can freely travel. In fact, an upward-curving behavior is typically observed for efficient quenchers (46). Up to a concentration of 2.0 M, Cs+ quenching showed little effect in the fluorescence of ALAS- and variant-bound PLP (data not shown), suggesting that a positively charged environment for the PLP cofactor prevents access of Cs+ to the fluorophore.



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  		FIG. 6.
A, KI-induced quenching of the PLP cofactor fluorescence of ALAS and circularly permuted variants. Wild type ALAS (•), 2X-ALAS ({circ}), L25 ({triangleup}), Q69 ({blacksquare}), N404 (+) and ({diamondsuit}) variants. B, acrylamide-induced quenching of the fluorescence of the PLP cofactor of the N404 variant. Experimental data were fitted to the Stern-Volmer equation (i.e. Equation 6 for A and Equation 7 for B) as described under "Experimental Procedures," where F0 corresponds to the fluorescence in the absence of KI (or acrylamide) and F is the measured fluorescence ({lambda}ex = 434 nm and {lambda}em = 515 nm).

 

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  		TABLE II
Parameters for the quenching of the fluorescence of the PLP cofactor in ALAS and circularly permuted variants by iodine and acrylamide

 

Analysis of the Active Site Structure of ALAS and Circularly Permuted Variants—To assess the effect of circular permutation of the ALAS polypeptide chain on the molecular surface of the dimer and architecture of the active site, secondary and tertiary structural models of ALAS and variant holoenzymes were examined vis á vis the results of the protein folding and PLP cofactor quenching experiments. As in our previous study (19), the present, and more refined, structural models were obtained using AONS as the homology template. Two major factors prompted us to use AONS as a structural model for ALAS. First, the 30.2% sequence identity between AONS and ALAS, which made it possible to obtain information on the protein backbone. Second, ALAS and AONS possess chemically similar substrates (i.e. an amino acid and a carboxylic acid CoA thioester) and similar proposed reaction mechanisms. The energy-optimized model of the wild type ALAS monomer revealed two major domains, the larger N-terminal domain (residues 1–390) and a C-terminal domain (residues 404–509) connected by a loop, which is probably unstructured (Fig. 7A). The N-terminal domain covers the PLP-binding core, which main structural feature is a seven-stranded {beta}-sheet with all, but one, strands parallel (Fig. 7B). Indeed, in all active, circularly permuted variants, this {beta}-sheet core remained as an intact unit, whereas the arrangement of the surrounding {alpha}-helices varied. Significantly, the two major N- and C-terminal domains match the two ALAS functional elements previously identified as the catalytic and the glycine-binding domains (19). Thus the seven-stranded {beta}-sheet and the overall two-domain organization were maintained in ALAS, L25, Q69, N404, and N408 variants (data not shown). However, these circular permutations, which at the primary structure level simply involved the transfer of the first 24, 68, 403, and 407 N-terminal residues to the wild type ALAS C terminus, brought changes in the domains, which mainly reflect different charge distributions at the molecular surface of the dimers (Fig. 7, C–F). In fact, cofactor-docking studies led us to predict that in wild type ALAS, PLP is cradled in a more positively charged pocket than in the case of L25 (Fig. 7, C and D). In the L25 variant, the solvent-accessible surface area was calculated to be larger than in the wild type protein, and it is plausible that the PLP cofactor was in a more relaxed conformation with the active site cavity wide open and readily accessible to solvent (Fig. 7D). In contrast, an inverted surface charge distribution was apparent in Q69 and N408 variants (Fig. 7, E and F), with the cofactor poorly shielded from the solvent in the N408 variant. The active site of the Q69 variant was defined less precisely, with the C-terminal domain more remotely located, resembling a groove where the PLP is bound (Fig. 7E).



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  		FIG. 7.
A, three-dimensional structure model of wild type ALAS. The model for the ALAS monomer was constructed using 7-amino-8-oxononanoate synthase (Protein Data Bank code 1BS0 [PDB] ) as the homology template. The approximate positions of the new termini produced by the circular permutations are indicated in red. B, protein topology schematic of the predicted three-dimensional structure of ALAS. The pointing triangles represent the {beta}-strands "out of" or "into" the plane of the diagram, and the circles represent {alpha}-helices. Images in A and B were produced with Molscript (61) and Tops (41, 42), respectively. C–F, molecular surface representation of the electrostatic potential of ALAS and circularly permuted variants (monomer). C, ALAS; D, L25; E, Q69; and F. N408. The PLP cofactor is depicted in ball-and-stick representation. Blue and red colors represent positively and negatively charged surfaces, respectively. Images were generated with Swiss PDB Viewer (39).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previously we demonstrated that active and circularly permuted ALAS variants could attain, and preserve, a wild type-like ALAS tertiary structure, secure a dimeric state, and bind the PLP cofactor, all despite the different arrangement of the secondary structure elements of their polypeptide chains (19). In order to elucidate whether alternative or multiple folding pathways govern the various permuted ALAS sequences toward an overall three-dimensional structure compatible with ALAS function, the Gdm-HCl-induced unfolding and conformational stability of active, circularly permuted variants were examined in relation to those of wild type ALAS. The ability of circularly permuted polypeptide chains to fold into wild type-like conformations has been interpreted to be due to 1) a small free energy gap between a stable, native form of the protein and alternative partially folded states (47–49) and/or 2) alternative folding mechanisms (50–53). The characterization of the Gdm-HCl-induced unfolding of ALAS and active, circularly permuted ALAS variants has permitted us to propose that, in addition to the low stability of ALAS (23.3 kJ mol–1), permuted variants are capable of folding into functional, native-like conformations as a result of alternative folding mechanisms.

The results presented in this study suggest the following model for the equilibrium Gdm-HCl-induced unfolding mechanism of ALAS and circularly permuted variants (i.e. L25, Q69, and N408) (Fig. 8). The dissociation of the dimer precedes the formation of a stable intermediate. We speculate that the loss of interactions between the domains forming the dimeric interface is the first event in the unfolding process. With the exception of the Q69 variant, this disruption of the dimeric interface affects the integrity of the active site and is accompanied with abolishment of enzyme activity. Complete unfolding of the stable intermediate, which retains substantial secondary structural content relative to its native counterpart, is the "energytaxing" step and reflects the major differences associated with the unfolding of ALAS and permuted variants.



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  		FIG. 8.
Proposed equilibrium folding/unfolding mechanism for wild type ALAS and circularly permuted variants. The first step of unfolding is the dimer subunit dissociation with minimal conformational change of the protomers. The conversion of the stable folding intermediate into a completely denatured protein is the highest energy-requiring step in the overall folding mechanism.

 

At low concentrations (i.e. lower than 0.5 M) of Gdm-HCl, ALAS and circularly permuted variants unfolded to inactive monomers (ALAS, N404, and N408) or inactive, disrupted dimeric intermediates (L25 and Q69) with minimal conformational changes. These conformational changes could not be detected by either far-UV CD or intrinsic fluorescence but could be detected by PLP cofactor fluorescence (Fig. 4). Significantly, size exclusion chromatography validated that the ALAS- and N404- to N408-derived species were monomeric at the unfolding transition (Fig. 5). As protein interfaces tend to contain more polar interactions than the interior of proteins (54), the facile Gdm-HCl-induced ALAS dissociation suggests that electrostatic interactions play a major role in maintaining ALAS as a stable dimer. Subunit interactions have been shown to be important for the folding stability of dimeric proteins (55, 56) with, for instance, dimeric triose-phosphate isomerase being stabilized predominantly by the association of monomers (55).

Although ionic interactions appear to be essential to stabilize the ALAS dimer, they do not appear to be the major contributing factor to overall protein stability. The loss of enzymatic activity of ALAS and circularly permuted variants for the same range of Gdm-HCl concentration (0.3–0.6 M) suggested comparable stabilities for the different dimers (Fig. 4). In fact, the similarity among the free energy values for the N {leftrightarrow} I transition () (Table I) corroborates this observation. Because PLP was present in our equilibrium unfolding studies, it is plausible that PLP could have stabilized the ALAS native structure and thus played a crucial role in restoring native ALAS-like structures for both unfolded ALAS and circularly permuted variants. Certainly, the apo forms of ALAS and permuted variants were unstable and precipitated under our experimental conditions. The idea that determinants of protein structure and stability arise as a response to (natural) ligands has been previously advanced (52, 57); indeed, the PLP cofactor contributes to the structural stabilization of several PLP-dependent enzymes, although its direct relevance to their folding mechanism varies (58–60). Accordingly, we postulate that interdomain interactions stabilize the dimeric interface, where the PLP cofactor is cradled and the active site resides. Furthermore, although there are differences among the PLP environments of wild type ALAS and active, circularly permutated variants (Figs. 6 and 7 and Table II) (19), their dimeric interfaces are similar. This view is consistent with the fact that by having the ALAS active site shared between the two subunits, functional, circularly permuted ALAS variants would require the correct positioning of the catalytic residues at the dimeric interface to maintain the "identity" of ALAS function.

The subsequent event (I {leftrightarrow} U), as monitored using either intrinsic fluorescence or far-UV CD, corresponded to the unfolding of the stable intermediate (I) into a completely denatured protein (U) (Fig. 8). Whereas the stable folding intermediate retained a significant secondary structural content of the native proteins (~30%), the calculated free energy values () associated with the I {leftrightarrow} U transition diverged among ALAS and circularly permuted variants (Table I). Because ALAS and circularly permuted variants have in common a seven-stranded {beta}-sheet core, which is also present in all fold type I PLP-dependent enzymes, but have different arrangements of its surrounding {alpha}-helices (19), it is tempting to speculate that the remaining secondary structural elements present in the unfolding stable intermediates of ALAS and variants varied, and thus different energies were required to disrupt their interactions.

In contrast to the equilibrium unfolding-refolding profiles of ALAS and L25, Q69, and N408 variants, which were independent of protein concentration and could be treated as simple, unimolecular reactions using a three-state model, the unfolding profile of N404 variant was protein concentration-dependent (Figs. 2 and 3). The protein concentration dependence for unfolding of the N404 variant indicates that unfolding is not unimolecular, and the N2 {leftrightarrow} 2U and 2I {leftrightarrow} 2U transitions are slow and fast, respectively. Thus, only the N2 and U protein species (and not the I species) are significantly populated. This observation leads to two alternative explanations. First, the N404 subunits unfold immediately after dissociation of the dimer. Second, the N404 circular permutation affects the unfolding kinetics with probable formation of additional kinetic folding intermediates. The first explanation does not seem plausible, because both intrinsic fluorescence and far-UV CD did not detect significant conformational changes in the N404 subunits. The second explanation, however, cannot be ruled out. Therefore, the non-unimolecular nature of the equilibrium unfolding of the N404 variant indicates an alternative folding mechanism. Remarkably, the N404 variant has the two previously identified functional elements (glycine binding and catalytic domains) swapped at the primary structure level (19). Together, these results suggest that the two functional elements do not fold independently, because their relocation within the polypeptide chain resulted in alteration of subunit stability and, most likely, kinetic folding mechanism.

In conclusion, the order of the secondary structure elements does not appear to be crucial in determining the folded ALAS structure. Despite the similar dimeric interface and energies required to stabilize the wild type and permuted ALAS variant dimers, the energy requirements for the folding of the individual subunits vary. Finally, the two functional elements, although predicted to be well defined structural domains, are not independent folding units of the ALAS polypeptide chain.


   FOOTNOTES
 
* This work was supported in part by the National Institutes of Health and the Chiles Endowment Biomedical Research Program of the Florida Department of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of an American Heart Association/Florida Division predoctoral fellowship. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir{at}hsc.usf.edu.

1 The abbreviations used are: ALA, 5-aminolevulinate; ALAS, 5-aminolevulinate synthase; AONS, 8-amino-7-oxononanoate synthase; Gdm-HCl, guanidinium chloride; PLP, pyridoxal 5'-phosphate. Back



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