Circular Permutation of 5-Aminolevulinate Synthase
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m207011200
ISSN1083-351X
AutoresAnton Cheltsov, Wayne C. Guida, Glória C. Ferreira,
Tópico(s)Protein Structure and Dynamics
ResumoThe 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. 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. In mammals, heme biosynthesis is initiated with the condensation of glycine with succinyl-CoA to form 5-aminolevulinic acid (ALA), 1The abbreviations used are: ALA, 5-aminolevulinate; ALAS, 5-aminolevulinate synthase; AONS, 8-amino-7-oxononanoate synthase; Gdm-HCl, guanidinium chloride; PLP, pyridoxal 5′-phosphate. CoA, and CO2 (1Ferreira G.C. Gong J. J. Bioenerg. Biomembr. 1995; 27: 151-159Crossref PubMed Scopus (81) Google Scholar, 2Ferreira G.C. Ferreira G.C. Moura J.J.G. Franco R. Inorganic Biochemistry and Regulatory Mechanisms of Iron Metabolism. Wiley-VCH, Weinheim, Germany1999: 15-34Google Scholar). 5-Aminolevulinate synthase (ALAS) (EC 2.3.1.37), 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 (3Bishop D.F. Henderson A.S. Astrin K.H. Genomics. 1990; 7: 207-214Crossref PubMed Scopus (126) Google Scholar) and the X chromosome (4Cotter P.D. Willard H.F. Gorski J.L. Bishop D.F. Genomics. 1992; 13: 211-212Crossref PubMed Scopus (58) Google Scholar) for the housekeeping and erythroid-specific enzymes, respectively. Erythroid, ALAS-produced heme accounts for 90% of the total heme synthesized in humans (5Sadlon T.J. Dell'Oso T. Surinya K.H. May B.K. Int. J. Biochem. Cell Biol. 1999; 31: 1153-1167Crossref PubMed Scopus (71) Google Scholar, 6Nakajima O. Takahashi S. Harigae H. Furuyama K. Hayashi N. Sassa S. Yamamoto M. EMBO J. 1999; 18: 6282-6289Crossref PubMed Scopus (99) Google Scholar), and mutations in human erythroid ALAS lead to X-linked sideroblastic anemia (7Bottomley S.S. Lee G.R. Wintrobe's Clinical Haematology. J. B. Lippincott/Williams & Wilkins, Baltimore, MD1999: 1022-1045Google Scholar, 8Bottomley S.S. May B.K. Cox T.C. Cotter P.D. Bishop D.F. J. Bioenerg. Biomembr. 1995; 27: 161-168Crossref PubMed Scopus (66) Google Scholar, 9Cotter P.D. May A. Li L. Al-Sabah A.I. Fitzsimons E.J. Cazzola M. Bishop D.F. Blood. 1999; 93: 1757-1769Crossref PubMed Google Scholar, 10Harigae H. Furuyama K. Kimura A. Neriishi K. Tahara N. Kondo M. Hayashi N. Yamamoto M. Sassa S. Sasaki T. Br. J. Haematol. 1999; 106: 175-177Crossref PubMed Scopus (25) Google Scholar), which is distinguished by inadequate heme synthesis and an overaccumulation of iron in erythroblast mitochondria (7Bottomley S.S. Lee G.R. Wintrobe's Clinical Haematology. J. B. Lippincott/Williams & Wilkins, Baltimore, MD1999: 1022-1045Google Scholar). ALAS is a pyridoxal 5′-phosphate (PLP)-dependent homodimer with 56-kDa subunits and the active site located at the dimer interface (11Tan D. Ferreira G.C. Biochemistry. 1996; 35: 8934-8941Crossref PubMed Scopus (31) Google Scholar). 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 (12Fanica-Gaignier M. Clement-Metral J. Eur. J. Biochem. 1973; 40: 19-24Crossref PubMed Scopus (22) Google Scholar). Along the catalytic pathway the PLP forms several intermediates (13Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar). In the absence of substrates or products, PLP is covalently bound via a Schiff base to the ϵ-amino group of Lys-313 of murine erythroid ALAS, forming an internal aldimine (14Ferreira G.C. Neame P.J. Dailey H.A. Protein Sci. 1993; 2: 1959-1965Crossref PubMed Scopus (64) Google Scholar). 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 (13Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar, 15Ferreira G.C. Vajapey U. Hafez O. Hunter G.A. Barber M.J. Protein Sci. 1995; 4: 1001-1006PubMed Google Scholar, 16Tan D. Barber M.J. Ferreira G.C. Protein Sci. 1998; 7: 1208-1213Crossref PubMed Scopus (19) Google Scholar, 17Gong J. Hunter G.A. Ferreira G.C. Biochemistry. 1998; 37: 3509-3517Crossref PubMed Scopus (39) Google Scholar, 18Tan D. Harrison T. Hunter G.A. Ferreira G.C. Biochemistry. 1998; 37: 1478-1484Crossref PubMed Scopus (24) Google Scholar). 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 (14Ferreira G.C. Neame P.J. Dailey H.A. Protein Sci. 1993; 2: 1959-1965Crossref PubMed Scopus (64) Google Scholar) and catalysis (13Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar), and Arg-439 is crucial in the binding of glycine substrate (18Tan D. Harrison T. Hunter G.A. Ferreira G.C. Biochemistry. 1998; 37: 1478-1484Crossref PubMed Scopus (24) Google Scholar). 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). 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. Reagents—The following reagents were from Sigma: DEAE-Sephacel, β-mercaptoethanol, Gdm-HCl, p-dimethylaminobenzaldehyde, acetylacetone, Tween 20, PLP, bovine serum albumin, α-ketoglutarate dehydrogenase, α-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 β-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 (20Huala E. Moon A.L. Ausubel F.M. J. Bacteriol. 1991; 173: 382-390Crossref PubMed Google Scholar) 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 21Ferreira G.C. Dailey H.A. J. Biol. Chem. 1993; 268: 584-590Abstract Full Text PDF PubMed Google Scholar). 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 (19Cheltsov A.V. Barber M.J. Ferreira G.C. J. Biol. Chem. 2001; 276: 19141-19149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 21Ferreira G.C. Dailey H.A. J. Biol. Chem. 1993; 268: 584-590Abstract Full Text PDF PubMed Google Scholar). 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 (21Ferreira G.C. Dailey H.A. J. Biol. Chem. 1993; 268: 584-590Abstract Full Text PDF PubMed Google Scholar), and the supernatant was brought to 25% ammonium sulfate. After stirring for 10 min at 4 °C, the solution was centrifuged at 27,000 × 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 (21Ferreira G.C. Dailey H.A. J. Biol. Chem. 1993; 268: 584-590Abstract Full Text PDF PubMed Google Scholar) with the following modifications: the DEAE-Sephacel resin was washed with buffer A until A 280 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 × 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 (22Hunter G.A. Ferreira G.C. Anal. Biochem. 1995; 226: 221-224Crossref PubMed Scopus (54) Google Scholar). 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 (θobs) 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 (23Lien L.F. Beattie D.S. Enzymes. 1982; 28: 120-132Crossref PubMed Google Scholar) 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 (24Mauzerall D. Granick S. J. Biol. Chem. 1956; 219: 435-444Abstract Full Text PDF PubMed Google Scholar) 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, F.U.=xi-xnativexunfolded-xnative(Eq. 1) where x i is the system parameter being monitored during the unfolding reaction and x native and x unfolded 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 ↔ I ↔ U where N, I, and U are native, intermediate, and unfolded states, respectively (25Ghelis C. Yon J. Protein Folding. Molecular Biology. Academic Press, New York1982: 223-295Google Scholar, 26Fan Y.X. Zhou J.M. Kihara H. Tsou C.L. Protein Sci. 1998; 7: 2631-2641Crossref PubMed Scopus (72) Google Scholar). As shown in Equation 2, if y represents the experimental variable being used to follow the transition, and y N, y I, and y U are the values of y for N, I, and U, respectively (27Tanford C. Adv. Protein Chem. 1968; 23: 121-282Crossref PubMed Scopus (2437) Google Scholar), then y=yN+fI(yI-yN)+fU(yU-yN)(Eq. 2) where f I and f U are fractions of I and U species, respectively, and can be expressed through equilibrium constants as shown in Equations 3 and 4, fI=KNI1+KNI+KNIKIU(Eq. 3) fU=KNIKIU1+KNI+KNIKIU(Eq. 4) When F.U. is used as the experimental variable, then y N and y U 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 (28Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2424) Google Scholar) as shown in Equations 5 and 6, KNI=expmNI[D]-ΔGNI0RT(Eq. 5) KIU=expmIU[D]-ΔGIU0RT(Eq. 6) By substituting Equations 6, 5, 4, and 3 into Equation 2, an equation relating y to denaturant concentration can be obtained. ΔGNI0 , ΔGIU0 , m NI, m IU, and y I 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 (29Eftink M.R. Ghiron C.A. Biochemistry. 1976; 15: 672-680Crossref PubMed Scopus (991) Google Scholar), where K SV 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.). F0F=1+KSV[Q](Eq. 7) F0F=(1+KSV[Q])exp(V[Q])(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) (30Alexeev D. Alexeeva M. Baxter R.L. Campopiano D.J. Webster S.P. Sawyer L. J. Mol. Biol. 1998; 284: 401-419Crossref PubMed Scopus (109) Google Scholar, 31Webster S.P. Alexeev D. Campopiano D.J. Watt R.M. Alexeeva M. Sawyer L. Baxter R.L. Biochemistry. 2000; 39: 516-528Crossref PubMed Scopus (110) Google Scholar) (Protein Data Bank accession code 1BS0) as template. AONS and ALAS share 30.2% sequence identity and 31.3% sequence similarity. According to Chothia and Lesk (32Chothia C. Lesk A.M. EMBO J. 1986; 5: 823-826Crossref PubMed Scopus (1988) Google Scholar), 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 (33Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55986) Google Scholar). 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 (34Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10632) Google Scholar). The obtained protein models were further optimized using MacroModel 7.2 (35Mohamadi F. Richards N. Guida W.C. Liskamp R. Lipton M. Caulfield C. Chang G. Hendrickson T. Still W.C. J. Comput. Chem. 1990; 11: 440-456Crossref Scopus (3933) Google Scholar) purchased from Schrödinger, Inc. (www.schrodinger.com). The models were also validated using PRO-CHECK (36Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The AMBER* (37Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11637) Google Scholar) 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 α-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 (38Kolossvary I. Guida W.C. J. Comput. Chem. 1999; 20: 1671-1684Crossref Google Scholar). 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 (39Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9638) Google Scholar). The secondary structures of the ALAS and circularly permuted variants were predicted using the DSSP program (40Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12413) Google Scholar). Protein topology schematics (TOPS) of the predicted three-dimensional structures were generated using the TOPS program (41Westhead D.R. Hatton D.C. Thornton J.M. Trends Biochem. Sci. 1998; 23: 35-36Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 42Westhead D.R. Slidel T.W. Flores T.P. Thornton J.M. Protein Sci. 1999; 8: 897-904Crossref PubMed Scopus (106) Google Scholar). 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 ex
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