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• W2054328275 abstract "The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the β-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal α-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal βαβ flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane. The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the β-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal α-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal βαβ flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane. Protein folding/unfolding is a highly cooperative process. It has been shown that the folding/unfolding of small globular proteins occurs via a two-state process, whereas the folding/unfolding of larger proteins (>100 amino acids) is complex and often involves the formation of intermediate(s) (1Dobson C.M. Karplus M. Curr. Opin. Struct. Biol. 1999; 9: 92-101Crossref PubMed Scopus (364) Google Scholar, 2Englander S.W. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 213-238Crossref PubMed Scopus (412) Google Scholar, 3Samuel D. Kumar T.K. Balamurugan K. Lin W.Y. Chin D.H. Yu C. J. Biol. Chem. 2001; 276: 4134-4141Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The most thorough investigations of protein folding and stability have been done with unusually small proteins, which are folded into single domains and display simple two-state unfolding processes. It is of interest, however, to extend studies to larger, more complex, and therefore more typical proteins. d-Amino acid oxidase (DAAO; EC 1.4.3.3) 1The abbreviations used are: DAAO, d-amino acid oxidase (EC 1.4.3.3); Cm, concentration of urea to give half-unfolded protein; ANS, 1,8-anilinonaphtalene sulfonic acid. has attracted our attention as a suitably more complex subject because it is considered the paradigm of the dehydrogenase/oxidase class of flavoproteins (4Massey V. Hemmerich P. Biochem. Soc. Trans. 1980; 8: 246-255Crossref PubMed Scopus (282) Google Scholar) and in particular of those in which the flavin is noncovalently bound. In fact, many proteins in nature require the noncovalent binding of cofactors to perform their biological activity, and these molecules fold in a cellular environment where their cognate cofactors are present. However, the manner in which cofactors affect the folding pathway remains poorly understood, because kinetic folding studies are frequently conducted in the absence of potentially complicating ligands. Furthermore, flavoproteins are often multisubunit proteins constituted either by identical or by different polypeptide chains. Up to now, deep and complete investigations have been restricted to small flavoproteins, such as flavodoxin (5van Mierlo C.P. van Dongen W.M. Vergeldt F. van Berkel W.J. Steensma E. Protein Sci. 1998; 7: 2331-2344Crossref PubMed Scopus (63) Google Scholar). To have insights on the relationships between cofactor uptake, the stable interaction between identical subunits (and on its significance), folding, and intracellular trafficking, we undertook a study of the stability of structural elements in the peroxisomal flavoenzyme DAAO from the yeast Rhodotorula gracilis. Proteins destined for the peroxisome are synthesized on free ribosomes of the cytoplasm and transported into peroxisomes post-translationally. There are two types of peroxisomal targeting signals (PTS); the first one (PTS1) is the SKL C-terminal sequence (or some conservative variants of it) (6Subramani S. Annu. Rev. Cell Biol. 1993; 9: 445-478Crossref PubMed Scopus (359) Google Scholar, 7Terlecky S.R. Fransen M. Traffic. 2000; 1: 465-473Crossref PubMed Scopus (44) Google Scholar). Proteins belonging to this group are made at their mature size, do not undergo cleavage of the targeting sequence upon transport into peroxisome, and have cytosolic receptors that mediate their association with the peroxisomal import machinery (6Subramani S. Annu. Rev. Cell Biol. 1993; 9: 445-478Crossref PubMed Scopus (359) Google Scholar, 7Terlecky S.R. Fransen M. Traffic. 2000; 1: 465-473Crossref PubMed Scopus (44) Google Scholar). It has also been demonstrated that stably folded proteins are substrates for peroxisomal import (8Walton P.A. Hill P.E. Subramani S. Mol. Biol. Cell. 1995; 6: 675-683Crossref PubMed Scopus (214) Google Scholar). DAAO catalyzes the dehydrogenation of d-isomers of amino acids, to give the corresponding α-keto acids, ammonia and hydrogen peroxide. DAAOs have been the object of extensive investigation (9Curti B. Ronchi S. Pilone Simonetta M. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Inc., Boca Raton, FL1992: 69-94Google Scholar, 10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar). In solution, DAAO from R. gracilis is a stable 80-kDa homodimer, with a molecule of FAD tightly (Kd = 2 × 10-8m) but noncovalently bound to each 40-kDa subunit. The three-dimensional structure of DAAO has been resolved at very high resolution, allowing investigators to find the rationale of its high catalytic efficiency (11Umhau S. Pollegioni L. Molla G. Diederichs K. Welte W. Pilone M.S. Ghisla S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12463-12468Crossref PubMed Scopus (173) Google Scholar, 12Pollegioni L. Diederichs K. Molla G. Umhau S. Welte W. Ghisla S. Pilone M.S. J. Mol. Biol. 2002; 324: 535-546Crossref PubMed Scopus (104) Google Scholar). In the “side to tail” model of monomer-monomer interaction (with a high buried surface area, 3049 Å2) (12Pollegioni L. Diederichs K. Molla G. Umhau S. Welte W. Ghisla S. Pilone M.S. J. Mol. Biol. 2002; 324: 535-546Crossref PubMed Scopus (104) Google Scholar), a large contribution to the interaction between monomers is given by a long (21 amino acids) loop connecting β-strands F5 and F6, that is unique to yeast DAAO. Recently, by rational design and site-directed mutagenesis, a stable monomeric holoenzyme form of DAAO has been obtained by partial elimination of this loop (from Ser308 to Lys321; SPLSLGRGSARAAK) (13Piubelli L. Caldinelli L. Molla G. Pilone M.S. Pollegioni L. FEBS Lett. 2002; 526: 43-48Crossref PubMed Scopus (24) Google Scholar). The Δ loop mutant DAAO shows slightly altered spectral and kinetic properties, a lower temperature stability, and a 5-fold increase in the Kd for FAD binding compared with the wild-type enzyme. We also demonstrated the possibility of obtaining a monomeric form of yeast DAAO by treatment with 0.5 m NH4SCN, without deletion of the βF5-βF6 loop (14Pollegioni L. Iametti S. Fessas D. Caldinelli L. Piubelli L. Barbiroli A. Pilone M.S. Bonomi F. Protein Sci. 2003; 12: 1018-1029Crossref PubMed Scopus (39) Google Scholar), as well as by removal of the coenzyme to yield the corresponding apoprotein (10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar). This latter result suggests a structural relationship between the FAD-harboring domain and the regions involved in dimerization. Recently, temperature ramp experiments following different probes allowed the identification of a clear sequence of events in the course of thermal unfolding of wild-type and Δ loop protein forms (14Pollegioni L. Iametti S. Fessas D. Caldinelli L. Piubelli L. Barbiroli A. Pilone M.S. Bonomi F. Protein Sci. 2003; 12: 1018-1029Crossref PubMed Scopus (39) Google Scholar). Apparently, a first, low temperature energetic domain relates to the unfolding of tertiary structure regions, whereas a second energetic domain relates to the loss of secondary structure elements and to the release of the cofactor at higher temperatures. In this paper, we expand the results obtained until now and report a novel, reversible step in the urea-induced unfolding pathway of R. gracilis DAAO that we believe may be relevant to intracellular trafficking of DAAO. Furthermore, to provide a basis for understanding the structure-function relationships in flavoproteins and the determinants of their stability, we have attempted to compare the chemical unfolding of dimeric DAAO with that of a monomeric form obtained by site-directed mutagenesis. Materials—Recombinant wild-type and Δ loop mutant DAAO were expressed and purified from E. coli cells as described previously (13Piubelli L. Caldinelli L. Molla G. Pilone M.S. Pollegioni L. FEBS Lett. 2002; 526: 43-48Crossref PubMed Scopus (24) Google Scholar, 15Molla G. Vegezzi C. Pilone M.S. Pollegioni L. Protein Expression Purif. 1998; 14: 289-294Crossref PubMed Scopus (65) Google Scholar). Starting from a 10-liter fermentation broth, 180 and 80 mg of pure enzyme with a specific activity of 110 and 86 units/mg protein were obtained for wild-type and Δ loop DAAO, respectively. The enzyme concentration was determined by using extinction coefficients at 455 nm of 12.6 mm-1 cm-1 for wild-type and 11.3 mm1 cm-1 for Δ loop DAAOs (10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar, 13Piubelli L. Caldinelli L. Molla G. Pilone M.S. Pollegioni L. FEBS Lett. 2002; 526: 43-48Crossref PubMed Scopus (24) Google Scholar). Urea was from Pierce, and the other reagents were of analytical grade. DAAO Activity Assay—DAAO activity was assayed with an oxygen electrode at pH 8.5, air saturation, and 25 °C, using 28 mm d-alanine as substrate in the presence of 0.2 mm FAD (10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar). The effect of urea concentration on enzyme activity of DAAO was determined using the oxygen-electrode assay on protein samples previously incubated at 15 °C for 40 min in the presence of different concentrations of urea. Spectroscopy—All fluorescence measurements were performed by using a 1-ml cell in a Jasco FP-750 instrument equipped with a thermostated cell holder. Tryptophan emission spectra were taken from 300 to 400 nm using excitation wavelengths of 280 and 298 nm. Flavin emission spectra were recorded from 475 to 600 nm using an excitation wavelength of 450 nm; 10- and 20-nm bandwidths were used for excitation and emission, respectively. Steady-state fluorescence measurements were performed at 15 °C and at 0.02 mg/ml protein concentration. All spectra were corrected by subtracting the emission of the buffer. ANS binding experiments were carried out at 15 °C and at 2.5 μm (0.1 mg/ml) protein concentration. Protein samples were incubated for 40 min in buffer containing different concentrations of urea, and ANS was added to a final concentration of 0.1 mm. Fluorescence emission spectra were recorded in the 450–600-nm range using an excitation wavelength of 370 nm. CD spectra were recorded on a J-810 Jasco spectropolarimeter and analyzed by means of Jasco software. The cell path was 1 cm for measurements above 250 nm and 0.1 cm for measurements in the 190–250-nm region. Proteins were in 50 mm potassium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol, and 2 mm EDTA. Equilibrium Unfolding Experiments—The unfolding equilibrium of DAAO was determined by following the changes in flavin and protein fluorescence as detailed above. To establish the time required to reach the equilibrium, the fluorescence intensity was measured as a function of time until no further changes were observed (40 min at 15 °C). Each point of the urea denaturation curves was determined on individual samples, prepared by mixing appropriate volumes of protein, 8 m urea in buffer, and plain buffer (50 mm potassium phosphate, pH 7.5, containing 5% (v/v) glycerol, and 2 mm EDTA). Refolding of DAAO—Wild-type and Δ loop DAAOs were incubated at various fixed concentrations of urea for 40 min at 15 °C. The enzymes were refolded by the dilution method (10× dilution at 15 °C) in 50 mm potassium phosphate, pH 7.5, 5% glycerol, and 2 mm EDTA. The refolding yield was determined by monitoring protein and flavin fluorescence, and the recovery of enzymatic activity (which was measured using the values for the native and fully denatured enzymes as reference). Data Analysis—Unfolding curves were usually analyzed using a two-state mechanism. Unfolding curves for the N ↔ D transition were normalized to the apparent fraction of the unfolding form, FD, using the following equation (16Tanford C. Adv. Protein Chem. 1968; 23: 121-282Crossref PubMed Scopus (2435) Google Scholar), FD=(Y−YN)/(YU−YN) (Eq. 1) where Y is the observed variable parameter, and YN and YU are the values characteristic of the native and fully unfolded conformations, respectively. The difference in free energy between the folded and the unfolded state, ΔG, was calculated by the following equation, ΔG=−RTlnK=−RTln[FU/(1−FU)] (Eq. 2) where K is the equilibrium constant, R is the gas constant, and T is the absolute temperature. The data were analyzed assuming the free energy of unfolding or refolding, ΔG, to be linearly dependent on the urea concentration (denoted here by C), as described in detail previously (17Pace C.N. Trends Biotechnol. 1990; 8: 93-98Abstract Full Text PDF PubMed Scopus (460) Google Scholar), ΔG=ΔGw−mC=m(Cm−C) (Eq. 3) in which ΔGw and ΔG represent the free energy of unfolding or refolding in the absence and presence of urea, respectively; Cm is the midpoint concentration of urea required for unfolding or refolding; and m stands for the slope of the unfolding or refolding curve at Cm. A least-squares curve fitting analysis was used to calculate the values of ΔGw, m, and Cm by a software routine. The same equation accounts for the free energy of formation of partly unfolded folding intermediates from native molecules. The urea-unfolding curves corresponding to a two-state model were analyzed using Equation 4, derived from Ref. 18Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1607) Google Scholar, that incorporates Equations 1, 2, 3. Y=YN+YUe−(ΔGw−mC)/RT1+e−(ΔGw−mC)/RT (Eq. 4) The analysis of the equilibrium unfolding transition using the flavin fluorescence data was performed according to a three-state denaturation pathway (N ↔ I ↔ U) according to Ref. 19Ayed A. Duckworth H.W. Protein Sci. 1999; 8: 1116-1126Crossref PubMed Scopus (21) Google Scholar, F=FN−1[urea]n1Cm1n1+[urea]n1+FI−U[urea]n2Cm2n2+[urea]n2 (Eq. 5) in which Cm1 and Cm2 are the midpoint concentration of urea for the N ↔ I and I ↔ U transitions, respectively; FN-I and FI-U represent the percentage change of flavin fluorescence associated to the N ↔ I and I ↔ U transitions, respectively; and exponents n1 and n2 reflect the steepness of the transition between states as a function of urea concentration. Liquid Chromatography/Mass Spectrometry Analysis after Limited Tryptic Digestion—Following incubation of 1 mg/ml wild-type DAAO with 0.1% trypsin at 15 °C for 30 min in the absence and in the presence of 2 m urea, the reaction mixture (50 μg) was fractionated by high pressure liquid chromatography using an HP 1100 modular system (Agilent Technology, Palo Alto, CA). Samples were loaded onto a Vydac 218TP52, 5-μm reversed-phase C18, 250 × 2.1-mm column. Solvent A was water containing 0.1% trifluoroacetic acid, and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. A linear gradient from 5 to 70% solvent B was applied over 90 min, after 5 min of isocratic elution at 5% solvent B, at a constant flow rate of 0.2 ml/min. UV detection was carried out at 220 nm. Effluents were directly injected, through a 100-μm inner diameter fused silica capillary, into the electrospray source of a Platform single quadrupole mass spectrometer (Waters, Manchester, UK). The ESI mass spectra were scanned, in the positive ion mode, from 2000 to 800 units at a scan cycle of 4.9 s/scan and 0.1-s interscan delay. The source temperature was 200 °C, and the capillary and orifice voltages were 3.6 kV and 40 V, respectively. Mass scale calibration was performed using the multiple charged ions from a separate injection of horse heart myoglobin (20Iametti S. Rasmussen P. Frøkiær H. Ferranti P. Addeo F. Bonomi F. Eur. J. Biochem. 2002; 269: 1362-1372Crossref PubMed Scopus (49) Google Scholar). Mass spectra were elaborated using the software MassLynx 2.0, furnished with the spectrometer. Mass values were reported as average masses. Wild-type and Δ loop DAAOs are purified as holoenzymes showing little difference in the visible absorbance spectra (10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar, 13Piubelli L. Caldinelli L. Molla G. Pilone M.S. Pollegioni L. FEBS Lett. 2002; 526: 43-48Crossref PubMed Scopus (24) Google Scholar). On the contrary, spectral properties depending on the protein folding (flavin and protein fluorescence and UV CD spectra) profoundly distinguish the monomeric and dimeric DAAO forms. Tryptophan emission fluorescence at 345 nm (following excitation at 280 nm), a marker of protein conformation, is 2–3-fold higher for native Δ loop than for wild-type DAAO, indicating a lower relevance of quenching interactions between tryptophan side chains (in particular that of Trp243 at the monomer-monomer interface) and nearby side chains in the monomeric form of yeast DAAO with respect to the dimeric form (14Pollegioni L. Iametti S. Fessas D. Caldinelli L. Piubelli L. Barbiroli A. Pilone M.S. Bonomi F. Protein Sci. 2003; 12: 1018-1029Crossref PubMed Scopus (39) Google Scholar). The emission fluorescence of the FAD cofactor at 520 nm, following excitation at 450 nm, is higher for Δ loop than for wild-type DAAOs, pointing to a different microenvironment surrounding the FAD coenzyme in the two proteins (14Pollegioni L. Iametti S. Fessas D. Caldinelli L. Piubelli L. Barbiroli A. Pilone M.S. Bonomi F. Protein Sci. 2003; 12: 1018-1029Crossref PubMed Scopus (39) Google Scholar). On the other hand, the protein and flavin emission fluorescence determined for the urea-unfolded forms of wild-type and Δ loop DAAOs are quite similar at 8 m urea (see Table I). For both proteins, unfolding by urea is accompanied by an increase in both flavin and tryptophan fluorescence.Table IComparison of thermodynamic parameters for urea-induced unfolding of native wild-type and Δ loop DAAOs at 15 °C, monitored by measurement of protein and flavin fluorescence (excitation (exc) at 280 and 450 nm, respectively) and CD DAAO samples were at a 0.02 mg of protein/ml concentration for fluorescence measurements and at a 0.5 mg of protein/ml concentration for CD measurements.ParameterWild typeΔ loopexc 280 nmaValues similar to those reported were also obtained using the protein fluorescence values determined following the excitation at 298 nm. In parenthesis are reported the values determined following the shift of protein fluorescence emission maximum (see Fig. 1)exc 450 nmCDexc 280 nmaValues similar to those reported were also obtained using the protein fluorescence values determined following the excitation at 298 nm. In parenthesis are reported the values determined following the shift of protein fluorescence emission maximum (see Fig. 1)exc 450 nmCDF → II → UbThese values have been estimated using a three-state model according to Ref. 19NearFarF → II → UbThese values have been estimated using a three-state model according to Ref. 19NearFarΔGw (kcal/mol)3.4 (1.2)2.85.03.61.9 (1.4)1.84.12.8m2.4 (0.7)1.66.21.81.11.8 (0.7)1.45.62.10.8YN93516916.4YI137144129130YU476210472230DY383132303112Cm (M)1.4 (1.8)1.82.83.71.0 (2.0)1.31.93.0a Values similar to those reported were also obtained using the protein fluorescence values determined following the excitation at 298 nm. In parenthesis are reported the values determined following the shift of protein fluorescence emission maximum (see Fig. 1)b These values have been estimated using a three-state model according to Ref. 19Ayed A. Duckworth H.W. Protein Sci. 1999; 8: 1116-1126Crossref PubMed Scopus (21) Google Scholar Open table in a new tab Far-UV CD spectra of monomeric and dimeric DAAOs did not reveal any major difference in the features related to the secondary structure of the two proteins, whereas the near-UV CD spectra of the wild-type and Δ loop mutant DAAO were different. The differences have been ascribed to a different contribution from aromatic amino acid residues, which are responsible for most transitions in the near-UV spectral region. Since the deleted portion does not contain aromatic residues, the different spectroscopic features of the two proteins have been explained in terms of an altered mutual relationships between nearby structural elements (14Pollegioni L. Iametti S. Fessas D. Caldinelli L. Piubelli L. Barbiroli A. Pilone M.S. Bonomi F. Protein Sci. 2003; 12: 1018-1029Crossref PubMed Scopus (39) Google Scholar). Following the addition of ≥6 m urea, the far- and near-UV spectra of both wild-type and Δ loop DAAOs were superimposible. The stability of wild-type and Δ loop DAAO forms was studied at first by equilibrium unfolding measurements. In the experiments reported below, different spectroscopic signals, catalytic activity, and the associative behavior of both proteins were monitored after equilibration in the presence of increasing urea concentration. Protein Fluorescence—Tryptophan emission fluorescence can be considered a sensitive marker of protein conformation and in particular of changes in hydrophobic regions of the structure. The intrinsic fluorescence of tryptophan residues was used as probe of the unfolding of DAAO. Because there are eight tryptophans in yeast DAAO (11Umhau S. Pollegioni L. Molla G. Diederichs K. Welte W. Pilone M.S. Ghisla S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12463-12468Crossref PubMed Scopus (173) Google Scholar), the overall changes in fluorescence reflect global changes in protein structure, and only the average microenvironments of tryptophans can be assessed. As stated above, tryptophan emission at ∼345 nm (following excitation at 280 or 298 nm) is significantly higher for Δ loop DAAO than for wild type. At increasing urea concentration, the two DAAO forms show a different increase in the intensity of the protein emission that is anyway complete at 2–3 m urea (Fig. 1). Treatment with urea also causes the emission maximum of tryptophan fluorescence in both DAAO forms to shift from 337 nm for wild-type and 339 nm for Δ loop DAAO to 352–353 nm at 5–6 m urea, a change that does not parallel the change in fluorescence intensity (Fig. 1). The fluorescence red shift stems from transfer of tryptophan side chains to a more polar environment upon protein unfolding (21Burstein E.A. Vedenkina N.S. Ivkova M.N. Photochem. Photobiol. 1973; 18: 263-279Crossref PubMed Scopus (1087) Google Scholar). Plots of changes in the intensity protein fluorescence at equilibrium as a function of the urea concentration apparently suggest a simple two-state transition (Fig. 1). The free energy of unfolding, ΔG, was calculated according to Equation 2. The free energy of unfolding in the absence of the denaturant (ΔGw) can be obtained by extrapolation of ΔG to zero denaturant concentration by using Equation 3. The energy value determined for the monomeric Δ loop enzyme is lower than the value for wild-type DAAO (Table I). The different dependence of the intensity of protein fluorescence and of the wavelength of emission maximum on the urea concentration indicates that the overall urea-induced denaturation of DAAO is a more complex process, pointing to the presence of a detectable intermediate. Flavin Fluorescence—As shown in Fig. 2, urea-dependent changes in flavin fluorescence are indicative of a two-step, three-state process for both proteins. An intermediate is formed at ∼3 m urea, and apparently it retains a bound cofactor, since its flavin fluorescence does not correspond to the much higher one typical of free flavin (10Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (182) Google Scholar). Considering the transition occurring at ≤3 m urea, increasing urea concentrations have a different effect on the two proteins. Flavin fluorescence of the Δ loop mutant starts to increase at lower urea concentration than that of wild-type DAAO. At ≥7 m urea, the fluorescence intensity of wild-type DAAO attains values similar to those of the Δ loop mutant and corresponds to that of the free flavin. Equilibrium values determined from changes in flavin fluorescence, analyzed by using Equation 5 according to a three-state mechanism, are reported in Table I. The increase in protein and flavin fluorescence at increasing concentrations of urea is different for the two DAAO forms (as confirmed by the Cm values in Table I), and changes in tryptophan fluorescence in both proteins appear to anticipate release of flavin cofactor (Figs. 1 and 2). Secondary and Tertiary Structure: CD Studies—To test whether the unfolding transition monitored by fluorescence measurements reflects a disruption of the overall protein structure or is just indicative of local unfolding and to have a better knowledge of the structural properties of the intermediate detected at ∼3 m urea, we analyzed urea-induced denaturation of both DAAO forms by CD spectroscopy. When urea-induced loss of secondary structure elements was monitored by following the changes in ellipticity at 223 nm (Fig. 3A), a different increase in the CD signal is observed for the two DAAO forms. The increase of the 223-nm signal (corresponding to the unfolding of the secondary structure elements) starts at 1 m urea for the monomeric Δ loop DAAO, but only at ≥2.0 m urea for the dimeric wild-type enzyme. In both cases, a two-state transition is observed, with Cm values significantly higher than those observed in protein fluorescence studies and for the first transition seen in flavin fluorescence studies and leading to formation of the intermediate (Table I). Thus, the observed transition may correspond to loss of the secondary structure of the intermediate (which accumulates at [urea] ≈ 2.5 m; see above) and to conversion of the intermediate into an unfolded state. Analysis of the CD spectra recorded on either protein at 3 m urea allowed a calculation of the residual content of secondary structures. α-Helix content decreased from 30.4% in the native wild-type protein to 25.2% (from 33.3 to 22.4% in Δ loop mutant DAAO), with a concomitant increase in random coiled structures (from 35.2 to 39.2% in wild-type DAAO and from 33.2 to 41.7% in Δ loop mutant DAAO). The addition of urea also has different effects on the tertiary structure of both wild-type and Δ loop mutant DAAO (as shown by the change in CD signals at 253 nm) (Fig. 3B). A two-state transition is observed at significantly lower denaturant concentration for the monomeric Δ loop mutant than for wild-type DAAO. This suggests that dimer dissociation sensibly altered the tertiary structure in each monomer and confirms a much higher sensitivity to urea for the Δ loop DAAO mutant with respect to the wild-type protein. For both proteins, changes in tertiary structu" @default.
• W2054328275 created "2016-06-24" @default.
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• W2054328275 date "2004-07-01" @default.
• W2054328275 modified "2023-10-16" @default.
• W2054328275 title "Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase" @default.
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