Structure-to-Function Relationship of Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with Lower Stability but Higher Enzymatic Activity
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m212122200
ISSN1083-351X
AutoresAlmerinda Di Venere, Maria Luisa Salucci, Guus van Zadelhoff, Gerrit A. Veldink, Giampiero Mei, Nicola Rosato, Alessandro Finazzi‐Agrò, Mauro Maccarrone,
Tópico(s)Polyamine Metabolism and Applications
ResumoLipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819–6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1–3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0–4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable. Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819–6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1–3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0–4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable. lipoxygenase guanidinium hydrochloride circular dichroism eicosatetraynoic acid 1-anilino-8-naphthalenesulfonate Lipoxygenases (Loxs)1form a homologous family of non-heme, non-sulfur iron containing lipid-peroxidizing enzymes, which catalyze the dioxygenation of polyunsatured fatty acids to the corresponding hydroperoxy derivatives. Mammalian Loxs have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1Kühn H. Borngraber S. Adv. Exp. Med. Biol. 1999; 447: 5-28Crossref PubMed Scopus (58) Google Scholar). They are also thought to have a role in atherosclerosis, brain aging, human immunodeficiency virus infection, kidney diseases, and terminal differentiation of keratinocytes, because they are key enzymes in the arachidonate cascade, together with cyclooxygenases (2Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3137) Google Scholar). In plants, lipoxygenases are active in germination, in the synthesis of traumatin and jasmonic acid, and in the response to abiotic stress (3Grechkin A. Prog. Lipid Res. 1998; 37: 317-352Crossref PubMed Scopus (269) Google Scholar). Recently, Lox activity has been shown to be instrumental in inducing irreversible damages to organelle membranes (4van Leyen K. Duvoisin R.M. Engelhardt H. Wiedmann M. Nature. 1998; 395: 392-395Crossref PubMed Scopus (260) Google Scholar, 5Grullich C. Duvoisin R.M. Wiedmann M. van Leyen K. FEBS Lett. 2001; 489: 51-54Crossref PubMed Scopus (70) Google Scholar), a process that might be the basis for the critical role of Loxs in programmed cell death induced by various pro-apoptotic stimuli (6Maccarrone M. Melino G. Finazzi-Agrò A. Cell Death Differ. 2001; 8: 776-784Crossref PubMed Scopus (160) Google Scholar). The biological activities of Loxs have attracted a growing interest on both their functional and structural properties. Plant and mammalian Loxs are made by a single polypeptide chain folded in a two-domain structure (7Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar). The N-terminal domain is a β-barrel of ∼110–115 (mammals) and 150 (plants) residues, whereas the larger C-terminal domain is mainly helical and contains the catalytic site. Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases from tissues of different species (7Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar). Lox-1 has a 30-kDa N-terminal domain and a 60-kDa C-terminal domain containing the catalytically active iron and the substrate-binding pocket. The function of the N-terminal β-barrel domain has been elusive for several years, for Lox-1 as well as for other Loxs (7Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar). Recently, the N-terminal domain has been shown to be essential for calcium binding and activation of 5-lipoxygenase activity (8Hammarberg T. Provost P. Persson B. Radmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and for nuclear membrane translocation of this Lox isoform (9Chen X.S. Funk C.D. J. Biol. Chem. 2001; 276: 811-818Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 10Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Equilibrium unfolding experiments on Lox-1 have shown that the C-terminal domain is less stable than the N-terminal domain, undergoing chemical denaturation in the early steps of the complex protein unfolding process (11Sudharshan E. Appu Rao A.G. J. Biol. Chem. 1999; 274: 35351-35358Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The smaller N-terminal domain seems to retain a large part of its β-barrel structure even at high urea concentration, suggesting that this portion of the protein structure might be important for the overall protein stability. Limited proteolytic cleavage of Lox-1 into the N- and C-terminal domains, and their subsequent isolation, have added new important structural and functional information (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). In particular, the electrophoretic, chromatographic, and spectroscopic analyses of the purified 60-kDa C-terminal domain of Lox-1 (termed "mini-Lox") have shown that the trimmed enzyme is still folded. Surprisingly, mini-Lox was shown to have a greater catalytic activity than intact Lox-1, thus suggesting a built-in inhibitory role for the N-terminal domain (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). In addition, mini-Lox displayed a higher binding affinity than Lox-1 for artificial membranes, attributable to the enhanced surface hydrophobicity exposed after removal of the 30-kDa N-terminal fragment. Interestingly, similar results have been recently reported about the effect of the N-terminal domain removal on the activity and membrane-binding ability of the reticulocyte-type 15-lipoxygenase (13Walther M. Anton M. Wiedmann M. Fletterick R. Kühn H. J. Biol. Chem. 2002; 277: 27360-27366Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A three-dimensional representation of mini-Lox can be generated from the Lox-1 crystallographic data (Fig. 1). The high number of trypthophans (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar) renders this enzyme spectroscopically very complex, as demonstrated by the great heterogeneity of the mini-Lox fluorescence spectrum (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). A somewhat greater solvent accessibility of aromatic chromophores of mini-Lox with respect to Lox-1 is apparent probably associated to a conformational change after proteolytic cleavage (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). In our study we have investigated the relationship between the activity of mini-Lox (e.g. enhanced enzymatic activity) and its structural features checked by circular dichroism, 1-anilino-8-naphthalenesulfonate (ANS) binding, steady-state, and dynamic fluorescence. Because the balance between the loss of hydrophobic interactions and the enhanced solvation upon the N-terminal removal is crucial to understand the mini-Lox properties, we have also studied the stability of mini-Lox using complementary techniques, such as chemical equilibrium unfolding measurements and denaturation by hydrostatic pressure. The results demonstrate that the removal of the N-terminal domain makes the mini-Lox more sensitive to denaturation by both guanidinium hydrochloride (GdHCl) and pressure. Furthermore, dynamic fluorescence measurements and ANS binding experiments provide new evidence that the active site is more accessible in the mini-Lox than in the Lox-1 and that quite different conformational changes follow the substrate binding in the two enzymes. All together these results provide a new structural rationale that might explain the peculiar mini-Lox features. Linoleic (9,12-octadecadienoic) acid and 5,8,11,14-eicosatetraynoic acid (ETYA) were purchased from Sigma. Ultrapure guanidinium hydrochloride and ANS were purchased from US Biochemical Corp. and Molecular Probes Inc., respectively. Lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC1.13.11.12; Lox-1) was purified from soybean (Glycine max[L.] Merrill, Williams) seeds as reported (14Finazzi-Agrò A. Avigliano L. Veldink G.A. Vliegenthart J.F.G. Boldingh J. Biochim. Biophys. Acta. 1973; 326: 462-470Crossref PubMed Scopus (128) Google Scholar), and mini-Lox was prepared as previously described (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). Briefly, Lox-1 was digested with trypsin at a Lox-1/trypsin 10:1 (w/w) ratio, which allowed completion of the reaction within 30 min. Chromatographic separation of the tryptic fragments was performed by high performance liquid chromatography gel-filtration on a Biosep-SEC-S3000 column (600 × 7.8 mm, Phenomenex, Torrance, CA). Fractions corresponding to the 60-kDa fragment eluted after 10 min as a single peak, and were pooled, dialyzed against water, and concentrated on Centricon 30 ultrafiltration units (Amicon, Beverly, MA) (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). This 60-kDa fragment, referred to as mini-Lox, was found to be electrophoretically pure on 12% SDS-polyacrylamide gels, and its N-terminal amino acid analysis showed the sequence STPIEFHSFQ, which corresponds to a unique trypsin cleavage site between lysine 277 and serine 278 (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). Such a cleavage should indeed remove a 30-kDa N-terminal domain of Lox-1, leading to a fragment of the expected molecular mass of 63,695 Da (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar). Protein concentration was determined according to Bradford (15Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222212) Google Scholar), using bovine serum albumin as a standard. Dioxygenase activity of mini-Lox in 100 mm sodium borate buffer (pH 9.0) was assayed spectrophotometrically at 25 °C by recording the formation of conjugated hydroperoxides from linoleic acid at 234 nm (16Schilstra M.J. Veldink G.A. Vliegenthart J.F.G. Biochemistry. 1994; 33: 3974-3979Crossref PubMed Scopus (96) Google Scholar). Except for lifetime measurements, all the experiments were performed dissolving Lox-1 and mini-Lox in 0.1 m Tris-HCl buffer (pH 7.2) at a final protein concentration of 1.5 μm. For lifetime measurements, Lox-1 and mini-Lox were used at 10 μm, to obtain a good signal to noise ratio. Protein denaturation by GdHCl was obtained after a 12 h incubation at 4 °C in the presence of different amounts of denaturant. Fluorescence and CD spectra were recorded at 20 °C after 30 min of incubation. Unfolding and refolding pathways were independent of protein concentration. Refolding of fully unfolded mini-Lox samples was achieved by diluting the denaturant concentration with buffer. The analysis of the unfolding transition was performed as described elsewhere (17Di Venere A. Rossi A. Rosato N. De Matteis F. Finazzi-Agrò A. Mei G. J. Biol. Chem. 2000; 275: 3915-3921Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), according to a two step denaturation pathway according to Scheme 1, N↔K1I↔K2USCHEME1where N, I, and U represent the native, intermediate, and unfolded protein fractions. They were directly evaluated as a linear combination from the fluorescence signal.K1 and K2 are the two equilibrium constants related to the respective free energy values ΔG1 and ΔG2, which are supposed to vary linearly with the denaturant concentration [D] as shown in Equation 1(where i = 1.2).ΔGi=ΔGH2O−m[D]Equation 1 A non-linear least-squares fit was used to evaluate the parameters reported in Table I, according to the minimum χ2 value. A single transition model was instead sufficient to fit both the CD and activity data.Table IPhysical Parameters Characterizing the Unfolding of Mini-LoxMini-Lox unfolding by GdHCIΔG1m1ΔG2m2ΔGTOT[D]12kcal/molkcal/molmkcal/molkcal/molmkcal/molmFluorescence1.5 ± 0.12.0 ± 0.12.5 ± 0.21.0 ± 0.14.0 ± 0.2Activity1.0 ± 0.22.2 ± 0.20.46Circular dichroism2.8 ± 0.20.9 ± 0.23.20Mini-Lox unfolding by pressureΔG1ΔV1kcal/molml/mol1.5 ± 0.1−69 ± 10The parameters have been obtained by fitting CD, enzymatic activity, and fluorescence data (Figs. 2 and 3) according to a two- or three-state denaturation process (see "Experimental Procedures"). The errors on the calculated parameters were directly obtained by the fitting routine. Open table in a new tab The parameters have been obtained by fitting CD, enzymatic activity, and fluorescence data (Figs. 2 and 3) according to a two- or three-state denaturation process (see "Experimental Procedures"). The errors on the calculated parameters were directly obtained by the fitting routine. CD spectra were recorded on a Jasco-710 spectropolarimeter, at 20 °C, using a 0.1 cm quartz cuvette. Steady-state fluorescence spectra have been recorded using an ISS-K2 spectrofluorometer (ISS), at 20 °C upon excitation at 280 nm. No differences were observed in fluorescence spectra when the excitation wavelength was varied from 280 to 295 nm due to a very efficient energy transfer from tyrosines to tryptophans. High pressure measurements were performed with the same instrument, using the high pressure ISS cell equipped with an external bath circulator. The analysis of the high pressure unfolding transition was performed assuming a two-state equilibrium model between native and intermediate species as shown in Equation 2 as follows, N↔K1IEquation 2 with ΔG = −RTlnK1 and ΔV = (∂ lnK1)/∂P). The fluorescence emission decay of both Lox-1 and mini-Lox was extrapolated from the phase-shift and demodulation data, obtained with the cross-correlation technique (18Gratton E. Limkeman M. Biophys. J. 1983; 44: 315-324Abstract Full Text PDF PubMed Scopus (417) Google Scholar) upon excitation at 280 nm, using the frequency-modulated light of an arc-xenon lamp, in the range 5–200 MHz. The emission was observed through a 305 nm cutoff filter to avoid the contribution of scattered light. ANS binding was measured as follows. Fluorescence spectra in the range 420–550 nm were recorded as a function of the amount of ANS (λexc = 350 nm), at fixed protein concentration, then each spectrum was resolved according to the linear combination in Equation 3 Sexp=CFSF+CBSBEquation 3 where Sexp, SF, and SB are column vectors corresponding to the experimental, ANS-free, and ANS-bound spectra. CF and CB represent the extrapolated linear correlation coefficients representing the percentage of free and bound ANS, respectively. The spectrum of the totally bound ANS (SB) was extrapolated, in a separate experiment, by varying the protein concentration, in the presence of a fixed amount of ANS (19Cardamone M. Puri N.K. Biochem. J. 1992; 282: 589-593Crossref PubMed Scopus (432) Google Scholar). The ANS binding data have been represented as Scatchard plots and fitted according to models for one or two independent classes of sites (20Cantor C.R. Schimmell P.R. Biophysical Chemistry. W. H. Freeman and Co., San Francisco1980: 849-866Google Scholar) as follows,ν[L]=∑iniki1+ki[L]Equation 4 where ν represents the moles of ANS bound per moles of protein, [L] is the concentration of free ANS,i = 1 or 2, and ni andki are the number of binding sites and the association constant of each class of sites. The fits were performed using the SPW 1.0 version of the Sigmaplot scientific graphic software (by Jandel Scientific). The stability of mini-Lox has been studied by equilibrium unfolding measurements at increasing GdHCl concentrations (Fig. 2). The non-coincidence of the fluorescence and CD measurements demonstrates that the unfolding transition was not cooperative and that the presence of stable intermediate species had to be taken into account. The simplest denaturation model, which successfully fitted the experimental data, was a three-state process, N ↔ I ↔ U (see "Experimental Procedures"), whose parameters are reported in TableI. As shown by the very early and steep increase of the fluorescence signal (Fig. 2), a small free energy of unfolding (Table I) characterizes the first transition (0–1.5 m GdHCl). No relevant change occurred in the CD signal at low GdHCl concentration, and the data could be fitted according to a two-state model (Table I). The overall stabilization energy obtained from the spectroscopic measurements (ΔGtot0 ≈ 4.0 ± 0.3 kcal/mol) demonstrated that mini-Lox is rather unstable, specially if compared with intact Lox-1 (ΔGtot0 ≈ 26 kcal/mol (11Sudharshan E. Appu Rao A.G. J. Biol. Chem. 1999; 274: 35351-35358Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar)). Combined refolding and proteolysis experiments have suggested that the C-terminal domain of Lox-1 is the less stable part of the intact enzyme. This domain, which roughly corresponds to the whole mini-Lox molecule, is probably fully unfolded in the intermediate state found in the Lox-1 denaturation pathway (11Sudharshan E. Appu Rao A.G. J. Biol. Chem. 1999; 274: 35351-35358Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The transition from the native structure to this intermediate species is characterized by a quite large free energy change (ΔG10≈ 14 kcal/mol), as compared with the total stabilization energy of mini-Lox (Table I). It can be therefore concluded that the low stability of mini-Lox is due to the removal of the 30-kDa N-terminal domain. As a matter of fact this hypothesis is indirectly supported by the mi values reported in Table I. Them parameter, which describes the cooperativity of the unfolding process, is strictly correlated to the change in protein-accessible surface area upon denaturation (21Myers J.K. Pace C.N. Scholtz J.M. Protein Sci. 1995; 4: 2138-2148Crossref PubMed Scopus (1704) Google Scholar). In particular, greater hydration has been found to correspond to larger mvalues and vice versa (21Myers J.K. Pace C.N. Scholtz J.M. Protein Sci. 1995; 4: 2138-2148Crossref PubMed Scopus (1704) Google Scholar). It is also well known thatm values obtained with GdHCl are about 2.2× larger than those obtained with urea (21Myers J.K. Pace C.N. Scholtz J.M. Protein Sci. 1995; 4: 2138-2148Crossref PubMed Scopus (1704) Google Scholar). Thus, even though different unfolding agents have been used in the equilibrium unfolding measurements of Lox-1 and mini-Lox (urea and GdHCl, respectively), it is possible to compare the different results obtained for the two proteins. In particular, from the results reported by Sudharshan and Appu Rao (11Sudharshan E. Appu Rao A.G. J. Biol. Chem. 1999; 274: 35351-35358Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), it can be expected that the GdHCl-induced denaturation of Lox-1 would yield m1 ≈ 4.4 kcal/mol m andm2 ≈ 3.5 kcal/mol, for the first and second step of its unfolding pathway. On the other hand, the totalm value for mini-Lox (Table I) is ≈ 3 kcal/mol,i.e. 30% less than that obtained in the first transition of Lox-1. This lower value stands for a smaller change in the solvent-exposed surface area, indicating that the native mini-Lox is more hydrated than the C-terminal domain of Lox-1. Thus, the N-terminal Lox-1 domain plays a fundamental structural role, shielding the other domain from the solvent and therefore enhancing its stability. Despite its low stabilization energy, the mini-Lox unfolding pathway was complex (Fig. 2), suggesting the presence of stable (or partially stable) intermediate species. Measurements of enzymatic activity demonstrated that the protein biological activity was progressively lost between 0 and 1.5m GdHCl (Fig. 2, inset). The corresponding free energy value (ΔG ≈ 1.0 ± 0.2 kcal/mol), very close to that calculated for the first fluorescence phase (Table I), reflects the high instability of the enzyme, especially around the active site region. In fact, in the same range a significant loosening of the protein tertiary structure was taking place, as revealed by the change (50%) of the protein intrinsic fluorescence signal (Fig. 2). These findings suggest that the intermediate state is partially unfolded, but it retains a native-like secondary structure. Such features resemble those of the so-called molten globule state, an unfolding intermediate species described in the denaturation pathway of several globular proteins (22Kuwajima K Proteins. 1989; 6: 87-103Crossref PubMed Scopus (1504) Google Scholar, 23Ptitsyn O.B. Pain R.H. Semisotnov G.V. Zerovnik E. Razgulyaev O.I. FEBS Lett. 1990; 262: 20-24Crossref PubMed Scopus (680) Google Scholar). The most relevant property of this structure is indeed a greater exposure of the protein hydrophobic moieties to the solvent. In the case of mini-Lox, the great heterogeneity of the fluorescence spectrum (12Maccarrone M. Salucci M.L. van Zadelhoff G. Malatesta F. Veldink G. Vliegenthart J.F.G. Finazzi-Agrò A. Biochemistry. 2001; 40: 6819-6827Crossref PubMed Scopus (58) Google Scholar), due also to a high number of tryptophan residues (Fig. 1), makes it impossible to dissect the contribution of each domain to the fluorescence of the intermediate state. In the last years, several studies have demonstrated that molten globule intermediates may be also produced by hydrostatic pressure, which may force solvent into the protein core (24Silva J.L. Foguel D. Royer C.A. Trends Biochem. Sci. 2001; 26: 612-618Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Fig. 3 reports the fluorescence spectra of mini-Lox at 1 and 2400 bar. A shift of the emission signal toward longer wavelengths at higher pressure values is observed (Fig. 3,inset), supporting the hypothesis of a progressive hydration of the internal tryptophylic residues. It almost perfectly overlaps the steady-state spectrum in the presence of 1.5 m GdHCl (Fig.3), demonstrating that physically induced unfolding may neatly reproduce chemical denaturation. Actually, the two-state fit reported in the inset of Fig. 3 yields the same free energy of unfolding (≈ 1.5 kcal/mol) characterizing the first denaturation transition in GdHCl (Table I). The corresponding volume change has also been evaluated (see "Experimental Procedures") and resulted to be quite small (−69 ± 9 ml/mol), despite the large protein size (≈ 63,000 Da) as usually found by compression of globular proteins (24Silva J.L. Foguel D. Royer C.A. Trends Biochem. Sci. 2001; 26: 612-618Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 25Royer C.A. Biochim. Biophys Acta. 2002; 1595: 201-209Crossref PubMed Scopus (366) Google Scholar). Recent studies on the pressure-induced molten globule state of cytochrome c (26Pryse K.M. Bruckman T.G. Maxfield B.W. Elson E.L. Biochemistry. 1992; 31: 5127-5136Crossref PubMed Scopus (36) Google Scholar), α-lactalbumin (27Kobashigawa Y. Sakurai M. Nitta K. Protein Sci. 1999; 8: 2765-2772Crossref PubMed Scopus (40) Google Scholar), apomyoglobin (28Vidugiris G.J.A. Royer C.A. Biophys. J. 1998; 75: 463-470Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and the Ras domain of RalGDS (29Inoue K. Yamada H. Akasaka K. Herrmann C. Kremer W. Maurer T. Doker R. Kalbitzer H.R. Nat. Struct. Biol. 2000; 7: 547-550Crossref PubMed Scopus (30) Google Scholar) reported ΔV values from 15 to 70 ml/mol, quite similar to that found here for mini-Lox, which, however, is a much larger molecule. In this line, the results obtained with point mutants of staphylococcal nuclease (30Frye K.J. Royer C.A. Protein Sci. 1998; 7: 2217-2222Crossref PubMed Scopus (137) Google Scholar) have suggested that the collapse of internal cavities under pressure might be the main source of the volume changes. Because the number and size of cavities in a protein have been found to be related to its molecular weight (31Rashin A.A. Iofin M. Honig B. Biochemistry. 1986; 25: 3619-3625Crossref PubMed Scopus (255) Google Scholar, 32Williams M.A. Goodfellow J.M. Thornton J.M. Protein Sci. 1994; 3: 1224-1235Crossref PubMed Scopus (271) Google Scholar, 33Hubbard J.S. Gross K.H. Argos P. Protein Eng. 1994; 7: 613-626Crossref PubMed Scopus (180) Google Scholar), the low ΔV value obtained for mini-Lox could be explained by assuming that most protein cavities are already solvated at ambient pressure. Not only this hypothesis is consistent with the chemical denaturation experiments, but it could also explain the easier resiliency of mini-Lox with respect to Lox-1. In fact, at variance with the native enzyme (11Sudharshan E. Appu Rao A.G. J. Biol. Chem. 1999; 274: 35351-35358Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 34Srinivasulu S. Appu Rao A.G. Biochim. Biophys. 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