Anke Hadasch

Alexander Sorokin

Alain Rabionb

Bernard Meunier

Sequential addition of pH and solvent eþ ects as key factors in H 2 O 2 , the oxidation of 2,4,6-trichlorophenol catalyzed by iron tetrasulfophthalocyanine

The efficiency of the H 2 O 2 oxidation of 2,4,6-trichlorophenol (TCP) catalyzed by iron tetrasulfophthalocyanine (FePcS) is highly dependent on the pH value of the reaction mixture, the local hydrogen peroxide concentration and the organization of FePcS molecules in solution. Among the several forms of FePcS in aqueous solutions (dimer or monomer), monomeric FePcS is proposed to be the catalytically active complex. The key role of the organic co-solvent (acetonitrile, acetone, alcohol, . . .) is to shift the dimer/monomer equilibrium toward monomeric FePcS, the efficient catalyst precursor. A stepwise addition of hydrogen peroxide signiÐcantly improves the conversion of TCP and allows a low catalyst loading, below 1% with respect to the pollutant, to be used.

Degradation of chlorinated aromatics is an important environmental research Ðeld since these compounds are among the most recalcitrant pollutants.1 Recently we found an efficient catalytic oxidation of 2,4,6-trichlorophenol (TCP) catalyzed by iron tetrasulfophthalocyanine (FePcS, see Fig. 1 for the structure).2h4

With hydrogen peroxide (" the clean peroxide Ï), FePcS has been shown to catalyze aromatic ring cleavage of trichlorophenol and pentachlorophenol.2h4 Chloromaleic acid has been demonstrated to be the main product of this aromatic ring cleavage, with chlorofumaric, maleic and fumaric acids being minor products. 2 The proposed mechanism of this aromatic ring degradation involves a nucleophilic iron(III) peroxo complex, PcSFeIIIOOH, as the key active species responsible for the epoxidation of intermediate quinones and for the attack of carbonyl groups of the oxidized quinones to explain the aromatic CwC cleavage via a Grob-type fragmentation.4 By using 14C-labeled TCP substrate, carbon dioxide was demonstrated to be the ultimate degradation product in this catalytic oxidative degradation of such a recalcitrant compound. 3 In this catalytic oxidation of TCP two out of the three chlorine substituents of the starting material were recovered as chloride ions, one carbon out of the six carbon atoms ended as carbon oxides and four other carbon atoms Ðnished as water-soluble diacids. 3 The Ðnal goal of this approach is not to burn the recalcitrant molecule to carbon dioxide, as in incineration processes, but to initiate a degradation of the pollutant to more biodegradable products. Here we wish to report new data on the key factors controlling the catalytic activity of the system in the oxidation of TCP : FePcSÈH 2 O 2 the sequential addition of and the pH and solvent H 2 O 2 e †ects on the monomer/dimer equilibria of the catalyst precursor(s). ). oxidations, performed in an acetonitrile-bu †ered water mixture (v/v \ 1 : 3) to dissolve the hydrophobic 2,4,6trichlorophenol, were initiated by a single addition of 5 equiv. of

Results and Discussion

In homogeneous conditions, a relatively high H 2 O 2 . catalyst-to-substrate ratio of 3.7% was necessary to obtain a full conversion of the pollutant within a few minutes. In the early stage of reaction we observed a gas release. GC analyses of the gas phase over the reaction mixture before and after reaction showed a slight increase in the dioxygen concentra- 1.

The two important criteria for optimal degradation of the pollutant are fast conversion of the substrate and a high number of chloride ions released per converted TCP molecule ; the latter is expected for an advanced degree of oxidation of the substrate. Complete substrate conversions were obtained with only 1% and 0.5% FePcS (runs 2 and 3) and the Ðnal conversion in the presence of only 0.1% FePcS was still 50% (run 5). These results show that fast and complete substrate conversion with a high dechlorination (2 Cl~per converted TCP) was obtained using 1% of catalyst (run 2 . a Products were quantiÐed by integration of the vinylic proton signals at 6.67 ppm (for 1), 7.30 ppm (for 2), 6.39 ppm (for 3), 6.75 ppm (for 4) and of the aromatic protons at 7.9È8.1 ppm for coupling products. All these data are yields in % with respect to TCP. b TCP conversion was only 80% in this run. c TCP conversion was 100% in runs 2È4. tion (the blue color of the starting iron phthalocyanine changes to blue-green after addition of the oxidant and gradually to a deep purple when quinone derivatives are generated).

Quantitative analysis of the oxidation products formed by the di †erent addition methods allowed us to compare H 2 O 2 the efficiency of the catalytic system to oxidize TCP. The products were quantiÐed by NMR and the results are reported in Table 2. The catalytic TCP oxidations were performed using 1 and 3 2) indicating a more complete TCP degradation. It should be noted that only the sequential-addition method allowed full substrate conversion at low catalyst loading to be reached. In the case of a 3.7% catalyst-to-substrate ratio, we observed a signiÐcant decrease in the yield of coupling products, from 37% in the case of the one-portion addition of (run 3, Table 2) to 25% (run 4, Table 2), suggesting H 2 O 2 again that the sequential addition of the solution H 2 O 2 favored the formation of ring-cleavage products in this catalytic TCP oxidation. The lower yields of dicarboxylic acids could be explained by their further oxidation in the reaction mixture because of a higher catalyst concentration compared to run 2, Table 2.

InÑuence of the pH value

We also studied the inÑuence of pH value of the bu †ered reaction mixture on the conversion of TCP at 60 min ; the data are reported in Fig. 2. The catalytic reaction is strongly dependent on the pH value of the solution. No catalytic activity was detected at pH 4 after 60 min, but for the same reaction time the substrate conversion at pH 5 and pH 6 increased to 24 and 66%, respectively. Full conversion of TCP was obtained at pH 7 and pH 8, which correspond to the optimal pH values of the catalytic reaction mixture. At pH 9 we observed a dramatic loss of catalytic activity.

The Ðrst step of the TCP oxidation is the one-electron oxidation of the phenolate anion.3,4 Since the value of TCP pK a is 6.2,6 only small amounts of TCP are present in the reaction mixture as phenolate (a more oxidizable form compared to the phenol itself) at pH 5 and 6, leading to poor substrate conversions. At pH 7 and pH 8, 100% of the TCP is present as phenolate, thus explaining the full substrate conversions as well as the better oxidation rates. The absence of catalytic activity at pH 9 can probably be explained by the basefavored formation of l-oxo dimeric species of FePcS.

It should be noted that the bu †er concentration inÑuenced the substrate conversion as well. Final bu †er concentrations of 0.125 M or 0.25 M were necessary to keep the catalyst in an Conversion of TCP (%) active form and to neutralize the acids formed during TCP oxidation.

InÑuence of the solvent on the catalytic oxidation reaction

In order to have a homogeneous solution when we worked with a reaction mixture containing 10 mM of TCP (2000 ppm), we used acetonitrile as a co-solvent to dissolve the hydrophobic TCP (acetonitrile : bu †ered water ratio of 1 : 3). Keeping in mind the possible development of the system for the oxidative depollution of aqueous FePcSÈH 2 O 2 effluents, we decided to study the inÑuence of acetonitrile content using 1 mM TCP (200 ppm) and a 3.7% FePcS-to-TCP molar ratio. Increasing the acetonitrile : water ratio leads to an increase in the substrate conversions (Table 3). The conversions of TCP after one hour were 13, 54, 96 and 100% in reaction mixtures containing 2.5, 10, 25 and 50% acetonitrile, respectively.

Several hypotheses can be proposed to explain the strong inÑuence of the acetonitrile content on this catalytic oxidation : (i) MeCN might act as an axial ligand on the iron center of FePcS to enhance its catalytic activity might be able to dissociate an inactive dimer of FePcS to a monomeric complex, which is catalytically active.

To check the role of acetonitrile, we replaced it by di †erent solvents. The conversions obtained after one hour in the presence of acetone, acetonitrile, ethanol and formamide were 100, 96, 86 and 69%, respectively (solvent : bu †ered water ratio of 1 : 3) (Table 4). The total dechlorination values decreased in the same order. The data in Table 4 also indicate that TCP conversions decrease when the solvent polarity9 increases, suggesting that these organic solvents might have a strong inÑuence on the dissociation of inactive dimeric forms of the catalyst precursors.

To verify the hypothesis of an organic solvent molecule acting as axial ligand to FePcS, we added 100 equivalents of 3-cyanobenzoic acid, a potentially good axial ligand, to 1 equivalent of FePcS (Table 3, run 1c). We did not observe a signiÐcant di †erence in the substrate conversions in the presence or in the absence of 3-cyanobenzoic acid (17% and 13%, respectively). This lack of inÑuence of the 3-cyanobenzoic acid suggests that the e †ect of the di †erent organic solvents is not related to their possible coordination on the axial position of FePcS.

The possible formation of a reactive intermediate between acetonitrile or formamide and by analogy to the Payne H 2 O 2 reaction8 was excluded, since a solvent unable to form this kind of peroxidic intermediate, such as ethanol, also gave a high conversion (86% within 60 min, Table 4). In addition, this experiment conÐrmed that free hydroxyl radicals (usually trapped by ethanol) do not play a key role in this oxi-H 2 O 2 dation reaction catalyzed by iron sulfophthalocyanine.

InÑuence of the solvent on the catalyst precursor(s)

The organic solvents caused a noticeable change in the UV-vis spectrum of FePcS. In pure water, we observed only one absorption maximum in the visible range at 632 nm. When adding an aqueous solution of this complex to a solvent mixture with a Ðnal organic solvent : water ratio of 1 : 3, we observe a decrease of the absorption maximum at 632 nm and the appearance of two absorption bands at 636 and 668 nm within 30 min (the co-solvents used were the same as in the catalytic reaction : acetonitrile, acetone or ethanol). The changes observed in the UV-vis spectra in the presence of acetonitrile with isobestic points at 554, 644 and 714 nm might be due to the dissociation of a dimer of FePcS to a monomeric complex. The dependence of the spectrum of FePcS on acetonitrile content is shown in Fig. 3.

The nature of the FePcS structure in aqueous solutions has already been the subject of many di †erent studies.10h18 There is a general agreement that FePcS exists in a dimeric form in aqueous solutions with an absorption maximum near 630 nm, whereas a monomeric form exhibits a peak in the region of 670 nm.10,13,14 FePcS obtained from di †erent preparations, 2 PcS G10 into two main fractions. 18 The Ðrst fraction, a dark green product, showed a strong peak at 670 nm with minimal absorption at ca. 630 nm. The second fraction, containing a FeIII low-spin product, was dark blue with a strong absorption at 632 nm and minimal absorption at 670 nm. The ratio between these two components was dependent on the preparation method. Materials prepared by these di †erent methods gave the same complex in the presence of imidazole. The exact nature of these green and blue products is still not available in the literature on metallophthalocyanine chemistry.

The magnetic moments of the solid FePcS obtained from the WeberÈBusch preparation were in the low-spin d5 region and Mo ssbauer parameters were consistent with a mixture of ferric S \ 1/2, 3/2, 5/2 spin states, with the low-spin species being the major component.12 FePcS has been prepared by the WeberÈBusch method in di †erent forms (H`,N a `,K `, Cs`) and studied by di †erent spectroscopic methods. 17 The authors concluded that FePcS contained a wide range of iron complexes with ratios depending on each preparation. The sodium salt of FePcS was a low-spin FeIII species in the solid state, whereas high-spin FeIII compounds were detected in bu †er solutions (pH 2.5È12.0). Fanning et al. found no evidence for l-oxo complexes in the solid state or in solution.17 One or two molecules of water were proposed to be axial ligands susceptible to be bound to the stacked high-spin FeIII complexes at high ionic strengths. By lowering the ionic strength and increasing the concentration a strong water interaction can occur to form a low-spin FeIII complex.17 However, the preparation and characterization of l-oxobis-[tetraalkylsulfonamidophthalocyanine iron(III)] complexes have been reported by Lever et al.11 The identity of this l-oxo dimer has been conÐrmed by UV-vis spectroscopy, magnetism and FTIR (the band at 810 cm~1 being attributed to the FewOwFe antisymmetric deformation). Gupta et al. published a FTIR spectrum for the solid FePcS complex in a KBr matrix having a band at 837 cm~1, in the typical frequency region of the FewOwFe deformation mode (800È900 cm~1).10 However, a similar band is also present in the FTIR spectra of CoPcS (831 cm~1) and (850 cm~1), indicat-H 2 PcS ing that the identiÐcation of the FePcS l-oxo dimer by IR is still a matter of debate.

Using the hydrophobic FePc complex, Ercolani et al. published the preparation and a well-documented characterization of two interchangeable l-oxo dimers. 15 The Ðrst l-oxo dimer showed two antisymmetric FewOwFe frequencies at 852 and 824 cm~1, which can be shifted to 806 cm~1 upon treatment, while the second l-oxo dimer did not show 18O 2 any band for the FewOwFe vibration. These authors proposed a bent FewOwFe structure for the Ðrst l-oxo species and a quasi-linear one for the second l-oxo dimer. In fact, the proposed bent l-oxo dimer might be a hydroxo bridge, as recently proposed by Scheidt and co-workers for iron and manganese porphyrin complexes.20,21 Hanack and coworkers have also prepared and characterized by UV-vis, FD mass, Mo ssbauer, NMR and EPR spectroscopy two isomeric l-oxo compounds for various substituted iron phthalocyanines.16 However, the nature of these two isomeric l-oxo compounds remains unknown. 16 One can conclude that despite a large number of studies the chemistry of l-oxo complexes of iron phthalocyanines, and in particular of FePcS, in aqueous solutions and the solid state is not presently fully understood.

We tried to evidence a l-oxo dimer structure of FePcS by using FTIR spectroscopy. We observed a band at 833 cm~1, which can be attributed to the FewOwFe deformation mode. Previously, a FewOwFe band at 810 cm~1 has been evidenced for the iron(III) complex of l-oxo-bis-tetra(dodecylsulfonamido)phthalocyanine.11 In order to check the hypothesis that the band at 833 cm~1 is the signature of a l-oxo dimer, we incubated FePcS in in the presence of H 2 18O 0.1 M acid in order to cleave the l-oxo dimer ; then the solution was neutralized with KOH to generate a Few18OwFe l-oxo dimer. We obtained practically the same IR spectrum to that of the starting FePcS with only a small shift of the 833 cm~1 band to 839 cm~1, which cannot be attributed to an isotopic shift. However, this disappointing experiment should not be taken as proof of the deÐnitive absence of a l-oxo dimer for the FePcS material that we are using as a catalytic precursor in the FePcS-mediated oxidation of TCP because of the complex nature of water-soluble metallophthalocyanines.

Nevertheless, the formation of strongly associated FePcS dimers in water is highly favorable since this process should reduce the unfavorable contacts between water and the apolar core of this aromatic macrocyclic molecule. In contrast, less polar organic solvents should disfavor the hydrophobic interactions between two di †erent planes of phthalocyanines, which is a driving force for dimer formation. Consequently, the presence of an organic solvent should diminish the tendency of dimerization. So co-solvents with a low polarity should strongly contribute to shift the dimer/monomer equilibrium towards monomer formation. In fact, this correlation was observed in the oxidation of TCP catalyzed by FePcS : acetone [ acetonitrile [ ethanol [ formamide [ water (see data in Table 4).

ModiÐcations of FePcS spectra in the presence of TCP, acids or bases

Addition of TCP to a water solution of FePcS resulted in the same changes in the UV-vis spectra as observed after an organic solvent addition (see above) with isobestic points at 552, 650 and 714 nm, suggesting the formation of a FePcS monomer.4 Spectrophotometric titration of a 20 lM FePcS aqueous solution with 50 mM HCl revealed a decrease of the band at 632 nm and the appearance of two new absorbances at 636 and 680 nm with quasi-isobestic points at 340, 564 and 650 nm (Fig. 4). Thus spectral changes (positions of new bands and isobestic points) are similar in all three cases : addition of acid, acetonitrile or TCP to a water solution of FePcS.

These data strongly suggest the formation of (a) monomeric form(s) of FePcS under all these conditions. We also noted a signiÐcant di †erence of 12 nm between the absorption bands in the presence of acetonitrile and TCP (668 nm) and HCl (680 nm). Upon HCl titration the spectrum reached a saturation level with maxima at 636 and 680 nm when the acid concentration was 2.5 mM. A further increase in acid When hydrochloric acid was replaced by sulfuric or phosphoric acid, the same phenomenon was observed. Therefore we concluded that the presence of di †erent anions of these acids, chloride, sulfate or phosphate, were not at the origin of the changes observed in the visible spectra of FePcS.

To check the reversibility of an equilibrium between di †erent forms of FePcS, a FePcS solution previously acidiÐed by was titrated with NaOH. In the beginning we H 2 SO 4 , observed the regular disappearance of the maximum at 680 nm without an increase of the absorption bands at 636 and 632 nm. At pH 6, the maximum at 680 nm nearly disappeared and further addition of NaOH produced a spontaneous reappearance of the initial maximum at 632 nm (Fig. 5). Further The hypothesis that a dimeric form of FePcS is transformed into a single monomer is not sufficient to explain all these di †erent experimental results. In the presence of a co-solvent or TCP, we propose that the l-oxo dimer (632 nm, compound A in Scheme 1) can be cleaved to a FePcS monomer with a hydroxide or phenolate residue as an axial ligand (668 nm, compound B in Scheme 1). Titration of FeIII(PcS)OH with an acid leads to another monomer Mcompound C, probably with a water axial ligand as pre-[FeIII(PcS)H 2 O]`N, viously observed for sulfonated iron porphyrin complexes. 22 The absorption band at 636 nm, observed with all the di †erent acids, can be attributed to the corresponding stacked monomers (compounds and B stack C stack ). When a 20 lM FePcS solution is titrated with acid, the ratio between the absorption bands at 680 and 636 nm is always equal to 0.8 and does not change when adding more acid. But when we titrate a 4 lM FePcS solution under the same conditions, the ratio between the two absorption maxima changes and is equal to 1.0. The higher monomer content at lower FePcS concentration supports the hypothesis of an equilibrium between the monomer form C and the corresponding stacked monomer Finally, the formation of C stack . a monomeric FePcS is a prerequisite for catalytic activity of the metallophthalocyanine in TCP oxidations and the key role of an organic solvent is to shift the dimer/monomer equilibrium towards a monomeric FePcS complex, the true catalyst precursor.

Lifetime of the FePcS catalyst in the oxidation of TCP H 2 O 2 One of the key questions in homogeneous catalytic oxidations is the lifetime of the catalyst. In order to answer this question we decided to monitor the catalytic activity in batch reactions after addition of new portions of pollutant and oxidant (in a one-portion addition or Ðve-aliquot addition fashion, see Fig. 6).

These experiments were performed under the conditions of run 4 of Table 3. To our surprise, we found that the TCP conversion was still 90% within 10 min in the third run [see Fig. 6(a)]. The half-conversion time of the third run was 3 min, very close to that measured for the Ðrst two runs (2 min) and the total dechlorination was 5.0 Cl~for 2.9 converted TCP molecules or an average value of 1.7 Cl~per TCP. The kinetics were slightly di †erent in the case of the Ðve-aliquot addition of hydrogen peroxide (half-conversion times were 9, 5 and 7 min for runs 1, 2 and 3, respectively), but the total dechlorination was 5.2 Cl~for 2.9 converted TCP molecules or an average value of 1.8 Cl~per TCP [see Fig. 6(b)]. It must be noted that the initial blue color of FePcS changed to green or violet after addition of hydrogen peroxide by aliquots or by the one-portion mode, respectively. But the color turned to brown during the second and third runs due to the formation of quinone derivatives, making the monitoring of the catalyst concentration by UV-visible spectrophotometry more difficult. Finally, these data are highly encouraging for a catalyst that is easily accessible and cheap.

Conclusion

Gradual addition of to the reaction mixture in oxida-H 2 O 2 tions of trichlorophenol, catalyzed by FePcS, results in a more "" economic ÏÏ consumption of the oxidant due to a decrease of the wasteful dismutation of As a consequence, higher H 2 O 2 . conversions occurred with more advanced TCP oxidations using a smaller charge of catalyst. The catalyst precursor is probably a non-stacked monomeric iron sulfophthalocyanine able to react with hydrogen peroxide to generate an ironperoxo complex, previously proposed as the active species in the oxidative degradation of chlorophenols. The key role of the miscible organic solvent used in these aqueous catalytic mixtures is related to the shift of the dimer/monomer equilibrium towards a non-stacked monomeric form of FePcS. At the present stage of development, this catalytic system requires an organic co-solvent to be efficient. This fact makes the system more adapted for the treatment of FePcSÈH 2 O 2 process waters still containing 10 to 20% of organic solvents than for the Ðnal treatment of aqueous effluents. Repetitive batch experiments conÐrmed that the catalyst is not destroyed at the end of substrate conversion, making this catalytic system based on a "" green oxidant ÏÏ and a low cost catalyst very attractive.

A further step in the development of the FePcSÈH 2 O 2 system would be to prepare a phthalocyanine complex that would exist in a monomeric form in water solution without addition of miscible organic solvents and to support this catalyst on materials compatible with water treatments. We are currently working in this direction.

Experimental Materials

The conversion of TCP was monitored by HPLC (Waters 510 pump, Waters 486 detector) equipped with a l-Bondapak C18 column, a methanol : water mixture (7 : 3, v/v) as the eluent at 1m Lm i n ~1 and detection at 294 nm. The UV-visible absorption spectra were recorded on a Hewlett Packard 8452 A spectrophotometer. 1H NMR spectra were obtained on a Bruker AM 250 MHz spectrometer. All solvents used were of analyti-cal grade. 2,4,6-Trichlorophenol was purchased from Janssen. Hydrogen peroxide was obtained from Acros as a 35 wt.% solution. 3-Cyanobenzoic acid was purchased from Aldrich. The concentrations of Cl~were determined by the mercuric thiocyanate method.23

Preparation of the iron tetrasulfophthalocyanine FePcS

Iron tetrasulfophthalocyanine (FePcS) was prepared according to the method of Weber and Busch. 19 We found that the yield is highly dependent on the addition rate of the solid reagent mixture to the hot nitrobenzene solution. Thus the well-ground mixture of the monosodium salt of 4sulfophthalic acid (17.28

Treatment of FePcS in the presence of H 2 18O

Two milligrams of FePcS were dissolved in 200 lLo fH 2 18O (97 atom%, Eurisotop, Gif-sur-Yvette) and 2.3 lLo f4 8 % HBr were added (Ðnal HBr concentration of 0.1 M). The resulting mixture was stirred for 1 h. Then HBr was neutralized with 2.3 lL of 8.84 M KOH and the reaction mixture was again stirred for 1 h. The sample was dried in vacuum. The IR spectrum of a KBr pellet showed the same bands as the initial FePcS material except that the 833 cm~1 band shifted to 839 cm~1.

Oxidation of TCP by FePcS and H 2

O 2 Di †erent concentrations of FePcS (Table 1). The reaction mixture of 2 mL contained 20 lmol of 2,4,6-trichlorophenol (500 lL of a 40 mM stock solution in acetonitrile), phosphate bu †er (1 mL of a 500 mM stock solution at pH 7) and di †erent catalyst concentrations : 740, 200, 100, 50, 20 nmol of FePcS for a 3.7, 1.0, 0.5, 0.25, 0.1% catalyst-to-substrate ratio (i.e., 500 lL of a 1.48, 0.4, 0.2, 0.1, 0.04 mM stock solution of FePcS in water, respectively).

( Quantitative analysis of the oxidation products by NMR (Table 2). The NMR analysis of the oxidation products was performed as for runs 1, 2 and runs 6, 7 of Table 1 ; the scale was 50 times larger (100 mL reaction volume). Acetonitrile and water were evaporated under vacuum at 50 ¡C and the residue was dried for 1 h at room temperature. Seven milliliters of 1 M HCl saturated with NaCl were added to the residue ; the solution was adjusted to pH 2 by dropwise addition of 12 M HCl. The products were extracted with diethyl ether (8 ] 60 mL). After evaporation of ether, the dark brown residue was dried under vacuum for 2 h at room temperature and dissolved in deuterated dimethyl sulfoxide for NMR analysis. We added 6 lL of (75 lmol) as an internal CHCl 3 standard to quantify the oxidation products. The total yields of coupling products were based on two protons per aromatic ring.

Di †erent pH values. The reaction mixture of 2 mL contained 20 lmol of 2,4,6-trichlorophenol (500 lLo fa4 0m M stock solution in acetonitrile), 0.2 lmol of FePcS (500 lLo fa 0.4 mM stock solution in water) and 500 lmol of bu †er (1 mL of the respective stock bu †er solution : pH 4 (0.25 M citric acidÈ0.5 M disodium hydrogen phosphate) ; pH 5, 6, 7, 8 (0.5 M phosphate bu †er) ; pH 9 (0.5 M boric acidÈsodium hydroxide).

(5 ] 20 lL of a 3.5% solution in H

2 O 2 H 2 O 2
water) was added to the reaction mixture and stirred at 18 ¡C.

Variation of the acetonitrile : water ratio (Table 3). The reaction mixture of 10 mL contained 10 lmol of 2,4,6-trichlorophenol (250 lL of a 40 mM stock solution in acetonitrile), 0.37 lmol FePcS (250 lL of a 1.48 mM aqueous stock solution) and 500 lmol of phosphate bu †er (1 mL of a 500 mM stock solution at pH 7). This solution was adjusted with the required volumes of acetonitrile and water to obtain the desired ratio (e.g., 4.75 mL of acetonitrile and 3.75 mL of water to obtain an acetonitrile : water ratio equal to 1 : 1).

( In the presence of di †erent co-solvents (Table 4). The concentrations used were the same as given above for Table 3. The stock solutions of TCP were prepared in the respective co-solvents. To obtain a co-solvent : water ratio of 1 : 3, the reaction mixture was adjusted with 2.25 mL of a co-solvent (acetone, acetonitrile, ethanol, formamide) and 6.25 mL of water.

Titration of FePcS

With di †erent co-solvents. A solvent mixture of 730 lLo f water and 250 lL of co-solvent (e.g., acetone, acetonitrile, ethanol or formamide) was prepared in a cuvette with a volume of 1 mL. Twenty microliters of a 1.48 mM stock solution of FePcS in water were added and the mixture was quickly shaken. The Ðrst UV-vis spectrum was recorded immediately ; further spectra were recorded every 2 min.

With TCP. One milliliter of a 20 lM FePcS solution in water was titrated every 5 min with 10 lLo fa4 0m MT C P solution in acetonitrile. The UV-vis spectra were recorded 4 min after every addition. To control the e †ect of acetonitrile, the TCP stock solution was replaced by acetonitrile.

With acetonitrile. Di †erent samples of acetonitrileÈwater mixtures with 0, 5,10,15,20,25,30 3 (i.e., 3.7% FePcS vs. TCP and 50% acetonitrile). The preparation of the reaction mixture is the same as mentioned above in the experimental part of Table 3 (the total volume being equal to 10 mL). New 10 lmol portions of 2,4,6-trichlorophenol (250 lL of a 40 mM stock solution in acetonitrile) were added at 24 and 49 min. In the case of a one-portion addition of corresponding to Fig. 6

Fig. 1

 1 Fig. 1 Structure of the iron complex of tetrasulfophthalocyanine (FePcS)

Sequential addition of H 2 O 2 A

 2 typical reaction mixture (Ðnal volume of 2 mL) contained the following Ðnal concentrations : 10~2 M of substrate (corresponding to 2000 ppm of TCP), 3.7 ] 10~4 or 10~4 M of catalyst (corresponding to 3.7 or 1% [FePcS]-to-[TCP] ratios, respectively) and 5 ] 10~2 M of oxidant All (H 2 O 2

Fig. 2

 2 Fig. 2 InÑuence of the reaction mixture pH on the oxidation of 2,4, 6-trichlorophenol (TCP) by the system FePcSÈH 2 O 2

O 2 ]

 2 \ 5 ratio \ 1:3. b Solvent parameter developed by Dimroth and Reichardt,9a see ref. 9(b) for a recent review article on solvent properties. c We used 2.5% acetonitrile in this reaction mixture to completely solubilize TCP.

Fig. 3

 3 Fig. 3 ModiÐcation of the UV-vis spectrum of FePcS in water by addition of acetonitrile

Fig. 4

 4 Fig. 4 ModiÐcation of the UV-vis spectrum of FePcS in water by addition of an HCl solution

Fig. 5

 5 Fig. 5 ModiÐcation of the UV-vis spectrum of an acidic aqueous solution of FePcS by addition of an NaOH solution

Fig. 6

 6 Fig. 6 Conversion of TCP in three consecutive oxidation reactions by in the same batch ; (a) one-portion addition of the FePcSÈH 2 O 2 oxidant, (b) Ðve-aliquot addition

  ] 20 lLo r1] 100 lL H 2 O 2 of a 3.5% solution in water, which was prepared by ten H 2 O 2 fold dilution of a commercial 35 wt.% solution) was H 2 O 2 added to the reaction medium and stirred at 18 ¡C. Aliquots of the reaction mixture were taken at deÐned times, diluted with an acetonitrileÈwater mixture to stop the oxidation reaction and analyzed by HPLC.

Table 1

 1 Oxidation of 2,4,6-trichlorophenol by FePcSÈH 2 O 2 Cl~ions released per converted TCP molecule. n.d. \ not determined. b Number of catalytic cycles. For very fast reactions (run 1 and run 6), the number of catalytic cycles cannot be compared with the other runs, since full conversion was achieved before 14 min. c Based on 14 min, the turnover rate corresponds to 31 cycles min~1.

		Catalyst : TCP	TCP
		substrate conversion	conversion	Dechlorinationa
	Run ratio (%) at 14 min (%)b at 60 min (%) at 60 min
	1	3.7	100 (27)	100		1.9
	2	1.0	100 (100)	100		2.0
	3	0.5	83 (166)	100		1.8
	4	0.25	64 (256)	84		n.d.
	5	0.1	44 (440)c	50		n.d.
	6	3.7	100 (27)	100		1.7
	7	1.0	38 (38)	38		n.d.
	Runs 1È5 : sequential addition of portion addition of H 2 O 2 a	H 2 O 2	(5 aliquots), runs 6È7 : one-

Table 2

 2 Quantitative analysis of oxidation productsa

		Chloromaleic Chlorofumaric Maleic Fumaric Coupling
	Run acid 1	acid 2	acid 3 acid 4	products
	1b	10	\1	\1	\12 0
	2c	21	3	1	2	24
	3c	13	2	1	2	37
	4c	741	2	2 5
	Run 1 : 1% FePcS, one-portion addition of 5 aliquots of run 3 : 3.7% FePcS, one-portion addition of run 2 : 1% FePcS, H 2 ; O 2 H 2 ; O 2 run 4 : 3.7% FePcS, 5 aliquots of H 2 O 2 ;H 2 O 2

Table 3

 3 Oxidation of TCP by

	InÑuence of the acetonitrile : bu †ered water ratio over the substrate conversiona FePcSÈH 2 . O 2
		Acetonitrile
		content	TCP conversion	Dechlorinationb
	Run	(%)	at 60 min (%)	at 60 min
	1	2.5	13	0.2 (1.5)
	1c	2.5	17	n.d.
	2	10	54	1.2 (2.2)
	3	25	96	2.0 (2.1)
	4	50	100	2.4 (2.4)

a [TCP] \ 1 mM, [FePcS] \ 0.037 mM, [phosphate bu †er] \ 50 mM (pH 7), mM, one-portion addition. b Cl~ions rel-[H 2 O 2 ] \ 5 eased per initial TCP molecule ; the data in parentheses correspond to the number of Cl~ions released per converted TCP molecule. c Addition of 3-cyanobenzoic acid ; 3-cyanobenzoic acid : FePcS ratio of 100 : 1.

Table 4

 4 Solvent inÑuence on TCP conversiona

		Conversion of TCP
	Co-solvent Acetone	at 60 min (%) 100	(30)b Polarity E T 42.2
	Acetonitrile	96	45.6
	Ethanol	86	51.9
	Formamide	69	56.6
	Waterc	13	63.1

a [TCP] \ 1 mM, [FePcS] \ 0.037 mM, [phosphate bu †er] \ 50 mM (pH 7), mM, one-portion addition, solvent : bu †er [H 2

  or 35% of acetonitrile containing 20 lmol of FePcS were prepared and the spectra recorded 10 min after preparation. HCl : water ratio to 10 : 890, 15 : 885, 20 : 880, 25 : 875, 30 : 870, 50 : 850. The spectra were recorded 15 min after preparation.Titration of an acidic solution of FePcS with NaOH. One hundred microliters of a 0.2 mM FePcS stock solution in water were added to a solution of 850 lL after 15 min. The acidiÐed FePcS solution was then titrated every 5 min with 10 lL of 0.05 M NaOH solution. The UV-vis spectra were recorded 4 min after every addition. Batch experiments were performed under the conditions used for run 4 in Table

	With HCl, 0.2 mM FePcS stock solution in water were added to a or One hundred microliters of a H 2 SO 4 H 3 PO 4 . mixture of 5 lL of 0.05 M HCl or and 895 (H 2 SO 4 H 3 PO 4 ) lL of To obtain di †erent acid concentrations, we H 2 O. changed the 0.05 M and 50 lLo f H 2 O 0.05 M HCl (or in a cuvette of 1 mL and the spec-H 2 SO 4 ) trum was recorded Lifetime of the FePcS catalyst in the	H 2	O 2	oxidation of TCP

Acknowledgements thanks the European Community for a PhD fellowship A.H. (TMR grant No. ERBFMBICT 950030). A.S. is indebted to ELF-Atochem for a postdoctoral fellowship. We are grateful to Christian Forquy and Laurent Fraisse (ELF-Atochem, Lacq) for helpful discussions. Financial support was provided by CNRS, Elf-Atochem and EC (Training and Mobility of Researchers Programme).


Staëlle Makamte

Amira Jabrani

Annick Paquelin

Anne Plessis

Mathieu Sanial

Aurélien Thureau

Olga Rudenko

Francesco Oteri

Marc Baaden

Valérie Biou
email: valerie.biou@ibpc.fr

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study

Keywords: Hedgehog signal, intrinsically disordered protein, small-angle X-ray scattering

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The conserved Hedgehog (Hh) signalling pathway is essential to specify cell and tissue fate during embryogenesis and later to regulate stem cell homeostasis [START_REF] Jiang | Hedgehog Signaling in Development and Cancer[END_REF]. Discovered in drosophila, where it ensures proper polarisation of the larva segments [START_REF] Nusslein-Volhard | Mutations affecting segment number and polarity in Drosophila[END_REF], the Hh pathway has since been identified in many animals where it controls multiple patterning events, and has rapidly emerged as a key player in oncogenesis by controlling tumour promotion or/and progression [START_REF] Pak | Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy[END_REF]. In the absence of Hh signal the G-Protein Coupled Receptor-related Smoothened (Smo) activity is inhibited by the Hh receptor Patched (Ptc). Binding of Hh releases this negative effect, triggering the activation of Smo, probably by favouring its association with intramembrane cholesterol (for review see [START_REF] Hu | The interplay of Patched, Smoothened and cholesterol in Hedgehog signaling[END_REF]). Smo activation also involves its relocalisation from intracellular vesicles to the plasma membrane in fly, and its rapid translocation from the base to the tip of the primary cilium in mammals [START_REF] Hooper | The Drosophila patched gene encodes a putative membrane protein required for segmental patterning[END_REF][START_REF] Ingham | Role of the Drosophila patched gene in positional signalling[END_REF] (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted June 14, 2021. ; https://doi.org/10.1101/2021.06.14.447554 doi: bioRxiv preprint SuFu disordered region is disclosed by solution studies 3 [START_REF] Marigo | Biochemical evidence that Patched is the Hedgehog receptor[END_REF][START_REF] Zhang | Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched[END_REF]. Via its interactions with a cytoplasmic protein complex, Smo activates kinases that change the phosphorylation state of several proteins of this complex and remodel their interactions. As a result, the transcription factor(s) of the Ci/Gli family are activated and transported into the nucleus where they in turn activate the expression of specific target genes, thus changing the cell fate. The molecular backbone of the pathway is conserved but it becomes more complex from flies to mammals. For example in vertebrates, three transcription factors are involved: Gli 1, 2 and 3; their activation takes place at the primary cilium and depends on intraflagellar trafficking and results in their translocation into the nucleus [START_REF] Huangfu | Hedgehog signalling in the mouse requires intraflagellar transport proteins[END_REF].

The proper function of the Hh pathway at the right time and place is so crucial that several levels of negative regulation exist to prevent inappropriate Hh activation. This includes the products of two tumour suppressor genes: Ptc at the cell surface and Suppressor of Fused (SuFu) inside the cell. Removal of SuFu has very different consequences in drosophila and mammals. In drosophila, the phenotype of sufu removal is only visible in the context of the absence of fused kinase [START_REF] Preat | Characterization of Suppressor of fused, a Complete Suppressor of the fused Segment Polarity Gene of Drosophila melanogaster[END_REF]. Conversely, SuFu KO mice die in utero and exhibit heart, brain and CNS defects due to a Hh constitutional activation [START_REF] Cooper | Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused[END_REF][START_REF] Svard | Genetic Elimination of Suppressor of Fused Reveals an Essential Repressor Function in the Mammalian Hedgehog Signaling Pathway[END_REF].

Discovered in drosophila [START_REF] Preat | Characterization of Suppressor of fused, a Complete Suppressor of the fused Segment Polarity Gene of Drosophila melanogaster[END_REF], where it interacts with a microtubule-associated complex including kinase Fused [START_REF] Monnier | Suppressor of fused links Fused and Cubitus interruptus on the Hedgehog signalling pathway[END_REF], SuFu is a 55 kDa protein that plays an inhibitory role via the sequestration of full-length Ci in the cytoplasm. In response to Hh, this negative effect of SuFu is suppressed by the kinase Fused. SuFu then accompanies Ci into the nucleus [START_REF] Sisson | Regulation of Ci and Su(fu) nuclear import in Drosophila[END_REF]. In zebrafish, SuFu regulates Gli transfer to the primary cilium [START_REF] Maurya | Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo[END_REF] and SuFu morpholino knock down affects eye, ear and muscle development, identifying SuFu as a medium-level inhibitor of the Hh pathway [START_REF] Koudijs | The Zebrafish Mutants dre, uki, and lep Encode Negative Regulators of the Hedgehog Signaling Pathway[END_REF]. In mammals, SuFu is essential for the proper differentiation of many cell types including neural cells [START_REF] Yabut | Suppressor of Fused Is Critical for Maintenance of Neuronal Progenitor Identity during Corticogenesis[END_REF] and it plays a role in white/brown adipocyte determination and obesity [START_REF] Pospisilik | Drosophila Genome-wide Obesity Screen Reveals Hedgehog as a Determinant of Brown versus White Adipose Cell Fate[END_REF]. Here also, SuFu acts upstream of the Gli transcription factors, controlling their fate. It was thus shown to sequester full-length Gli 1 and 2 transcription factors in the cytoplasm in the absence of Hh. Moreover, SuFu also plays nuclear roles with the repressor forms of Gli as it was shown to recruit the SAP18-mSin3 repressor complex to the Gli binding sites [START_REF] Paces-Fessy | The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins[END_REF]. Hh binding to Ptc represses this negative effect, allowing the Gli proteinsaccompanied by SuFu-to enter the nucleus. The present state of research describes SuFu as a hub, interacting with many partner proteins, and the interactions are remodelled when Hh is activated, mostly

SuFu disordered region is disclosed by solution studies

4 via post-transcriptional modifications, such as phosphorylation and ubiquitylation, of SuFu [START_REF] Chen | Dual phosphorylation of Suppressor of fused by PKA and GSK3beta regulates its stability and localization in the primary cilium[END_REF][START_REF] Raducu | SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development[END_REF][START_REF] Wang | Nek2A phosphorylates and stabilizes SuFu: A new strategy of Gli2/Hedgehog signaling regulatory mechanism[END_REF].

Figure S1 shows an amino acid sequence alignment of SuFu from three species belonging to different phyla: Homo sapiens (hSuFu), Danio rerio (zebrafish) (zSuFu) and Drosophila melanogaster (dSuFu). The crystal structures of hSuFu and dSuFu [START_REF] Cherry | Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation[END_REF][START_REF] Zhang | Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci[END_REF] harbour two ordered domains corresponding to the regions framed in blue and pink boxes in the Figure S1, with respectively 41% and 24% identity between hSuFu, zSuFu and dSuFu. The N-terminal domain is also found in prokaryotic proteins, amongst which antitoxin systems, another process involving protein-protein interactions [START_REF] Zhang | A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems[END_REF], and the C-terminal domain is specific to SuFu proteins. In addition to those two domains known from the crystal structures, Figure S1 shows that SuFu amino acid sequence contains two additional regions predicted to be intrinsically disordered by the PONDR server (http://www.pondr.com). The N-terminus disordered region IDR1 (in a dotted grey box) and the second region IDR2 (in a dotted black box) stemming from the C-terminal domains are the sequences that harbour the most striking differences between drosophila and vertebrates with 6% identical residues.

The N-terminal domain has a 7-stranded twisted beta sheet followed by a 3-helix bundle, and spans residues 30-263 (as numbered in hSuFu). The C-terminal domain has two beta sheets with 6 and 4 strands respectively and two helices; it consists of residues 267-280 and 356-457. The structure of free hSuFu (pdb code 4KM9/4BL8) is extended and the two domains become close to each other when a peptide from Gli is bound to hSuFu (pdb code 4KMD/4BLB), showing a potential tendency of the two domains to move with respect to each other. The drosophila SuFu crystal structure pdb code 4KMA spans residues 13-259 for the N-terminal domain and 265-454 for the C-terminal domain. Both domains have very similar folds to the human protein, but their relative orientations are different. The IDR2 region, which is disordered in the human structure, has only five disordered amino acids in the drosophila crystal structure. The rest of the sequence corresponding to IDR2 is structured and caps the C-terminal domain, but it has no regular secondary structure.

Due to the absence of structural information for full-length vertebrate SuFu, we used circular dichroism (CD) and small angle X-ray scattering (SAXS) to investigate the solution structures of SuFu proteins from Drosophila melanogaster, Danio rerio and Homo sapiens. We also studied hSuFuD30, a truncated mutant of hSuFu devoid of its IDR1 that allowed us to better analyse the effect of IDR2 alone. We found that the solution structure of the drosophila protein is very similar to the crystal structure, and

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted June 14, 2021. ; https://doi.org/10.1101/2021.06.14.447554 doi: bioRxiv preprint SuFu disordered region is disclosed by solution studies 5 investigated its breathing movements by normal mode analysis. Human and zebrafish SuFu show similar dimensions and exhibit an N-terminal IDR1 region that seems to remain close to the N-terminal domain, while the IDR2 loop in the C-terminal domain spans a large volume and can thus fetch its partner protein a long way from its core. Our present studies show that, even though their crystal structures are quite similar, the solution structures of vertebrate and drosophila SuFu are rather different and we discuss the specificity of IDR2 in the context of SuFu interaction with partner proteins.

Experimental procedures

1) Protein expression and purification.

dSuFu and hSuFu were cloned and expressed as previously described [START_REF] Jabrani | Biophysical characterisation of the novel zinc binding property in Suppressor of Fused[END_REF].

The cDNA encoding dSuFu was cloned in the pDEST17 (Invitrogen) expression vector according to standard Gateway TM protocols. The resulting plasmid encoded an N-terminal hexahistidine tag and a TEV protease cleavage site before the gene of interest. E. coli strain C41(DE3) [START_REF] Miroux | Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels[END_REF] were grown in 2YT medium at 37 °C until the optical density reached 0.6. Protein expression was induced with 0.2 mM isopropyl β-thiogalactoside overnight at 20 °C. Bacterial cells were recovered by centrifugation at 5000 g for 15 min and frozen at -80°C. Similar procedures were used for the cDNAs encoding hSuFu and zSuFu respectively, which were cloned in a pACYDuet vector. hSuFuD30 construct was produced by truncating the nucleotides coding for the first 30 aminoacids of hSuFu. Cells were lysed in 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 10% glycerol using a Constant Cell disruptor at 2.3 kbar, then centrifuged for 1 hour at 9000g. The supernatant was loaded onto a Nickel affinity column (Roche), washed in the loading buffer and eluted in a buffer containing 200mM imidazole. The eluate was collected and the buffer exchanged against 10 mM HEPES pH 7.5, 100 mM NaCl, 10 % glycerol, 2.5 mM MgCl2 and 2 mM β-mercaptoethanol on a concentrator. Then the protein solution was incubated with Tobacco Etch Virus protease for 24 hours at 10°C to cleave the 6-Histidine tag and the associated linker. The cleavage mixture was then dialysed back into the lysis buffer and incubated with Nickel affinity gel (Roche) for one hour to separate the cleaved protein from the TEV protease and tag. The flow through was then concentrated and injected on a Superdex 200 10/300 gel filtration column equilibrated with HEPES 50mM pH 7.5, NaCl 200mM, glycerol 10%, Dithiothreitol 5mM or bis-tris 50 mM pH 5.5, NaCl 50mM, glycerol 10% and loaded onto a HiTrapQ column, washed and eluted in a NaCl gradient. The fractions corresponding to pure SuFu protein were concentrated and used for future studies.

CD studies were performed in the protein in two different buffers: A) HEPES HCl 50mM pH 7.5, NaCl 200mM, glycerol 10%, Dithiothreitol 2mM or B) bis-tris 50 mM pH 5.5, NaCl 200mM, glycerol 10%. SAXS studies were performed in buffer A only.

2) SRCD measurements

Synchrotron radiation circular dichroism (SRCD) was measured at the DISCO beamline (SOLEIL synchrotron, Gif sur Yvette, France). All sample concentrations were measured using the absorbance at 280nm, after centrifugation and just prior to measurement, using a theoretical extinction coefficient of 59400 M -1 .cm -1 for dSuFu, 67775 M -1 .cm -1 for hSuFu and 66390 M -1 .cm -1 for zSuFu. Concentrations were found to be between 4 and 10 mg/ml.

The beamline monochromator was calibrated using a camphorsulfonic acid solution prior to measurements. One microlitre of sample was deposited on one face of a calcium fluoride circular cuvette (Hellma), then the second face was carefully positioned and the cuvette closed by capillary force.

Interferometry measurement showed the optical path to be 2.3 µm. The cuvette was placed in an airtight, metal sample holder that was positioned in the beamline Peltier temperature-controlled chamber, allowing for quick temperature changes with very little evaporation. Spectra were measured between 280 and 170 nm, each final spectrum being averaged from three repeated measurements. Thermal unfolding measurements were performed by averaging three spectra collected in steps of 5°C between 10 and 90 °C. Buffer spectra were taken in the same conditions for subtraction from the protein spectra. Spectra were then processed for buffer subtraction and scaling using the CDTools program [START_REF] Lees | CDtool-an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving[END_REF]. The melting temperature was evaluated by fitting the curve displaying the CD at 208nm with a sigmoidal function in Kaleidagraph (Synergy Software), as a function of temperature.

3) Small angle X-ray scattering measurements

The SAXS measurements were done at the SWING beamline (Thureau et al., submitted.) at the SOLEIL synchrotron (Gif sur Yvette, France). The sample was injected at a flux of 0.3 ml/minute into an Agilent Bio SEC-3-300 size exclusion column pre-equilibrated in the indicated buffer and mounted on an HPLC system, prior to its analysis in the X-ray beam. The X-ray energy was 12 keV. For dSuFu, measurements were carried out with an Aviex charge coupled device detector, the system being programmed to measure 100 images of the buffer then 240 images of the protein, using a 1-second exposure time interspaced by 500 ms. For hSuFu, hSuFuD30 and zSuFu measurements, an Eiger 4M pixel detector was used. 600 images were measured with 990ms exposure and 10 ms dead time each. The sample to detector distance is listed in Table 2.

The images were processed using the FOXTROT software (https://www.synchrotronsoleil.fr/en/beamlines/swing) developed at the SOLEIL synchrotron. The curves derived from buffer measurement were averaged and subtracted from the buffer plus protein curves. Then the radius of gyration Rg was calculated using a Guinier plot on the most intense curve and extended to all scattering curves, thus producing a plot of Rg and scattered intensity, I0, for all images. Curves from hSuFu and zSuFu were processed with US-SOMO [START_REF] Brookes | US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data[END_REF] to deconvolute the contribution from oligomers. The curves obtained from dSuFu did not indicate higher order species and consequently did not need deconvolution. The subtracted frames, corresponding to the deconvoluted peak with a stable Rg, were selected to produce the protein scattering curve of scattered intensity as a function of the momentum transfer q= 4πsin(Ɵ)/λ, where 2Ɵ is the diffusion angle and λ is the wavelength. Further analysis (Guinier and Kratky plots, Radius of gyration Rg and intensity at origin I0 calculation) was performed with the ATSAS program suite [START_REF] Franke | ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions[END_REF].

Five ab initio envelope calculations were carried out using the DENSS software [START_REF] Grant | Ab initio electron density determination directly from solution scattering data[END_REF] using the RAW interface [START_REF] Hopkins | BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis[END_REF]. These were input into the DAMMAVER [START_REF] Volkov | Uniqueness of ab initio shape determination in small-angle scattering[END_REF] suite to produce an averaged protein envelope.

4) Molecular modelling of dSuFu.

The dSuFu crystal structure 4KMA was modified into an open conformation using Rosetta, [START_REF] Kaufmann | Practically Useful: What the Rosetta Protein Modeling Suite Can Do for You[END_REF] based on the hSuFu structure (pdb ID 4KM9), as a template for relative domain orientation. This model structure, and the crystal structure 4KMA for dSuFu, were used as the initial and final frame, respectively, to guide a linear morphing obtained through the program Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF].

Each of the 20 morphing frames has been minimised without any constraint using the AMBER force field as implemented in Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF]. The 20 frames have then been analysed through the program Foxs [START_REF] Schneidman-Duhovny | Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments[END_REF] in order to calculate the corresponding scattering curve and its agreement with experimental data. The frame with the curve having the lowest chi value with respect to the experimental data was retained as the experimental model.

5) Molecular modelling of hSuFu missing regions

hSuFu residue numbering is according to Uniprot entry Q9VG38. Our SAXS data was measured on fulllength hSuFu. The crystal structure of hSuFu PDB code 4KM9 was used as a starting point. It shows two ordered domains, but the first 20 aminoacids and 76 residues in the C-terminal domain are [START_REF] Weinkam | Structure-based model of allostery predicts coupling between distant sites[END_REF] was used to build missing residues and the structure was further refined using YASARA [START_REF] Krieger | YASARA View-molecular graphics for all devices-from smartphones to workstations[END_REF] to ensure a good geometry, and the chi 2 of the calculated scattering curve with respect to the experimental curve was 12.2. The model was then submitted to Dadimodo server https://dadimodo.synchrotron-soleil.fr [START_REF] Rudenko | Evolutionary refinement of the 3D structure of multidomain protein complexes from small angle X-ray scattering data[END_REF] for a flexible refinement against the SAXS data by introducing flexibility in the HDR1 and the HDR2. Five models were generated and ranked according to the chi value. The two ordered domains: residues 30-255 for the N-terminal domain and residues 259-268 and 356-472 for the C-terminal domain were kept rigid while residues 1-29, 256-258 and 269-355 were left flexible. Another test was performed with a closed model starting from structure 4KMD on which the HDR1 and HDR2 regions obtained from the Yasara-refined hSuFu structure were grafted.

6) Molecular modelling of zSuFu

zSuFu was modelled by homology using the Phyre2 server [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF]. The best-ranked model was based on hSuFu structure PDB code 4KMH. Then missing residues in the IDR2 loop (73 residues) were added with the AllosModFoxs program [START_REF] Weinkam | Structure-based model of allostery predicts coupling between distant sites[END_REF], yielding Chi2 values between 22 and 80. The best model was processed with Dadimodo, with residues 18 -255 for the N-terminal and residues 259 -268 and 356 -472 for the C-terminal domain being kept rigid while residues 1-19, 256-258 and 279-355 were left flexible. Five models were generated and ranked according to the chi value.

Results

1) Synchrotron radiation circular dichroism

(SRCD) shows that truncated hSuFuD30 is well structured, but less stable than hSuFu.

We measured SRCD spectra between 10 and 90°C for dSuFu, hSuFu, hSuFuD30 and zSuFu in two buffers: A) HEPES HCl 50mM pH 7.5, NaCl 200mM, glycerol 10%, Dithiothreitol 2mM or B) bis-tris 50 mM pH 5.5, NaCl 200mM, glycerol 10%. All proteins showed spectra typical of structured proteins at low temperature, and the spectra became shallower when the temperature was increased, as exemplified in Figure 1A for hSuFu. The spectra cross at an isobestic point characteristic of a two-state transition. Table 1 shows the unfolding temperatures evaluated from the variation of CD at 208 nm as a function of temperature fitted to a sigmoid function with Kaleidagraph. For hSuFu and hSuFuD30 the Tm was measured at pH 7.5 with DTT and at pH 5.5 without DTT. Both forms of hSuFu showed an increase in their Tm in the first condition, but hSuFu is more stabilised by pH 7.5 and DTT than hSuFuD30. Figure 1B shows the evaluation of secondary structure content using the BestSel program [START_REF] Micsonai | Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy[END_REF] at 10 and 60°C. At low temperature, our SuFu constructs contain between 40 and 50% of regular secondary structure and the other residues are in random coil. All studied SuFu constructs become richer in beta strand and lose helical structure when the temperature is raised from 10 to 60°C. This trend is observed in other helix-containing proteins such as myoglobin [START_REF] Fändrich | Myoglobin forms amyloid fibrils by association of unfolded polypeptide segments[END_REF].

2) SEC-SAXS and molecular modelling show that dSuFu is an elongated monomer in solution

with a closed conformation.

The SAXS measurements were conducted at the SWING beamline at the SOLEIL synchrotron, using a High Pressure Liquid Chromatography injection system (Thureau et al., submitted). In view of the higher stability at pH 7.5 with DTT detected with SRCD, we decided to perform our SAXS studies in those conditions. The sample migrated on the size exclusion column, and produced a peak with a homogeneous Radius of gyration (Rg) (Supplementary Figure S2) which allows us to say that the resulting SAXS curve (Figure 2A, green curve) corresponds to a monodispere particule. The estimation of the Rg using the Guinier approximation indicates an absence of aggregates and gives an Rg value of 27.2 Å. The Shannon sampling formalism indicates that the data is accurate at least up to a scattering vector of 0.4 Å -1 . Table 2 gives a list of all measured values. The dimensionless Kratky plot (Figure 2B, grey curve) has a bell shape and does not rise at high angles, which denotes that dSuFu has a mostly compact structure with very little disorder. The distance distribution function, P(r), calculated using an indirect Fourier transform, yielded a well-behaved curve with a maximum particle dimension, Dmax, of 90Å and (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted June 14, 2021. ; https://doi.org/10.1101/2021.06.14.447554 doi: bioRxiv preprint SuFu disordered region is disclosed by solution studies 10 a maximum of the curve at r=35Å (Figure 2C, grey curve). The calculated mass of 51 kDa, obtained with Primus and Vc calculation [START_REF] Rambo | Accurate assessment of mass, models and resolution by smallangle scattering[END_REF], and the Rg values of 27.2 Å and 27.3 Å calculated from the Guinier and P(r) functions, respectively confirm that the particle is a monomer and is monodisperse in solution. The average envelope resulting from ab initio calculations encloses most of the crystal structure of dSuFu 4KMA with the exception of the 274-282 loop, which is mobile in the crystal structure, as shown by the thinner radius of the ribbon representation (Figure 3A). This figure highlights the IDR2 region that lines the C-terminal domain (residues 308-340 on the bottom left of the figure). It is ordered but it has no regular secondary structure.

The scattering curve calculated from the 4KMA crystal structure fits rather well with the SAXS spectrum, however the chi value is 7.3 and there is some disagreement between 0.15 and 0.25 Å -1 q value (Figure 3B, blue experimental curve, compare with 4KMA curve). We performed a modelling study in order to better characterise possible movements in dSuFu and find a model that fits the SAXS data better.

The following two-step procedure was applied. First, we analysed the possible deformations in the SuFu structures. Wide rearrangements likely accounting for a conformational change in the dSuFu crystal structure have been inferred through hinge detection and compared with hSuFu crystal structures. For each structure, the Hingeprot program [START_REF] Emekli | HingeProt: automated prediction of hinges in protein structures[END_REF] shows the two slowest modes of movement.

For the 4KMA crystal structure, the slowest mode describes the bending of the two domains around residues Q252-D253 (Fig 3C). The second mode is described by three rigid blocks, namely N-terminal (M1 to C151 and Q177 to C194), central (Y152 to A176, and Q195 to A263) and C-terminal (G264 to E454), coloured in red, white and blue, respectively, in Figure 3D. The N-and C-terminal domains also describe a bending motion, but here the domains do not pivot on the same hinge because of the rigidity of the central block (see magenta arrows in Fig. 3D). Additionally, a wide fluctuation is observed in the loop A105-P128.

In the next step, we sampled the movements of the two domains along the direction of the bending mode in order to check if a better agreement with our SAXS results can be obtained by modelling an intermediate on this bending pathway. Two extreme open and closed conformations were generated for dSuFu, using the open (4KM9) and closed (4KMD) conformations of hSuFu as templates. The conformational change from one state to the other was modelled using linear morphing, and twenty additional intermediate conformations were generated, each of which was regularized through energy minimization, (see materials and methods section). The SAXS profiles were then simulated for each of these energy-minimised conformations and compared to the experimental profile. Chi values calculated up to a q value of 0.4 Å -1 are as follows: open model, 5.0; closed model, 7.7; 4KM9, 6.5; 4KMA, 7.3; best model, 3.8. The intermediate model whose diffusion curve was closest to the SAXS data was selected as the best solution (Figure 3B purple curve). This best model and the 4KMA structure of dSuFu have an RMSD of 0.7 Å for all backbone atoms. The differences mostly concern the C-terminal domain whose RMSD, with respect to the crystallized counterpart, is 0.9Å versus 0.4 Å for the N-terminal domain. Although the secondary structure is globally conserved between the proposed solution model and the crystal structure, the residues ranging from A294 -D332 in the latter are more closely packed in the domain core. The higher flexibility of these residues, containing the unresolved loop D296 -K308, accounts for a widening of the cleft between the two domains as shown by the minimum distance between the two domains (5 Å between G133-Q302 C-alpha of 4KMA structure versus 11 Å of the equivalent G147-R309 in the model). Finally, we used Dadimodo server to refine models for dSuFu starting from our best model and defined a flexible linker between the two domains consisting of residues 256-265, leaving the C-terminal domain free to move with respect to the N-terminal domain. The five resulting structures are highly similar to each other and have an average chi 2 of 1.81±0.04 (Figure 4 A-C and Table 3).

In summary, SAXS measurements show that the solution structure of dSuFu is slightly more open than the 4KMA dSuFu crystal structure, but is nevertheless different from the free human protein 4KM9. In addition, hinge detection shows that the clamp mode likely characterises SuFu motion. Finally, the region corresponding to IDR2 caps the C-terminal domain and is not disordered in the drosophila protein.

Next, we decided to study the solution structure of vertebrate SuFu.

3) SEC-SAXS shows that the IDR2 disordered region spans a large volume for hSuFu and zSuFu.

We chose to compare proteins from vertebrates that are far from each other in evolution: zebra fish and human. hSuFu shows 36% identity with dSuFu and 81% identity with zSuFu, while zSuFu has 35% identity with dSuFu (Fig S1). We measured SEC-SAXS data on purified hSuFu, hSuFuD30 and zSuFu.

A reducing agent was necessary to remove a dimer-like species at pH7.5, coherent with the neutral pKa of cysteine protonation. We suspect that, at high pH, a disulphide bridge is formed involving Cysteine 292 located in the IDR2 (data not shown).

The scattering curves are shown on Figure 2A and the Radii of gyration (Rg) values are listed in Table 2. The Shannon sampling formalism shows that all curves can be used at least up to 0.4Å -1 [START_REF] Shannon | the mathematical theory of communication[END_REF]. Associated to the dimensionless Kratky curves shown on Figure 2B, our data show that hSuFu (dark blue curve), hSuFuD30 (black curve) and zSuFu (tuquoise curve) span a much larger volume than dSuFu (green curve). Indeed, for comparable molecular weights, the radius of gyration is 25% higher in hSuFu and zSuFu than for dSuFu. This is in agreement with the presence of the IDR2 in zebrafish and human, which is ordered in drosophila. A comparison between the results of full-length hSuFu and truncated hSuFuD30 indicates the contribution of the IDR2 to the total volume. The largest interatomic distance Dmax is 140Å, only slightly decreased by the truncation, showing that the IDR2 contributes to the majority of the Dmax difference between drosophila (90Å) and human (150Å).

We went on to generate molecular models of SuFu whose calculated scattering curves fit the experimental curve well.

For full-length hSuFu, as explained in the Methods section, we started from free hSuFu 4KM9 and built the missing regions using AllosModFoxS [START_REF] Weinkam | Structure-based model of allostery predicts coupling between distant sites[END_REF]. We ran YASARA [START_REF] Krieger | YASARA View-molecular graphics for all devices-from smartphones to workstations[END_REF] to regularise the geometry and then used the Dadimodo software [START_REF] Rudenko | Evolutionary refinement of the 3D structure of multidomain protein complexes from small angle X-ray scattering data[END_REF] to find models that fit the experimental curve better. In addition, as dSuFu domains are more closed even in the absence of the Gli peptide ligand, we wanted to check if hSuFu could harbor a closed conformation in solution. We thus generated a second starting model using the Gli peptide-bound conformation of hSuFu 4KMD on which the missing regions were attached similarly to the extended model.

For the elongated models, the average chi 2 value is 1.73 ±0.01 (Table 3). Figure 4D shows the superposition of the five models calculated. The N-terminal and C-terminal domains are in open conformation with respect to each other and the N-terminal disordered residues seem to have a rather compact structure. This is different from the IDR2 loops that protrude a long way into the solvent, conferring a very elongated structure to hSuFu. A series of ab initio envelopes were calculated with the DENSS program and averaged with the DAMAVER suite. The elongated envelope is in agreement with the hypothesis that IDR2 spans a large volume in solution (figures 4E). However, we consider that the most significant validation is the fit between the model and the experimental scattering curves (figure 4F). In order to further assess the conformation of the IDR2 domains we ran Dadimodo starting from the second model that had a closed conformation of the two domains. The resulting models have an average chi 2 of 2.80±0.3. Their fit is significantly less good than those of the elongated models, showing that this set of conformations is less probable in solution. However, we cannot exclude that the protein spends part of its time in this closed conformation.

SuFu disordered region is disclosed by solution studies 13

In summary, hSuFu has a very elongated conformation in solution, compatible with an open conformation of the two ordered domains and an extended IDR.

We then compared our results with those obtained with N-terminal truncated hSuFuD30 using the elongated YASARA model as a starting point. Dadimodo provided five models with an average chi 2 of 1.77±0.017 (Table 3). As shown on Figure 4G, the models are slightly more variable than those of fulllength hSuFu. The C-terminal domain moves more with respect to the N-terminal one and the IDR2 loop has different relative orientations. This observation may be due to a noisier scattering curve (Figures 2A and4H), but probably unrelated to its lower stability, as the Tm's obtained with SRCD (see above) are comparable. Finally, The DENSS envelope has a shape similar to that of full-length hSuFu (figure 4I), in agreement with a 9% lower Dmax value.

The zebrafish SAXS data confirm that the solution structure of zSuFu is very similar to that of human SuFu, both in terms of dimensions (Table 2), models and ab initio envelope (Figure 4J-K), and significantly different from those of dSuFu. The five Dadimodo refined models have chi 2 values of 1.18±0.06 (Figure 4L and Table 3) and have extended IDR2 loops similarly to hSuFu models.

In summary, our SAXS measurements provide structural data that the crystal structures do not supply.

They show that drosophila SuFu has a very different shape in solution than vertebrate SuFu. This difference is mainly due to the presence of an extended IDR2 similar in both human and zebrafish species.

Discussion and conclusion

Our solution investigation disclosed differences between drosophila and vertebrate SuFu that could not be anticipated from their respective crystal structures. Our SAXS results show that the closed conformation of dSuFu is not due to crystal packing effects but is intrinsic to dSuFu, and our modelling results indicate that this may be due to different hierarchy in the hinge motions, leading to a different equilibrium position. In addition, the sequence corresponding to the Intrinsically Disordered Region IDR2 is ordered in drosophila, thus stabilising the two domains in this closed conformation.

The hSuFu protein has an elongated monomeric structure and SAXS data allowed us to build models for the missing residues at the N-terminal and in the C-terminal domain. Those models suggest that the IDR2 forms an elongated loop that protrudes into the solvent. This is comforted by the study of a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted June 14, 2021. ; https://doi.org/10.1101/2021.06.14.447554 doi: bioRxiv preprint SuFu disordered region is disclosed by solution studies 14 truncated mutant hSuFuD30 and of zSuFu, which is 81% identical in amino acid sequence to hSuFu and whose SAXS-derived size and shape is very similar to that of hSuFu.

The IDR2 loop thus contributes strongly to the large values of Rg and Dmax of those proteins, and we investigated its amino acid composition in order to predict how the IDR2 of other species might behave.

The IDR2 is 76 and 73 residue-long in human and zebrafish, respectively.

In the sequences aligned in Figure S1, the IDR2 starts with an aromatic residue W/F (W275 in human) belonging to the first strand of the first ß-sheet of the C-terminal domain. This aromatic establishes contacts that stabilise the interaction with conserved hydrophobic residues in the second ß-sheet. The last residues in the IDR2 are the first ones of this ß-sheet and they ensure the stabilisation of the loop ends. Disorder prediction using the Prediction of Natural Disorder PONDR server (http://www.pondr.com) shows that all sequences have a disorder tendency. Figure 5A shows the PONDR plot for the three proteins studied in this work. It confirms that dSuFu has a shorter disordered segment than zSuFu and hSuFu. In fact, if we consider the recommended disorder index threshold of 0.5, the PONDR metaserver predicts a stretch of 60 disordered residues while the crystal structure of dSuFu 4KMA shows only 11 disordered residues, confirmed by our SAXS data. Only when raising the threshold to 0.9 does the PONDR result almost predict the regions disordered in the crystal. On the other hand, with a threshold of 0.6, PONDR predicts a 90-residue-long disordered stretch for the human and zebrafish sequences, which is very close to the missing region in the crystal structures of hSuFu. This is in agreement with the observation that drosophila IDR2 region is much less disordered than the vertebrate one.

The elongation of IDR2 loop in vertebrates might be associated with an increased complexity in the Hh pathway from flies to vertebrates. This is exemplified by the transcription factor Cubitus interruptus (Ci) that is unique in drosophila, while three Gli proteins are simultaneously present in mammals: Gli1, Gli2 and Gli3 [START_REF] Ruppert | The GLI-Kruppel family of human genes[END_REF], allowing fine-tuning of the gene expression pattern via other partner proteins.

The function of the IDR2 region is not well known. However, Cherry et al. have studied the effect of its removal on the Hedgehog pathway activation. They found that the IDR2 is dispensable for Gli binding and Hh pathway activation with a luciferase reporter in HEK cells. However, activation of the Hh pathway in mouse embryonic fibroblast cells was repressed in the presence of full-length hSuFu but not in the presence of IDR2-truncated hSuFu. Moreover, a chimeric human protein harbouring drosophila IDR2 failed to repress the pathway [START_REF] Cherry | Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation[END_REF]. This confirms that IDR2 of vertebrate SuFu has a role in the regulation of the Hh pathway, likely linked to post-translation modifications and nuclear trafficking. The amino acid composition of SuFu sequences as displayed in Table 4 shows that the IDR2, as compared to the overall sequence, is richer in acidic residues and, to a lesser extent, in basic and polar residues, and poorer in hydrophobic residues, as could be expected from the nature of IDRs. The "Phosphosite" data base [START_REF] Hornbeck | PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation[END_REF] indicates that ten out of sixteen post-transcriptional modifications of hSuFu occur in the 80 aminoacids of the IDR2. Six serine and threonine residues are predicted to be phophorylated and three lysines are predicted to be ubiquitylated or acetylated. Moreover, Zhang et al. showed that the mammalian SuFu harbours a nuclear export signal spanning residues 308-318 and two phosphorylatable serines 342 and 346 [START_REF] Zhang | Suppressor of Fused chaperones Gli proteins to generate transcriptional responses to Sonic Hedgehog signaling[END_REF]. As shown on Figure 5B, those residues are located in the IRD2 making them very accessible to interact with kinases and the nuclear pore complex. This property makes the IRD2 a very good candidate to interact with exportins similarly to the complex between exportin CRM1, Ran-GTP and import adapter snurportin1 (SPN1), that involves disordered regions of SPN1 in contact with CRM1 [START_REF] Monecke | Crystal Structure of the Nuclear Export Receptor CRM1 in Complex with Snurportin1 and RanGTP[END_REF]. It is known that SuFu interacts with Ci/Gli and several other partners such as SAP18, galectin3 or p66ß [START_REF] Lin | Regulation of Sufu activity by p66ß and Mycbp provides new insight into vertebrate Hedgehog signaling[END_REF][START_REF] Paces-Fessy | The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins[END_REF]. The difference between vertebrate and drosophila SuFu is further underlined by the observation that phosphorylated serines in dSuFu do not seem necessary for Hh pathway repression [START_REF] Oh | A Comparison of Ci/Gli Activity as Regulated by Sufu in Drosophila and Mammalian Hedgehog Response[END_REF].

In conclusion, our biophysical studies show that the disordered region of Suppressor of Fused has different lengths and properties in insects and vertebrates. In vertebrates, it is highly dynamic and spans large areas, allowing it to interact with post-translational modifying enzymes. Looking for partners specifically able to bind the IDR2 would help understand the role of this peculiar appendix. , 301, 342, 346, 349, 352 and threonines 305 and 353 are coloured black ; ubiquitylated lysines 303, 321 and 344 are colored orange [START_REF] Akimov | UbiSite approach for comprehensive mapping of lysine and Nterminal ubiquitination sites[END_REF] and nuclear export sequence (residues 308-318) is coloured blue [START_REF] Zhang | Suppressor of Fused chaperones Gli proteins to generate transcriptional responses to Sonic Hedgehog signaling[END_REF]. q(Å) q(Å) q(Å) q(Å)

Tables

Supplementary figures legends

Table 4 :

 4 amino acid composition of the three SuFu sequences displayed in Figure S1. The percentages are calculated using the Protparam server (https://web.expasy.org). The first column corresponds to all amino acids of the sequences; the second column corresponds to the IDR2 aminoacids as shown in the dotted black box on Figure S1. The third column is the difference between the IDR2 and the overall composition.

Figure 1 :

 1 Figure 1: SRCD measurements of SuFu from different species. A, CD spectra showing thermal unfolding of hSuFu at pH 7.5. B, secondary structure evaluation from SRCD spectra for all studied proteins at 10 and 60°C.

Figure 2 :

 2 Figure 2: SAXS studies of hSuFu WT and D30, dSuFu and zSuFu. Green, dSuFu; dark blue, hSuFu WT; black, hSuFuD30; light blue, zSuFu. A, SAXS scattering curves B, dimensionless Kratky plots; C, pair distance distribution P(R).

Figure 3 :

 3 Figure 3: SAXS structure of drosophila SuFu. A, superposition of the crystal structure 4KMA and the envelope calculated by GASBOR and DAMAVER suite. The structure is displayed using a worm-like representation with a radius inversely proportional to the B-factor in the PDB file. B, scattering curves superposed using Crysol (top) and their differences (bottom). The experimental curve is in light blue, the PDB models 4KM9 (hSuFu) in red, 4KMA (dSuFu) in mustard green, the three models used in the normal mode simulation: closed in green, open in orange and best model in purple. C, D, the two slowest modes of movement calculated by Hingeprot. The colours distinguish the domains moving relative to each other and the arrows show the direction of movements.

Figure 4 :

 4 Figure 4: Modelling of dSuFu (A-C) hSuFu WT (D-F), hSuFuD30 (G-I), zSuFu (J-L) and from SAXS data. A, D, G, J: Five best models from Dadimodo; B E, I, K, rotated models superimposed with envelope from DENSS; C, F, H, L, fitting of experimental and model scattering curves.

Figure 5 :

 5 Figure 5: Intrinsically disordered region of SuFu: disorder prediction and structural features. A, PondR prediction of protein disorder for the three sequences studied in this work; B, hSuFu model obtained from our SAXS data showing phosphorylatable serines 290, 301, 342, 346, 349, 352 and

Figure S1 :

 S1 Figure S1: alignment of SuFu aminoacid sequences from human, danio rerio and drosophila melanogaster, showing aminoacid numbering on the sides. Top line= conservation histogram; dotted grey box= IDR1; dotted black box=IDR2 for human and zebrafish; dotted red box = IDR2 for drosophila; N-terminal domain is in light blue boxes and C-terminal domain is in pink boxes. the wavy blue box shows the nuclear export signal and arrows show phosphorylable serines in the vertebrate IDR2. Green background= alpha helix; yellow background = beta strand

Figure

  Figure S3: hSuFu I0, Rg SAXS profile as a function of image index after baseline subtraction with US-SOMO.

Figure

  Figure S4: zSuFu I0, Rg SAXS profile as a function of image index after baseline subtraction with US-SOMO.

  Figure 1

  Figure 3

  Figure 5

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2021.06.14.447554	this version posted June 14, 2021.	The copyright holder for this preprint

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2021.06.14.447554	this version posted June 14, 2021.	The copyright holder for this preprint
				SuFu disordered region is disclosed by solution studies

8

disordered. AllosModFoxs program

Table 1

 1

	Protein	Buffer

Table 1 :

 1 transition temperature for studied proteins measured by SRCD (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 2

 2

	Sample	dSuFu	hSuFuD30	hSuFu	zSuFu
	Data collection parameters
	Beamline		Swing beamline, SOLEIL synchrotron (France)
	Energy (keV)	12	12	12	12
	Sample-detector distance	1804	2087	2000	2000
	(mm)
	q-range (Å-1)	0.008-0.5	0.008-0.5	0.008-0.5	0.008-0.5
	Exposure time	1s	990ms	990ms	990ms
	detector	Aviex charge coupled		Eiger 4M
		device
	software
	Primary data reduction	Foxtrot
	Data processing	US-SOMO
	Ab initio analysis	GNOM V5.0, DENSS
	Computation of model	Crysol
	intensities
	Model building	Modeller	4km9+	4km9+	Phyre +
			AllosModFoxS	AllosModFoxS	AllosModFoxS
	Model refinement	Dadimodo	Dadimodo	Dadimodo	Dadimodo
	Structural parameters
	Rg frm Guinier (Å)	27.2	31.5	36.0	33.7
	I0 (cm-1)	0.017	0.011	0.03	0.05
	qRgmax	1.27	1.24	1.2	1.2
	Dmax frm P(r) (Å)	90	140	150	150
	Rg from P(r) (Å)	27.3	31.9	38.1	36.1
	I0 from P(r) (cm-1)	1.747	0.00472	0.03	0.05
	Ch i2 Gnom	2.0	2.22	1.56	1.68
	Dmax/Rg	3.3	4.8	4.2	4.4
	qRgmax (Kratky)	1.6	2.1	2.0	2.1
	qopt (Å -1 ) (Shannon)	0.61	0.52	0.49	0.53

Table 2 :

 2 SAXS data for dSuFu, hSuFu and zSuFu

Rg= radius of gyration; Dmax= maximum interatomic distance; qRgmax= abscises of the maximum value for the normalised Kratky plot shown in Figure

3B

.

Table 3

 3

	sample	dSuFu	hSuFuD30	hSuFu	zSuFu
	CRYSOL on Dadimodo output (5
	structures)
	q range (Å -1 )	0.008-0.4	0.008-0.4	0.008-0.4	0.008-0.4
	Chi 2	1.80±0.04	1.77±0.02	1.73±0.01	1.18±0.06
	Hydration shell contrast Dro (e-Å 3 )	0.030	0.045	0.03	0.03
	Ra Å	1.617	1.614	1.615	1.615

Table 3 :

 3 atomistic modelling of structures from SAXS data

	Table 4
	aminoacid % overall % IDR2 %IDR2-%overall
	A	5.38	5.12	-0.26
	C	1.61	2.33	0.72
	D	6.50	8.84	2.34
	E	7.62	9.30	1.69
	F	3.84	1.40	-2.45
	G	7.55	3.26	-4.29
	H	3.14	1.86	-1.28
	I	4.82	6.51	1.69
	K	3.91	6.05	2.13
	L	10.76	7.44	-3.32
	M	2.10	1.40	-0.70
	N	3.00	3.72	0.72
	P	8.04	8.37	0.34
	Q	5.17	6.51	1.34
	R	5.31	8.84	3.53
	S	6.92	13.95	7.04
	T	5.24	3.72	-1.52
	V	5.10	1.40	-3.71
	W	1.82	0.00	-1.82
	Y	2.17	0.00	-2.17

Acknowledgements

We are very grateful to Marie-Hélène Le Du, Dominique Durand and Patrice Vachette (Institut de Biologie Intégrative de la Cellule, CEA, CNRS, Université Paris-Saclay, France.) for help with data processing software, the SOLEIL synchrotron for access to beamlines, Javier Perez at Swing beamline, and Frank Wien and Mathieu Refrigiers at Disco beamline. Molecular graphics figures and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.

Funding

AJ was financed by a thesis grant from Ministère de l'Enseignement Supérieur et de la Recherche and

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SuFu disordered region is disclosed by solution studies

16 by a fourth year thesis grant from Association pour la Recherche contre le Cancer (ARC). SM was financed by a thesis grant from Ministère de l'Enseignement Supérieur et de la Recherche -IDEX Paris Sciences Lettres. This work was supported by the Centre National de la Recherche Scientifique, and by the "Initiative d'Excellence" program from the French State (Grant "DYNAMO", ANR-11-LABEX-0011-01) and Fondation ARC pour la recherche sur le cancer (grant 1112) to APl and MS.

Author contribution

SM, AJ and APa cloned and purified proteins. SM, AJ and VB measured and processed CD and SAXS data. VB, MB and FO performed molecular modelling and analysis. AT and OR helped with Dadimodo modeling and data processing. APl and MS provided constructs and background about Hh pathway. VB designed the research, conducted research and wrote the manuscript with the help of all authors.

Abbreviations

Ci, cubitus interruptus Hh, Hedgehog IDR, intrinsically disordered region Ptc, Patched SAXS, small angle X-ray scattering Smo, Smoothened SRCD, synchrotron radiation circular dichroism SuFu, Suppressor of Fused


Camille Oger

Zhenan Bao

Ken Xie

An evening about Young Chemists in Politics at la Maison de la Chimie. From left to right

Chemistry International January-March 2020 by Sophie Carenco et al.

F

or 100 years, IUPAC has supported chemists worldwide in all facets of their craft: industrial development, academic research, teaching, and many more. Mindful of nurturing its future leaders, since 2020 the Union ensured the attendance of the younger generation of chemists at the General Assembly and Congress in part through their "Young Observers" program [1]. It provides an opportunity for scientists below 40 to acquaint themselves with the work of the Divisions and Committees. The IUPAC Congress, which attracts several thousand attendees every two years, was not finely tuned to the expectations and contributions of the rising generation of chemists. However, the paradigm has begun to change. For the 47 th Congress organized in Paris, France, 5-12 July 2019, the scientific executive committee, chaired by Clément Sanchez, enthusiastically received the proposal from a team of young chemists to build a "Young Chemists Symposium", organized by younger chemists for the benefit of all the attendees. In addition to an opening networking session with poster presentations, six sessions were hosted in the symposium:

• "From the bench to the market" with testimonies from entrepreneurs, was especially appreciated as it unraveled the do's and don'ts of a start-up business; • "Scientifi c writing" with contributions from the European Research Council (ERC) executive agency, a journal editor and a writing coach, was well attended by attendees at all stages of their career; • "Teaching" was enlightening to all by demonstrating new, stimulating practices in countries such as Singapore, Netherlands and France; • "New channels and challenges for scientifi c outreach" illustrated how Youtube and the digital world could rejuvenate the fi eld; An IYCN-centered session brought together younger chemists from around the world and was focused around professional development and the personal success stories of empowered young leaders in chemistry;

"Redefi ning scientifi c excellence in Africa" highlighted, from the point of view of three researchers from this continent, how research could be better appreciated in countries where it is still not enough developed.

The symposium was complemented by other events that brought young chemists together. This included a tour of Paris' historical sites around the life of Marie Skłodowska-Curie as a social event (provided by the association "Parcours des Sciences" [6]). The IYCN off ered a CV-clinic during one of the lunch time sessions that featured Robert Bowles of the RSC leading the discussion, and a Q&A style discussion with Michelle Lucas and Georg Wießmeier from Sibelco. Lastly, a special evening event was organized to foster discussions between the younger chemists from the conference and these from the Paris area. Held in the grand auditorium at la Maison de la Chimie, it dealt with "young chemists and politics", with two VIPs: Zhenan Bao from Stanford University and Ken Xie from the Australian Embassy in Paris. During the evening, the winners of the "Chemistry Rediscovered" video contest organized by EYCN were presented [START_REF] Oger | Société Chimique de France[END_REF].

Overall, more than 20 speakers contributed to

Young Chemist Symposium at the Palais des Congrès, Paris

From Young Attendees to Young Actors:

21 Chemistry International January-March 2020 the symposium, which gathered a large number of attendees from academia, industry and teaching, in the 100-seat modern room of the Palais des Congrès. It was covered live through a dedicated twitter account (@YoungSci_IUPAC) whose audience rapidly reached 600 followers. It also effectively served as a meeting point for active members of younger chemists' networks, for young observers, as well as for the IYCN delegates who participated in the congress, thus fostering unique opportunities for the younger generations to connect with the IUPAC community.

As the first of its kind, gathering funding was challenging for such an innovative event. Fortunately, several partners contributed to allow the symposium to take place, including IUPAC, Fondation de la Maison de la Chimie, UdPPC, Sibelco, Evonik, ACS, RSC, SCF, GDCh and IYCN.

In conclusion, the "Young Chemists Symposium" managed to address topics not covered by the rest of the congress (e.g. entrepreneurship), and to provide original viewpoints on other issues (e.g. teaching, outreach, young chemists in politics) for the benefit of all attendees. Let us hope that this is the beginning of a new era for IUPAC Congresses and similar program will be staged in Montreal during the 2021 Congress, in one form or another.

  Organization of the symposium started two years earlier under the leadership of Camille Oger, assistant professor at the University of Montpellier and a former board member of the RJ-SCF (the French young chemists network) [2] and the European Young Chemists Network (EYCN) [3]. In order to foster international collaboration and intercultural cooperation, other networks were invited to contribute to the symposium organization: the International Younger Chemists Network (IYCN) [4], EYCN, and Afriscitech [5]. The sessions were open to all attendees.