Emilio Salceda

Javier Pérez-Castells

Blanca López-Méndez

Anoland Garateix

Hector Salazar

Omar López

Abel Aneiros

Ludger Ständker

Lászlo Béress

Wolf- Georg Georg Forssmann

Enrique Soto

Jesús Jiménez-Barbero

Guillermo Giménez- Gallego
email: gimenez_gallego@cib.csic.es

CgNa, a

Keywords: site 3 toxin, sodium channel inactivation, Condylactis gigantea, sea anemone toxin, NMR, solution structure

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

Introduction

Sea anemones are sessile coelenterates that protect themselves against predators by releasing different types of polypeptide toxins. These toxins mainly affect ion channel activity, although some of them exhibit cytolytic activity [START_REF] Aneiros | Bioactive peptides from marine sources: pharmacological properties and isolation procedures[END_REF]. The first polypeptide toxins purified from Anemonia sulcata and Condylactis gigantea were reported to affect voltage activated sodium channels [START_REF] Norton | Cardiotonic polypeptides from Anthopleura xanthogrammica (Brandt) and A. elegantissima (Brandt)[END_REF][START_REF] Loret | Positively charged amino acid residues located similarly in sea anemone and scorpion toxins[END_REF]. Indeed, these voltage gated sodium channels represent an important target for different natural toxins and at least six receptor sites have been identified for these kinds of neurotoxins.

Sodium channel toxins from the sea anemone can be classified as type I and II according to their sequence homology [START_REF] Norton | Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel[END_REF], and these types of toxins exhibit comparable electrophysiological properties. Type I toxins elicit important neurotoxic (repetitive firing) and cardiotoxic (arrhythmia) effects, delaying the inactivation of mammalian voltage gated sodium channels. Moreover, these type I toxins target receptor site 3 located at the extracellular face [START_REF] Rogers | Molecular determinants of high affinity binding of α-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel α subunit[END_REF].

There are many toxins that target site 3 of mammalian voltage gated sodium channels that constitute a structurally diverse group of peptides isolated from the scorpion [START_REF] Meves | Interactions of scorpion toxins with the sodium channel[END_REF][START_REF] Possani | Scorpion toxins specific for Na+-channels[END_REF], sea anemone [START_REF] Norton | Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel[END_REF][START_REF] Narahashi | Condylactis toxin: interaction with nerve membrane ionic conductances[END_REF], spider [START_REF] Fletcher | The structure of versutoxin (δ-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel[END_REF][START_REF] Little | δ-Atracotoxins from australian funnel-web spiders compete with scorpion α-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels[END_REF], and wasp [START_REF] Sahara | A new class of neurotoxin from wasp venom slows inactivation of sodium current[END_REF][START_REF] Kinoshita | Novel wasp toxin discriminates between neuronal and cardiac sodium channels[END_REF]. These toxins are important tools to study the mechanism of sodium channel inactivation and to emulate certain important periodic pathologies such as paramyotonia congenita and hyperkalemic periodic paralysis. Indeed, the effects of such toxins are similar to those caused by the mutations responsible for these diseases [START_REF] Cannon | Loss of Na+ channel inactivation by anemone toxin (ATX II) mimics the myotonic state in hyperkalaemic periodic paralysis[END_REF][START_REF] Lehmann-Horn | Voltage-gated ion channels and hereditary disease[END_REF][START_REF] Jurkat-Rott | Voltage-sensor sodium channel mutations cause hypokalemic periodic paralysis type 2 by enhanced inactivation and reduced current[END_REF][START_REF] Blumenthal | Voltage-gated sodium channel toxins: poisons, probes, and future promise[END_REF].

We recently isolated and performed a preliminary characterization of CgNa, the main peptide toxin from Condylactis gigantea [START_REF] Ständker | A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation[END_REF]. Based on sequence homology, CgNa should be included in the type I toxin group along with ATXI, ATXII and ATXV from Anemonia sulcata [START_REF] Wunderer | Amino-acid sequence of toxin I from Anemonia sulcata[END_REF][19][START_REF] Scheffler | the Anthopleura xanthogrammica heart stimulant, anthopleurin A[END_REF], AftI and AftII from Anthopleura fuscoviridis [21], ApA and ApB from Anthopleura xanthogrammica [22][START_REF] Reimer | Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin-B[END_REF][START_REF] Kelso | Identification and characterization of novel sodium channel toxins from the sea anemone Anthopleura xanthogrammica[END_REF], ApC from Anthopleura elegantissima [START_REF] Salceda | Effects of APC, a sea anemone toxin, on sodium currents of mammalian neurons[END_REF] and BgII and BgIII from Bunodosoma granulífera [START_REF] Goudet | Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels[END_REF][START_REF] Salceda | The sea anemone toxins BgII and GgIII prolong the inactivation time course of the tetrodotoxin-sensitive sodium current in rat dorsal root ganglion neurons[END_REF]. We have resolved the three-dimensional structure of CgNa in solution by 1 H-NMR spectroscopy, and we have characterized its electrophysiological effects in mammalian DRG neurons in detail. Moreover all this information has been correlated with that available for other closely related type I and II anemone toxins. The results may contribute to further define the structural basis underlying the electrophysiological properties of this family of sea anemone sodium channel toxins.

Materials and Methods

Biological materials

Animal care and all the experimental procedures were carried out in accordance with the Helsinki Declaration. The number of animals used in this study was kept to the minimum necessary for a meaningful interpretation of the data. CgNa was isolated and purified from the sea anemone Condylactis gigantea [START_REF] Ständker | A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation[END_REF]. Aliquots of a stock solution in deionized water were prepared and stored in a freezer (-20 ºC). Prior to every experiment, each one of these aliquots was diluted in the corresponding perfusion solution.

DRG neurons were isolated and cultured from Wistar rats (P5-9) of either sex according to the procedure described by Salceda et al. [START_REF] Salceda | The sea anemone toxins BgII and GgIII prolong the inactivation time course of the tetrodotoxin-sensitive sodium current in rat dorsal root ganglion neurons[END_REF].

Electrophysiological measurements

Whole cell recording was carried out using an Axopatch-1D amplifier (Axon Instruments, Union City, CA). The generation pulse and the data sampling was controlled by Pclamp 8.0 software (Axon Instruments) using a 16-bit data acquisition system (Digidata 1320A, Axon Instruments). All experiments were performed at room temperature (23-25 ºC). DRG neurons are known to express tetrodotoxin-resistant (TTX-R) and tetrodotoxinsensitive (TTX-S) sodium channels [START_REF] Kostyuk | Ionic currents in the somatic membrane of rat dorsal root ganglion neurons-I. Sodium currents[END_REF][START_REF] Roy | Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons[END_REF]. Thus, the types of sodium currents present in the cell under study were determined before each experiment. Only cells in which the tetrodotoxinresistant (TTX-R) sodium current was less than 10% of the total current, as defined from a steady-state inactivation profile, were used to determine the effects of the toxin on TTX-S sodium currents [30].

Leakage and capacity currents were digitally subtracted using the P-P/n method, and the capacitance and series resistance (80%) were electronically compensated. Experiments were rejected when the voltage error exceeded 5 mV after compensation of series resistance at the maximum peak current. No corrections were made for smaller values.

Toxin was ejected under pressure using a microinjector (Baby Bee, Bass, West Lafayette, IN, USA) from a micropipette positioned in the vicinity of the cell being recorded.

The solutions employed in the experiments are detailed in Table 1. Unless otherwise stated, all numerical data are presented as the mean±S.E.M (number of values used in calculating the mean) of at least four measurements. A more detailed description of the methods and data analysis has been presented previously [START_REF] Salceda | The sea anemone toxins BgII and GgIII prolong the inactivation time course of the tetrodotoxin-sensitive sodium current in rat dorsal root ganglion neurons[END_REF].

NMR spectroscopy

For acquisition of NMR spectra, the concentration of CgNa in 90% H 2 O/10% 2 H 2 O and in 100% 2 H 2 O was 1.4 mM. 1 H-NMR spectra were recorded at 500 MHz on a Bruker AVANCE-500 spectrometer, using an inverse multinuclear probehead fitted with gradients along the Z-axes. Spectra were referenced to external TSP, and they were recorded using the States-TPPI method incorporating

NOESY cross peak assignment and structure calculation

Peak lists for the NOESY spectra recorded with a 0.25 s mixing time were generated by interactive peak picking using the XEASY software [START_REF] Bartels | The Program Xeasy for Computer-Supported Nmr Spectral-Analysis of Biological Macromolecules[END_REF]. NOESY cross peak volumes were determined by the automated peak integration routine, peakint [32] implemented in XEASY.

The three-dimensional structure of CgNa was determined using the standard protocol of The MOLMOL program [START_REF] Koradi | MOLMOL: a program for display and analysis of macromolecular structures[END_REF] was used to build the coordinates for the non-standard amino-acid residue 4-hydroxyproline. The topological information for this residue has been reported previously [START_REF] Wang | Automatic atom type and bond type perception in molecular mechanical calculations[END_REF], and it was used here without any further modification. No additional force field parameters for 4-hydroxyproline were needed.

The MOLMOL program was used to visualize the three-dimensional structures.

CYANA was used to obtain statistics on target function values, restraint violations and

Ramachandran plots according to PROCHECK-NMR conventions [START_REF] Laskowski | AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR[END_REF], etc. RMSD values were calculated with CYANA for superpositions of the backbone N, C α and C' atoms, or the heavy atoms over the whole protein or over its structured regions. To obtain the RMSD of a structure represented by a bundle of conformers, all conformers were superimposed on the first one and

Results

NMR structural analysis of CgNa

Protein resonance assignments were obtained using standard strategies based on 2D-NMR experiments. In total, 98.7% of the amide and 1 H shifts of the side-chain moieties CH n were unambiguously identified and assigned. 1 H assignments were also obtained for the indole NH of Trp 31 and for the side-chain NH2 resonances of all Asn and Gln residues.

Structure determination

The statistics regarding the quality and precision of the 20 energy-minimized conformers that represent the solution structure of CgNa are summarized in Table 2. The structures are well defined and they are in perfect agreement with the experimental data. Of the 1618 cross peaks identified in the 2D NOESY spectrum, 1554 (96%) were unambiguously assigned by the CYANA program. Aditionally, standard upper and lower limit distance constraints were applied to enforce the disulfide bridges. The disulfide arrangement (CysI-CysV, CysII-CysIV, CysIII-CysVI) was predicted on the basis of sequence homology and the conservation of cysteine spacing with another related type I sea anemone toxin. Test calculations with alternative disulfide bond pairings were also assessed, in all cases generating structures that deviated more from the experimental NMR data than those described above, and with less favorable conformational energies. Furthermore, 292 long-range distance restraints between protons that were separated in the sequence by five or more residues were used in the final calculations with CYANA and in the subsequent energy-refinement with the AMBER program. No violations of NOE distance restraints greater than 0.2 Å were found in the final bundle of structures. The low RMSD values of 0.31 Å for the mean coordinates of the backbone and of 0.58 Å for all heavy atoms except the flexible loop of residues 8-16, reflected the good convergence of the ensemble.

Additionally, the quality of the structures was also reflected by the presence of 67% of the (Φ,Ψ) backbone torsion angle pairs in the most favored regions, and 31% of these were found within the regions additionally permitted by the Ramachandran plot, according to the PROCHECK-NMR program.

Solution structure

The final 20 structures calculated for CgNa have been superimposed over the backbone heavy atoms (Figure 1). The structure consists of four β-strands from antiparallel β-sheets (involving residues 1-3, 21-23, 32-33, 43-46), and three β-turns at residues 13-16, 27-30 and 31-34. The first β-strand is connected to the second one by a long poorly defined loop exposed to the solvent (Asp 7 -Ser 19 ). The peptide bonds at Hyp 3 and Pro 11 were predominantly determined to be in a major trans orientation due to the presence of α i -δ i+i NOE cross peaks.

Nonetheless, there was also evidence of isomers with a cis peptide bond at Hyp 3 identified by one NOE between the Hα protons of Val 2 and Hyp 3 . The relative slow isomerization between cis and trans forms of the prolyl peptide bond usually induces additional cross peaks for nearby residues. In the CgNa spectra, two sets of Val 2 cross peaks were observed in the TOCSY fingerprint region.

Comparison with other long sea anemone polypeptides

Other polypeptides with three-dimensional structure similar to that of CgNa were identified in a search of the DALI three-dimensional protein structure database [START_REF] Holm | Protein structure comparison by alignment of distance matrices[END_REF] using CgNa as the query. Only sea anemone toxin peptides classified as sodium channel blockers were retrieved and a pairwise comparison was made between the averaged three-dimensional structures of the toxins singled out by DALI and CgNa (Table 3). The highest DALI Z-scores correspond to the anthopleurins (ApA, ApB) and the ATX Ia neurotoxin, proteins that display more than 50% sequence identity to CgNa in structure-based sequence alignments (Figure 2).

Although CgNa should be classified as a group I sodium-toxin, type II Sh I was also retrieved in this search and while it only shows 44% identity to CgNa, it was associated with a significant DALI Z-score (Figure 2 and Table 3, RMSD column). While the similarity of folding between CgNa, ApB and Sh I is evident (Figure 3A), important differences at the primary structure level reflect the distinct distribution of electrostatic charge at functionally important regions of the protein surface between CgNa, ApB and Sh I (Figures 3B). Moreover, these differences extend to the geometry of a surface-exposed hydrophobic patch (Figure 3C) that is thought to be involved in the recognition of the toxin by the channel [START_REF] Blumenthal | Ionic channels as targets for toxins, drugs and genetic diseases[END_REF].

Electrophysiology in mammalian DRG neurons

The mean capacitance of the neurons examined in this study was 47±1.9 (83) pF, which corresponds to a cell diameter of about 38 µm. In current clamp experiments the neurons had a mean resting membrane potential of -53.7±2.4 (3) mV. Action potentials were evoked by a depolarizing current pulse of 5 ms and 100 pA with an 8 s interpulse interval (Figure 4A). At -60 mV, the duration of the action potential was 3. In voltage clamp experiments the inactivation of TTX-S Na + currents was well fitted by a single exponential function (correlation coefficient ≥ 95%). Accordingly, CgNa slows the inactivation of the TTX-S Na + current. The mean time constant of inactivation (τ h ) was studied using a 40 ms test pulse at -20 mV from a holding potential (V h ) of -100 mV. Under control conditions, τ h was 0.5 ± 0.7 ms while it increased to 0.85 ± 0.15 ms (9) in the presence of CgNa (10 µM, Figure 4B). Moreover, the maximum effect was reached within the first minute following exposure to the toxin. The relationship between the current at the end of the pulse and the current measured at its peak showed an increase about 100% after toxin application. The effect of CgNa on inactivation kinetics were partially reversible after washing the preparation and at the highest concentration used (10 µM), 71% of recovery was reached following a 5 min washout (not shown).

The effect of CgNa on the inactivation time course of the TTX-S Na + current was concentration dependent and thus, the dose-response curve was constructed using the fixedpulse protocol described above. The data were fitted by a dose-response function as follows:

y=A 1 +(A 2 -A 1 )/1+10 (logIC 50 -x)P
, where A 1 is the y value of the bottom plateau, A 2 is the y value of the top plateau, log IC 50 is the concentration at which the response is halfway between A 1 and A 2 , and P is the Hill slope. The dose-response relationship for the effect of CgNa on the inactivation of the TTX-S Na + current was fitted with a curve that had an IC 50 of 1.34±0.4 [START_REF] Holm | Protein structure comparison by alignment of distance matrices[END_REF] µM and a Hill slope coefficient of 0.6±0.2 (41) (Figure 4B). The effect of CgNa (10 µM) on the peak Na + current amplitude was not significant at any of the concentrations tested (paired Student´s t test, p > 0.05).

From the current-voltage relationships, the current density versus voltage curves were obtained by normalizing the ionic current amplitudes as a function of the membrane capacity (Figure 4C). Under control conditions the maximum current density was reached at -20 mV and perfusion of CgNa (10 µM) did not produce any significant change in the Na + current density, or its reversal potential, at any of the voltages tested (6, paired Student´s t test, p > 0.05).

To study the effects of CgNa on the conductance and voltage dependence of steady-state TTX-S Na + current inactivation, a two pulse protocol from a V h of -100 mV was used. In this protocol, a 100 ms conditioning pre-pulse from -120 to +70 mv in 10 mV increments, was The TTX-S Na + conductance was calculated from the equation: g Na = I Na /(E-E Na ) where E is the test pulse voltage and E Na is the reversal potential for sodium. Thus, the Na + conductance was plotted as a function of the membrane potential, and the ratio was fitted by a Boltzmann equation both in controls and after exposure to CgNa (10 µM). No difference was observed in the potential at which activation is 0.5 (V½ act ) under control conditions (-29.5±0.6)

or after the application of CgNa (-31.0±0.6 mV, Student's t test, p > 0.05). The slope factors for these curves were 6.3±0.5 (6) mV and 6.9±0.5 (6) mV, respectively (data not shown).

To study the action of CgNa on the rate of recovery from inactivation, a two pulse protocol was used with an increment in the duration of the interpulse interval. In this protocol, the V h was either -100 or -80 mV, the test pulses (40 ms) were up to -20 mV and the interpulse interval (∆t) varied from 2.5 to 1000 ms. Using a V h of -100 mV, the time course of recovery We explored the state of the Na + channel on which CgNa exerts its action with a pair of depolarizing trains of 200 pulses (pulse duration = 40 ms; pulse interval = 200 ms) to -20 mV from a holding potential of -100 mV. The first (control) and second (test) trains were separated by a rest period of 1 min during which the cell was held at a holding potential of -100 mV and perfused with 10 µM CgNa. The inactivation time constant (τ h ) of the Na + current evoked by each pulse was plotted against the pulse number. In the absence of the toxin, τ h remained constant during the control train. In contrast, the time constant of the ionic current produced by the first test pulse increased by 59.1±14.6(4) % following CgNa application, indicating that the toxin preferentially binds to the channel in the resting state. The effectiveness of CgNa did not augment when the channels were activated by trains of repetitive stimuli, indicating that its action was not dependent on channel usage (Figure 6).

The effect of CgNa on the TTX-R Na + currents was studied using the same solutions used for TTX-S Na + currents but with the addition of TTX (300 nM) to the external solution (n = 5). CgNa showed no significant effect on TTX-R Na + currents (Figure 7A). Finally, the effect of CgNa (10 µM) on total potassium (I K , n = 4, Figure 7B) and total calcium currents (I Ca , n = 4, Figure 9C) was studied. Similarly, CgNa did not modify either the peak amplitude or the sustained component at the end of the pulse of these currents.

Discussion

Electrophysiology

CgNa increases the duration of the action potential due to the slowing down of tetrodotoxin-sensitive sodium current inactivation. This action seems to be very selective for the TTX-S Na + channel since the highest toxin concentration that we used (10 µM) did not produce any significant effect on other ion currents. The effect of CgNa on the inactivation process takes place within the first minute of its application and it was partially reversible following its removal and wash out. The reversibility of the effects of sea anemone toxins upon vertebrate or insect Na + channels has been reported by many authors [START_REF] Salgado | Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels[END_REF][START_REF] Cestele | Biochemical and pharmacological characterization of a depressant insect toxin from the venom of the scorpion Buthacus arenicola[END_REF][45], in contrast to the irreversible toxicity on crustacean Na + channels [START_REF] Salgado | Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels[END_REF]. Interestingly, while the effects of ATX I and ATX II in crayfish giant axons were irreversible [START_REF] Rathmayer | Sea anemone toxins: tools in the study of excitable membranes[END_REF], the activity of ATX III in the same preparation was reversible. The different properties of these toxins may be caused both by the characteristics of the compounds themselves and also through the structural and functional differences of sodium channels in various tissues and animal phyla.

Toxin application significantly modified the steady state inactivation of TTX-S Na + channels. The slowing of the inactivation kinetics and the reduced voltage dependence of the steady-state availability curve indicate that CgNa modifies the transition rate of the Na + channel

both from the open to the inactivated state and from the inactivated to the open state. CgNa did not increase the peak currents of Na + channels at the concentrations used here, despite the marked effect of the toxin on sodium-current inactivation which would increase current density.

Thus, we can not exclude the possibility that CgNa may also produce some degree of channel occlusion.

In presence of the toxin, the τ h on the first pulse after a rest period increased and this value remained within a narrow range during successive pulses in the test train. These features are consistent with the action of the toxin not being dependent on channel use. Indeed, these observations indicate that CgNa has a high affinity for the closed state of the channel and that accordingly, the toxin shows no preference for the Na + channel in the open state. The time course of recovery from Na + current inactivation was not significantly affected by CgNa when studied using a V h of -100 mV. However, while the fast time constant was not affected by CgNa at a V h of -80 mV the slow time constant was enhanced in the presence of CgNa. Previous reports indicate that ApB also significantly enhances the recovery from inactivation of both cardiac and neural Na + channel isoforms [START_REF] Benzinger | Augmentation of recovery from inactivation by site-3 Na channel toxins -A single-channel and whole-cell study of persistent currents[END_REF]. On the basis of kinetic modifications of the Na + channel, this has been explained as an increase of the direct rate of transition back to the open state or as the enhancement of the normal transition through the different inactivation states.

Since the effect of CgNa on the recovery rate from inactivation was voltage dependent we think that in our case the binding of CgNa to Na + channels displayed some type of voltage dependence, as previously suggested for α-scorpion toxins [START_REF] Strichartz | Rapid voltage-dependent dissociation of scorpion α-toxins coupled to Na channel inactivation in amphibian myelinated nerves[END_REF].

Structural studies

Structural studies analyzing the effects of modifying sea anemone toxins have been carried out by several laboratories, mainly focusing on mutated ApB. Through such studies, the Gly 10 and Gly 15 residues of ApB appear to be important for toxin affinity and Gly 20 is an essential folding determinant [START_REF] Seibert | Arg-14 loop of site 3 anemone toxins: effects of glycine replacement on toxin affinity. Biochemistry 42, 14515-arginine 12 and lysine 49 in the activity of the cardiotonic polypeptide anthopleurin B[END_REF]. Indeed, these three Gly residues are conserved in CgNa. In addition, Arg 12 , Leu 18 , Trp 33 , Lys 37 , Ile 43 and Trp 45 significantly contribute to the tight binding affinity of ApB [50][START_REF] Dias-Kadambi | The role of exposed tryptophan residues in the activity of the cardiotonic polypeptide anthopleurin B[END_REF][START_REF] Dias-Kadambi | Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltagesensitive sodium channel[END_REF][START_REF] Benzinger | A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B[END_REF]. Only two of these residues are strictly conserved in CgNa (Leu 18 and Trp 33 , 31 in CgNa numbering (Figure 2).

According to previous analyses, the overall alignment of type I and type II toxins identifies 11 identical residues and 5 others that are subject to conservative substitution along the peptide [START_REF] Norton | Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel[END_REF]. Of those, CgNa lacks the invariable Arg 14 (substituted by His) and the conserved hydrophobic residue at position 41 (substituted by Ala). Overall, CgNa has a greater percentage of exposed negatively charged residues and a lower percentage of exposed hydrophobic residues than the typical type I and II toxins. These differences are mainly derived from the lack of cationic residues in the flexible loop 8-16 of CgNa and the unique amino acids Asp 36 , Glu 37 and Glu 43 in CgNa (instead of Trp 45 in ApB) which create an important negatively charged patch in conjunction with the conserved residues Asp 7 and Asp 9 . This patch occupies part of a strongly cationic surface that builds up around Lys 37 in ApB [START_REF] Khera | Multiple cationic residues of anthopleurin B that determine high affinity and channel isoform discrimination[END_REF][START_REF] Khera | Importance of highly conserved anionic residues and electrostatic interactions in the activity and structure of the cardiotonic polypeptide anthopleurin B[END_REF]. As it has been demonstrated that Lys 37 directly interacts with Asp 1612 of the rat cardiac Na + channel [START_REF] Benzinger | A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B[END_REF], the partial substitution of such a positive patch could be one factor that explains the lower potency of CgNa (10 µM towards ganglion neurons expressing TTX-R Na + channels vs. 9 nM towards N1E-115 cells in the case of ApB). Substitution of Arg 12 (Ser in CgNa) may further weaken this interaction, as mutation of Arg 12 has been shown to reduce the affinity of ApB for both neuronal and cardiac channels [50]. Moreover, there is a substitution of the conserved hydrophobic residue at position 43 by Ala in CgNa (residue 41 of its primary structure), and an Asp residue is found at position 43 in this protein rather than an aromatic residue in the other type I toxins with a defined threedimensional structure. These changes also disrupt a surface-exposed hydrophobic patch that involves residues previously shown to be relevant for the binding of the toxin to the channel [START_REF] Dias-Kadambi | The role of exposed tryptophan residues in the activity of the cardiotonic polypeptide anthopleurin B[END_REF][START_REF] Dias-Kadambi | Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltagesensitive sodium channel[END_REF]. Despite these differences, the protein is still active and thus, neither the cationic residues in the 8-16 loop, Lys 37 , nor the small surface-exposed hydrophobic patch that includes Ile 43 of ApB are absolutely essential for binding to the neuronal isoform of the mammalian voltage-gated Na + channels. Future mutational analysis of CgNa, based on the entire set of structural comparisons discussed above, should further contribute to elucidating the structural characteristics underlying the biological properties of CgNa and related toxins. These data were obtained from a single cell and as can be seen, not only is the effect of CgNa already evident from the first pulse after the rest period but this is its maximum effect. Bar indicates perfusion with 10 µM CgNa.

  Figure 4D). The half maximal inactivation in control neurons of -65.8±0.6[START_REF] Meves | Interactions of scorpion toxins with the sodium channel[END_REF] mV was shifted to -73.7±1.4[START_REF] Meves | Interactions of scorpion toxins with the sodium channel[END_REF] mV in the presence of CgNa. The slope values augmented significantly from 7.5±0.3 (6) mV in control conditions to 11±0.8[START_REF] Meves | Interactions of scorpion toxins with the sodium channel[END_REF] mV in the presence of CgNa (paired Student´s t test, p < 0.05).

  from inactivation was fitted by a second order exponential function with a fast time constant (τ rec fast ) in control conditions of 5.5±0.3 ms and a slow time constant (τ rec slow ) of 26.4±1.2 ms. These figures were not significantly different to those found after application of CgNa: τ rec fast = 5.1±0.3 ms and τ rec slow = 25.4±1.1 ms (Figure 5A). In contrast, at a V h of -80 mV, the time course of the recovery from inactivation of TTX-S Na + current was enhanced by CgNa. While Biochemical Journal Immediate Publication. Published on 16 May 2007 as manuscript BJ20070130 © 2007 The Authors Journal compilation © 2007 Biochemical Society 13 the τ rec fast did not significantly change from 6.3±1.4 ms in control conditions to 5.8±0.6 ms in the presence of CgNa, the τ rec slow diminished upon exposure to CgNa from 49.6±8.2 ms in control conditions to 20.8±3.5 ms in the presence of CgNa (Figure 5B).

Figure 1 .

 1 Figure 1. Stereo view of the 20 representative structures of CgNa superimposed over the backbone atoms N, C α and C' of residues 1-47. The three disulfide bridges are colored in grey.

Figure 2 .

 2 Figure 2. Structure-based sequence alignment of the CgNa toxin with type I (upper) and type II (lower) sea anemone polypeptides of known structure. Following the notation from Norton [4], identical residues in each class are boxed and those that are conservatively substituted are shaded in grey. Asterisks indicate residues that are identical in all type I and type II sequences and daggers those which are conservatively substituted. The residue numbering scheme is based on the CgNa sequence. ApA(B) have an Pro-Ser insert following Gly25 and therefore, the numbering of the residues after this position is shifted by two units in relation to CgNa.

Figure 3 .

 3 Figure 3. (A) Superimposed ribbon representation of the CgNa and ApB (left) and CgNa and Sh I (right) structures. CgNa is colored in green and dark grey. For CgNa and ApB the structure that is closest to the average is shown, while for Sh I the closest to the average refined minimized structures of Wilcox et al. [56] is shown. (B) Solvent accessibility surface representations of ApB (left), CgNa (middle) and Sh I (right), that represents an approximately 90° counterclockwise rotation around the vertical axis of the structures shown in Figure 3A. Electrostatic potential at the surface is represented by graded colors (blue most positive; red most negative). (C) Solvent accessibility surface of ApB (left), CgNa (middle) and Sh I (right). Hydrophobic residues are colored in yellow, and positively and negatively charged amino acids in blue and red, respectively. The views are related to those in Figure 3A by a counterclockwise rotation of approximately 180° around the vertical axis.

Figure 4 .

 4 Figure 4. Current and voltage clamp experiments on TTS-S Na + . (A) CgNa increases the length of the action potential. Action potentials of a DRG neuron recorded under control conditions, in the presence of CgNa (10 µM) and after washout (5min). In this particular cell a small depolarization of about 2 mV was observed. The dotted line indicates the zero voltage level. Inset: data for the entire experimental group in which current-clamp experiments were carried out. Exposure to the toxin increased the duration of action potentials measured at 50% of its amplitude by 463% (n = 3). (B) CgNa increased the inactivation time course of TTX-S I Na . Inset: representative experiment showing the effect of CgNa (10 µM) 1 min after commencing

Figure 5 .

 5 Figure 5. CgNa enhanced the time course of the recovery from inactivation. (A) Recovery curves obtained from the protocol in the inset (V h = -100 mV). Curves were fitted (solid lines) by a second order exponential function with a fast (τ rec fast ) and a slow (τ rec slow ) time constant. CgNa (10 µM, filled circles) did not produce significant changes in these parameters. (B) Using a V h = -80 mV, the τ rec slow decreased significantly from 49.6±8.2 ms in control to 20.8±3.5 ms in the presence of the toxin.

Figure 6 .

 6 Figure 6. CgNa acts on TTX-S Na + channels in the closed state. (A) Post-rest protocol employed to obtain the records. The toxin was applied (bar) at the beginning of the rest period (one minute) represented by the break. (B) Temporal course of the inactivation time constant τ h .

Figure 7 .

 7 Figure 7. CgNa did not alter TTX-R Na + , K + or Ca + currents. In all cases two superimposed traces and two current densities versus voltage curves are presented, one under control conditions (white circles) and the other approximately 2 min after toxin perfusion (filled circles). (A) TTX-R sodium currents were isolated in the presence of 300 nM TTX. (B) Potassium currents. Current density versus voltage curves were obtained measuring the maximum current (circles) or the current at the end of the trace (squares) and normalizing these data to membrane capacity. (C) Calcium currents.

Figure 2 . 31 Figure 3 . 34 Figure 6 . 35 Figure 7 .

 2313346357 Figure 2.

  the watergate 3-9-19 pulse sequence for water suppression. In general, 256 equally spaced evolution-time period t 1 values were acquired, averaging 16 transients of 2048 points, with a

spectral width of 7002.8 Hz (14.00 ppm). Time-domain data matrices were all zero-filled to 4K in both dimensions, thereby yielding a digital resolution of 3.41 Hz/pt. Prior to Fourier transformation, a Lorentz-Gauss window with different parameters was applied for to both the t 1 and t 2 dimensions for all the experiments. NOESY spectra were obtained with different mixing times (0.15 and 0.25 s) and TOCSY experiments were recorded using 0.025 and 0.07 s DIPSI2 mixing pulses. Both NOESY and TOCSY experiments were performed at 298 K.

  3 ± 1.1 ms when measured at 50% of the peak amplitude. Perfusion with CgNa (10 µM) increased the duration of the action potential duration to 18.6±3.8 ms after 1 min, producing a 4.6 fold increase with respect to control values. The resting membrane potential was not significantly affected by the toxin (paired Student´s t test, p > 0.05). After a 5 min washout, the duration of the action potential returned to 3.7±1.2 ms, which represents a recovery of about 80% with respect to the maximum effect.

Table 2 .

 2 Statistics for the NMR solution structure of CgNa in 90% H 2 O/10% 2 H 2 O at 298 K.Average values over the 20 energy-minimized CYANA conformers. The final CYANA target function value was calculated for the structure before energy minimization with AMBER 8.0.

	NOE cross peaks:	1618
	assigned (%)	96
	NOE upper distance limits:	829
	short-range, |i -j| ≤ 1	435
	medium-range, 1 < |i -j| < 5	99
	long-range, |i -j| ≥ 5	295
	Maximal violation (Å)	0.19
	Violations > 0.2 Å	0
	CYANA target function (Å 2 )	0.84
	AMBER energy (kJ/mol, kcal/mol)	-18.26•10 4 (-4.38•10 4 )
	RMS deviations from idealized geometry:
	bond lengths (Å)	3.26
	bond angles (°)	0.05
	RMSD to mean coordinates (Å):
	backbone N,Cα,C' of residues 1-47	1.20
	all heavy atoms of residues 1-47	1.45
	backbone N,Cα,C' of residues 1-7,17-47	0.31
	all heavy atoms of residues 1-7, 17-47	0.58
	Ramachandran plot statistics (%):
	residues in the most favoured region	67
	residues in the additionally allowed regions	31
	residues in the generously allowed regions	2
	residues in the disallowed regions	0

Biochemical Journal Immediate Publication. Published on 16 May 2007 as manuscript BJ20070130 © 2007 The Authors Journal compilation © 2007 Biochemical Society 28Table 3 .

 3 Statistical comparison of the CgNa structure with those of type I and type II toxins . PDB ID codes: 1AHL for ApA; 1APF for ApB; 1ATX for ATX Ia; 1SHI for Sh I. b PDB residue numbers of equivalent residues. On the left, the numbers corresponding to the different toxins already reported in the literature and on the right, the equivalent residue in CgNa. c RMSD between mean structures of the superimposed backbone atoms N, C α and C' of equivalent fragments d including and e excluding the unstructured flexible loop 8-16, respectively.

		Aligned residues b	RMSD to CgNa (Ǻ) c
	Type I a
	ApA	1-25,28-49 <=> 1-25,26-47	d 3.00/ e 1.30
	ApB	1-25,28-49 <=> 1-25,26-47	2.09/1.62
	ATX Ia	1-34,42-46 <=> 1-34,42-46	3.22/2.02
	Type II a
	Sh I	1-46 <=> 2-47	2.35/1.25

a

Biochemical Journal Immediate Publication. Published on 16 May 2007 as manuscript BJ20070130 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

This article is dedicated to the memory of the late Abel Aneiros. This work was supported by a CONACyT grant (J200.812-2004), a VIEP-BUAP grant (6/I/SAL/05) and the Dirección General de Investigación Científica y Técnica of Spain (Grants BIO2002-0374 and BQU2003-03550-C03-01) is also acknowledged. Supercomputing time from the CESGA is also acknowledged.


Mariarita De Felice
email: m.defelice@ibp.cnr.it

Valentina Aria

Luca Esposito

Mariarosaria De Falco

Biagio Pucci

Mosè Rossi

Francesca M Pisani

Dr Mariarita De

A novel DNA helicase with strand annealing activity from the crenarchaeon Sulfolobus solfataricus

Keywords: Genome stability, DNA recombination, DNA helicase, DNA pairing activity, Archaea, Sulfolobus solfataricus RQC, RecQ

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In this study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5 . The corresponding protein, named by us Hel112, was produced in Escherichia coli in soluble form, purified to homogeneity and characterised. Gel filtration chromatography and glycerol gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterisation of the two oligomeric species revealed that only the monomeric form has an ATPdependent 3'-5' DNA helicase activity, whereas, unexpectedly, both the monomeric and dimeric form possess DNA strand annealing capability. The Hel112 monomeric form is able to unwind forked and 3'-tailed DNA structures with high efficiency, whereas it is almost inactive on bluntended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

INTRODUCTION

The ability to faithfully duplicate genetic information is essential to all cells. To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. This is the reason why many human disorders are caused by defects in proteins involved in these processes. The importance of genome stability is evidenced by the difficulties that cells face when these mechanisms break down. There is a symbiotic relationship between DNA replication and recombination: the replication forks stalling due to damage on the genome is rescued through recombination events that ensure completion of the DNA replication process. Many aspects of DNA metabolic pathways, including both DNA replication and recombination, often involve steps that require unwinding of double-stranded DNA to form transient single-stranded intermediates, by the action of specific DNA helicases. They possess a distinct polarity, either 3' to 5' or 5' to 3' with respect to the strand on which the protein is presumed to translocate, deriving the energy from nucleotide triphosphate hydrolysis [START_REF] Delagouette | Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: Structures and properties of isolated helicases[END_REF]. Particular attention has been given to the DNA helicases of the RecQ super-family [START_REF] Hickson | RecQ helicases: caretakers of the genome[END_REF]. They are named after the 3'-5' DNA helicase RecQ of Escherichia coli and are evolutionarily conserved from bacteria to humans [START_REF] Bennett | Structure and function of RecQ DNA helicases[END_REF]. RecQ-like DNA helicases share a centrally located helicase domain of about 400 amino acids that includes seven signature motifs characteristic of a wide variety of nucleic acids helicases. Included among these conserved sequences are the Walker A and B motifs, which are required for binding and hydrolysis of ATP.

Outside of the helicase domain there is much less sequence similarity among the RecQ superfamily members. Nevertheless, there are two additional identifiable domains that are found in some, but not all, RecQ-like proteins. A conserved region of about 80 residues, apparently unique to the RecQ super-family, is located C-terminally to the helicase domain and is designed as RecQ Conserved (RQC, [START_REF] Morozov | A putative nucleic acidbinding domain in Bloom's and Werner's syndrome helicases[END_REF]). This domain has been suggested to be involved in protein interaction [START_REF] Brosh | Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity[END_REF] and DNA-binding [START_REF] Von Kobbe | Werner syndrome protein contains three structure-specific DNA binding domains[END_REF][START_REF] Liu | The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding[END_REF]. X-ray crystallographic analysis of E. coli RecQ protein has shown that the RQC region consists of an -helical Zn 2+ -binding module followed by a winged-helix (WH) domain [START_REF] Bernstein | High-resolution structure of the E. coli RecQ helicase catalytic core[END_REF]. This latter acts as a double-stranded DNA-binding motif in several proteins including the catabolite gene activator protein [START_REF] Gajiwala | Winged helix proteins[END_REF]. A second sequence feature found in certain RecQ-like DNA helicases is the Helicase and RNase D Conserved (HRDC) motifs that is located C-terminally to the RQC domain. The HRDC motif is poorly conserved at the primary sequence level but has a well-defined structural fold comprising five -helices and a number of basic residues that constitute a putative DNA-binding surface [START_REF] Liu | The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins[END_REF]. Whereas unicellular organisms express a single RecQ-like protein, more complex organisms express two or more. There are five human RecQ DNA helicases: 1) WRN, defective in Werner syndrome; 2) BLM, defective in Bloom syndrome, 3) RTS, defective in Rothmund-Thomson syndrome; 4) RecQ1; and 5) RecQ5 [START_REF] Harrigan | Human diseases deficient in RecQ helicases[END_REF]. Only the biochemical properties of BLM [START_REF] Mohaghegh | The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases[END_REF], WRN [START_REF] Brosh | Enzymatic mechanism of the WRN helicase/nuclease[END_REF], RecQ1 [START_REF] Cui | Characterization of the DNA-unwinding activity of human RecQ1, a helicase specifically stimulated by human replication protein A[END_REF], a Drosophila variant of RecQ5 [START_REF] Ozsoy | Analysis of helicase activity and substrate specificity of Drosophila RECQ5[END_REF], Saccharomyces cerevisiae Sgs1 [START_REF] Bennett | Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae[END_REF] and E. coli RecQ [START_REF] Harmon | Biochemical characterization of the DNA helicase activity of the Escherichia coli RecQ helicase[END_REF] have been carefully characterised. They all unwind DNA with a 3' to 5' polarity, but their substrate specificity is not identical. Several mechanisms have been proposed by which RecQ-like DNA helicases can help to prevent the breakdown of replication. These enzymes are thought to promote resolution of D-loops and double Holliday-junctions which originate when replication forks collapse due to the presence of a block in lagging strand [START_REF] Wu | The Bloom's syndrome helicase suppresses crossing over during homologous recombination[END_REF]. In addition, RecQ-like DNA helicases have been implicated in reverse branch migration resolving the four-way junctions (or "chicken-foot" structures) that originate when a DNA lesion in the leading strand produces regression of the replication fork [START_REF] Karow | The Bloom's syndrome gene product promotes branch migration of Holliday junctions[END_REF]. Unwinding of these secondary structures results in the leading strand bypassing the damage and avoids their processing by the action of Holliday junction resolvases and subsequent homologous recombination. On the other hand, BLM has been very recently proposed to promote the formation of the "chicken-foot" structures in vivo on the basis of its ability to catalyze regression of a model replication fork in vitro in an ATP-dependent manner [START_REF] Ralf | The Bloom's syndrome helicase can promote the regression of a model replication fork[END_REF].

Biochemical
Archaea, the third domain of life, share many similarities with Eukarya in their genetic information processing pathways even if they have a bacterial-like cellular structure [START_REF] Kelman | Archaeal DNA replication and repair[END_REF]. Therefore, they represent an attractive model system to understand the more complex mechanisms of the eukaryotic cell. Whereas the DNA replication machinery of both Archaea and Eukarya has been extensively investigated, still little is known about the biological function of the various DNA helicases that are involved in repair or restart at damaged replication forks and no clear RecQ homolog has been identified so far in Archaea.

Here we report the identification and biochemical characterization of a novel archaeal DNA helicase. This protein, referred to as Hel112, has been identified as a putative homolog of human RecQ5 by sequence similarity searches of the Sulfolobus solfataricus genome database.

Recombinant Hel112 shows two predominant stable oligomeric states: monomer and dimer. Only the monomeric form of Hel112 has a 3'-5' DNA helicase activity, preferring a forked DNA as the substrate. Both the monomer and the dimer possess strand annealing activity. The biochemical properties of purified Hel112 are described and discussed. transformed with the plasmid pET28a-Hel112 were grown at 37 °C in 5 litres of LB medium containing 30 g/ml chloramphenicol and 100 g/ml kanamicin. When the culture reached an A 600 nm of 0.8, protein expression was induced by adding isopropyl-1-thio--D-galactopyranoside at 0.2 mM. The bacterial culture was incubated at 37 °C for additional 2 hours. Then cells were harvested by centrifugation and the pellet (10 g) was stored at -20 °C until use. The cell pellet was thawed and suspended in 50 ml of buffer A (25 mM Tris-HCl, pH 8.0, 2.5 mM MgCl 2 , 500 mM NaCl, 15% glycerol) supplemented with a protease inhibitors cocktail (Sigma). Cells were broken by two consecutive passages through a French pressure cell apparatus (Aminco Co., Silver Spring, MD) at 1500 p.s.i. The resulting cell extract was treated with DNase I (at 0.25 mg/ml) and Rnase A (at 0.1 mg/ml) for 30 min on ice with shaking. Then, protamine sulfate (at 2 mg/ml) was added and incubation was continued for additional 30 min on ice with shaking. The sample was centrifuged for 30 min at 30,000 rpm (Beckman rotor 70.0 Ti) at 10 °C. The supernatant was subjected to heattreatment at 80 °C for 20 min, then kept in ice for 10 min. The thermo-precipitated proteins were removed by centrifugation for 30 min at 30,000 rpm (Beckman rotor 70.0 Ti) at 10 °C. The supernatant was passed through a 0.22-m filter (Millipore), mixed with Ni 2+ -nitrilotriacetic acid Superflow-agarose resin (Qiagen) pre-equilibrated in buffer A and incubated for 1 hour in ice with gentle shaking. The resin was washed with buffer A and elution was carried out with 100 ml of an imidazole gradient (25-500 mM) in buffer A. 4-ml fractions were collected and analysed by SDS-PAGE to detect the Hel112 polypeptide. Fractions containing the recombinant protein were pooled and concentrated using an Amicon system up to 6 mg/ml. The protein sample was dialysed overnight against buffer A containing NaCl at 100 mM. The sample was loaded onto an Heparin Sepharose column (column volume: 8 ml) at 0.5 ml/min using an AKTA system (GE Healthcare Life Sciences). The column was washed using the same buffer and bound protein was eluted using DNA substrates -The sequence of the synthetic oligonucleotides used to prepare the DNA substrates is reported in Table 1. Oligonucleotides were labelled using T4 polynucleotide kinase and [ - 32 P]ATP after the labelling reaction they were purified using a Micro Bio-Spin P-30 Tris

Biochemical

Chromatography Columns (Bio-Rad Laboratories), according to the manufacturer's instructions.

To prepare duplexes DNA molecules, mixtures were incubated for 5 min at 95 °C which contained the labelled and the complementary unlabelled oligonucleotides at a 1:2 molar ratio. Then, the samples were slowly cooled at room temperature. mM EDTA, 0.5 mg/ml proteinase K, 20 % glycerol, 0.1% bromophenol blue). Reaction products were separated on a 8% polyacrylamide gel in TBE containing 0.1% SDS at a constant voltage of 150 V. After the electrophoresis the gel was soaked in 20% trichloroacetic acid and analyzed by a phosphorimager. Reaction products were quantified and any free oligonucleotide in the absence of enzyme was subtracted.

DNA band-shift assays

Strand annealing assay -Reaction mixtures (10 l) contained partially or fully complementary synthetic oligonucleotides (each at a concentration of 2.5 nM), one of which was labelled at the 5'end with T4 polynucleotide kinase and [ - 32 P]ATP, and the indicated amounts of Hel112 in helicase assay buffer. Reaction mixtures were assembled on ice. Then, the protein was added to each mixture and the samples were incubated for the indicated times at 75 °C in a heated-top PCR machine to prevent evaporation and reactions were stopped by addition of 2.5 l of 5 x Stop Solution. Samples were subjected to gel electrophoresis as described for the DNA helicase assay.

Reaction products were quantified and any annealed oligonucleotide in the absence of enzyme was subtracted.

For the three-strand junction, samples (10 µl) contained a mixture of oligonucleotides (2.5 nM C80, 5 nM G80 and 5 nM G80bub; the C80 was labelled at the 5'-end with T4 polynucleotide kinase and

RESULTS

Identification of a novel Sulfolobus solfataricus DNA helicase -

The aim of this study was to identify a DNA helicase belonging to the RecQ super-family in the hyper-thermophilic archaeon Sulfolobus solfataricus and investigate its biochemical properties. Since the archaeal molecular machineries responsible for DNA replication/recombination/repair are evolutionarily related to the eukaryal counterparts [START_REF] Kelman | Archaeal DNA replication and repair[END_REF], we carried out BlastP [START_REF] Altschul | Gapped BLAST and PSI_BLAST: a new generation of protein database search programs[END_REF] searches of the S. solfataricus genome database using each of the five human RecQ-like DNA helicases as query sequence in order to identify putative RecQ homologs in this archaeal species. Only when human RecQ5 was used as the query sequence in these searches, ORFs putatively coding for DNA helicases (SSO 0112, 0017, 0965, and 0394) were identified. Among these sequences, SSO0112 (875 amino acid residues; named by us Hel112) gave the highest similarity score with human RecQ5 . The most conserved region between the two proteins is the N-terminal portion which contains the sequence similarity motifs typically found in the Super-Family 2 DNA helicases ( [START_REF] Gorbalenya | Helicases: amino acid sequence comparisons and structure-function relationships[END_REF]; sequence similarity 41 %). Differently from other RecQ proteins, Hel112 does not seem to contain the RQC and the HRDC) sequence motifs that are located C-terminally to the conserved DNA helicase domain.

Hel112 was over-produced in E. coli as a protein containing an hexa-Histidine tag at the Nterminus using the vector pET28a. The protein was found to be expressed at high level in soluble form and was purified using a procedure that included a thermal treatment of the cell extracts, chromatographic steps on a Ni + -chelate resin, Heparin Sepharose, and gel filtration on a Superdex 200 column, as described under Experimental. The fractions of the gel filtration chromatography, carried out in a buffer containing 200 mM NaCl, were analysed by SDS-PAGE (Fig. 1A); this analysis revealed that Hel112 possesses two different oligomeric states with an apparent molecular mass of about 65 and 140 kDa. Since Hel112 theoretical molecular weight is calculated to be 100 kDa, we suppose that the two oligomeric states correspond to the monomeric and dimeric form of the protein in solution. In order to determine whether the two forms of Hel112 were In order to analyse the biochemical properties of Hel112, DNA unwinding assays were carried out using different kinds of DNA duplex molecules. The activity assays were performed at 60 °C (and not at 75 °C) to reduce the spontaneous melting of the probe. We first analyzed the pool recovered from the Ni + -chelate column but no DNA helicase activity was detected even using 1.5 µg of the protein sample on different substrates (data not shown). Then, we decided to test the DNA unwinding activity of the peak fractions eluted from the gel filtration column using two partially complementary oligonucleotides forming forked DNA molecules as the substrates (see Table 1).

As shown in Fig. 2A, an evident displacement of the probe was observed when Hel112 in the monomeric state (gel filtration fraction # 74, see Fig. 1) was assayed. In contrast, the dimeric species (corresponding to the gel filtration # 65, see Fig. 1) did not display the ability to melt the fork DNA molecules (Fig. 2B-C). Surprisingly, the fraction # 65 seemed to catalyse annealing instead of unwinding of DNA molecules (Fig. 2B-C). Furthermore, DNA pairing activity was detected when fraction # 74 was assayed. To better investigate this issue strand annealing assays were carried out on both the gel filtration peak fractions using the fork DNA as the substrate. To avoid melting of double-stranded DNA due to the DNA unwinding activity of the protein, ATP was not present in the reaction mixtures. This analysis revealed that Hel112 in either monomeric or dimeric form possesses DNA pairing activity (see Fig 2 D-F).

To perform a more accurate analysis of Hel112 oligomeric state, glycerol gradient sedimentation analyses were carried out. Samples of the gel filtration peak fractions were subjected to ultracentrifugation through a preformed glycerol gradient. As shown in Fig. 1B, the protein of the peak fraction # 74 behaves as a monomer (expected molecular size of 100 kDa) and the protein of the peak fraction 65 was detected in a position of the gradient (between catalase and aldolase protein markers) compatible with a dimeric structure.

The monomeric form of Hel112 has ATP-dependent 3'-5' DNA helicase activity -The gel

filtration peak fraction corresponding to Hel112 monomeric form (fraction # 74 in Fig. 1) was used to more deeply characterise the DNA helicase activity. To determine the directionality of Hel112 helicase DNA unwinding assays were carried out using a substrate with a single-stranded region at either 3'-or 5'-end (3'-mis and 5'-mis, respectively). As shown in Fig. 3A, Hel112 is able to utilise only the substrate bearing a 3'-tail suggesting that the protein is able to translocate onto the DNA in the 3' to 5' direction. In addition, in the absence of ATP Hel112 was found to be devoid of any DNA unwinding capability indicating that the 3'-5 DNA helicase activity is ATP-dependent (Fig. 3B).

To analyse the substrate preference of Hel112, various kinds of DNA substrates were used in the unwinding assays, such as fork, bubble and blunt-ended molecules. As shown in Fig. 4A, fork and orthophosphate released was determined by means of a phosphorimager system and plotted versus the amounts of Hel112 used as either a monomer or a dimer (Fig. 5B). As indicated by the plots, the two oligomeric forms of Hel112 have a comparable level of ATPase activity. The ATPhydrolysis function is not stimulated in the presence of either single-or double-stranded DNA (data not shown).

Hel112 has strand annealing activity -The Hel112 strand pairing activity was tested on different DNA substrates. An initial kinetic analysis was carried out using partially complementary oligonucleotides forming the fork DNA and a fixed amount of monomeric Hel112 (100 ng). As reported in Fig. 6A-B, Hel112 promoted annealing of about 50% of the single-stranded molecules by 2 min. On the other hand, in the absence of protein spontaneous melting of the paired oligonucleotides was observed, since samples were incubated at 75 °C due to the thermophilic nature of Hel112. To verify that the observed strand pairing was truly due to a specific catalytic function of Hel112, we carried out parallel control assays using S. solfataricus MCM DNA helicase, produced and characterised in our laboratory [START_REF] Carpentieri | Physical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sulfolobus solfataricus[END_REF]. In the control reactions performed with this hexameric DNA helicase no difference was observed with the sample not containing protein (data not shown). In addition, DNA pairing assays were performed with increasing amounts of either monomeric or dimeric Hel112 at 75 °C for 2 min. Plots of annealed probe versus the amount of protein used are shown in Fig. 6D-E. It can be noticed that the strand annealing activity of Hel 112 monomeric and dimeric form is comparable on all the substrates used in these enzymatic assays (Fig. 6C).

Then, we decided to determine whether Hel112 was able to combine DNA pairing and unwinding to catalyse strand-exchange reactions, as recently reported for RecQ-like DNA helicases [START_REF] Machwe | RecQ family members combine strand pairing and unwinding activities to catalyze strand exchange[END_REF]. To this end, we utilised a fork substrate containing a 54-bp duplex region and 26-nt single-stranded 5'and 3'-tails, which was made from a 5'-labelled 80-mer oligonucleotide (named C80) annealed to an unlabeled partially complementary 80-nt oligomer (named G80F). Assays were carried out with the Hel112 monomeric form using this fork substrate in combination with a third unlabeled 80-nt oligonucleotide (named G80B). The G80B oligonucleotide is complementary to both ends of C80, apart from a central non-complementary stretch (21-nt), and its 5'-end can pair with the 3'-tail of C80 to form a three-strand junction structure, as previously described [START_REF] Machwe | RecQ family members combine strand pairing and unwinding activities to catalyze strand exchange[END_REF]. Displacement of the G80F oligonucleotide from the three-strand junction structure together with annealing of G80B 3'end to C80 5'-end produces a blunt-ended duplex with a central 21-nt bubble. In these assays, equimolar amounts of the above described fork substrate and the partially complementary G80B detected corresponding to a three-strand junction structure. On the other hand, the formation of the bubble-containing duplex was not observed as the result of enzyme-catalysed strand-exchange, even when the G80B was present in molar excess with respect to the forked DNA substrate and the reaction mixtures were incubated up to 30 min after the addition of ATP (data not shown).

Therefore, Hel112 can promote strand-annealing also on DNA substrates with a more complex structure, but it seems unable to catalyse strand-exchange reactions, as recently reported for RecQ-like DNA helicases [START_REF] Machwe | RecQ family members combine strand pairing and unwinding activities to catalyze strand exchange[END_REF].

Hel112 monomer and dimer show different DNA binding affinity -To further biochemically

characterise Hel112, gel mobility shift assays were performed using either the monomeric or the dimeric form of the protein and various kinds of DNA ligands. As shown in Fig. 7, Fork and 3'-mis DNA are bound by Hel 112 in either monomeric or dimeric state with higher affinity. On the other hand, a clear difference in the DNA-binding capability is observed between the two forms of the protein on both single-and double-stranded DNA: the monomer binds these molecules with a very low affinity, whereas the DNA binding capability of the Hel 112 dimeric form is negligible on both these ligands.

Hel112 in the monomeric state is able to bind single-stranded DNA with an affinity lower than the one observed for the fork and 3'-mis DNA. In contrast, Hel112 in the dimeric form binds singlestranded DNA with very low affinity (only about 10% of the input DNA is shifted using 200 ng of protein, Fig. 7B-C). The difference in the DNA-binding capability between the two forms of the protein is more evident on double-stranded DNA: the monomer is able to bind these DNA molecules with low affinity (only 40 % of the total DNA is shifted by 400 ng of protein), whereas the dimer does not display any DNA-binding capability on this ligand.

Biochemical

DISCUSSION

The Hel112 protein described in this study has been identified by a sequence similarity search of the Sulfolobus solfataricus genome database using human RecQ5 as the query sequence. Our biochemical analysis revealed that Hel112 possesses catalytic properties shared by RecQ superfamily members. First of all, Hel112 can exist in two stable oligomeric states (monomer and dimer), but only the monomeric form displays a DNA unwinding activity which operates in the 3' to 5' direction. Similarly, RecQ proteins are monomeric, although E. coli RecQ, human BLM and WRN were also reported to form oligomeric assemblies in certain experimental conditions [START_REF] Harmon | Biochemical characterization of the DNA helicase activity of the Escherichia coli RecQ helicase[END_REF][START_REF] Karow | Oligomeric ring structure of the Bloom's syndrome helicase[END_REF][START_REF] Xue | A minimal exonuclease domain of WRN forms hexamer on DNA and possesses both 3'-5' exonuclease and 5'-protruding strand endonuclease activities[END_REF].

Hel112 is able to unwind forked and 3'-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. These properties are shared by human BLM and WRN and Drosophila and human RecQ5 . In contrast, E. coli RecQ is able to unwind blunt-ended DNA as well as bubble-containing structures [START_REF] Bennett | Structure and function of RecQ DNA helicases[END_REF]. Furthermore, S.

solfataricus Hel112 is endowed with an efficient strand annealing activity that promotes the formation of various duplex DNA molecules starting from fully or partially complementary synthetic oligonucleotides. This peculiar feature has been recently described for RecQ-like proteins of metazoans, including WRN, BLM, RecQ1 and RecQ5 [START_REF] Garcia | Human RECQ5 , a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF][START_REF] Sharma | Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1[END_REF][START_REF] Cheok | The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA[END_REF].

Nonetheless, Hel112 lacks both the QRC and HRDC sequence motifs that are typically found in RecQ super-family members [START_REF] Bennett | Structure and function of RecQ DNA helicases[END_REF]. The sequence similarity between human RecQ5 is limited to the to the N-terminal half of the polypeptide chain which contains the sequence similarity boxes of the Super-Family 2 DNA helicases [START_REF] Gorbalenya | Helicases: amino acid sequence comparisons and structure-function relationships[END_REF]. All that considered, Hel112 can not be strictly classified within the RecQ Super-Family, although it displays biochemical features and catalytic properties that are hallmark of the RecQ-like DNA helicases. Despite the proposed critical cellular role and their ubiquitous distribution among Bacteria and Eukarya, clear RecQ homologs have not yet been identified in Archaea. Nevertheless, proteins responsible for Holliday junction migration were characterized from Pyrococcus furiosus (Hjm, [START_REF] Fujikane | Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon Pyrococcus furiosus[END_REF]) and M. thermoautotrophicus (ORF Mth810,

  Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society EXPERIMENTAL Materials -All chemicals were of reagent grade. Restriction and modification enzymes were from New England Biolabs. Radioactive nucleotides were purchased from GE Healthcare Life Sciences. Oligonucleotides were synthesised by Proligo (Paris, France).Production of Hel112 in E. coli -The Hel112 gene was amplified from S. solfataricus P2 genomic DNA by PCR using the Vent polymerase (New England Biolabs) with the oligonucleotide Hel112 NheI (5'-GGTTGCTAGCTTGAGCAATACTTACTTCTACTCTGATGAAG -3') as the 5' primer (the engineered NheI site is underlined and the Hel112 initiation codon is in bold) ) as the 3' primer (the engineered BamHI site is underlined). The PCR product was cloned into NheI/BamHI-linearised E. coli expression vector pET28a (Novagen) and sequenced. E. coli Rosetta cells (Novagen)

-

  For each substrate, 10 µl mixtures were prepared which contained: 200 fmol of [ 32 P]-labelled DNA in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.5 mM MgCl 2 , 0.7 mM 2-mercaptoethanol, and the indicated amounts of Hel112. Following incubation for 10-15 min at room temperature, complexes were separated by electrophoresis through 5% polyacrylamide/bis gels (37.5:1) in 0.5 x TBE. Gels were dried down and analyzed using a Storm PhosphorImager (Molecular Dynamics; Image Quant software). DNA helicase assay -Reaction mixtures (10 l) contained 50 fmol of [ 32 P]-labelled substrate (about 1 x 10 3 cpm/fmol) and the indicated amounts of Hel112 (gel filtration peak fractions corresponding to the monomeric or the dimeric form of the protein) in helicase assay buffer (25 mM Tris-HCl, pH 8.0, 2.5 mM 2-mercaptoethanol, 50 mM sodium acetate, 5 mM MgCl 2 ) containing 5 Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society mM ATP. Samples were incubated for 30 min at 60 °C in a heated-top PCR machine to prevent evaporation and reactions were stopped by addition of 2.5 l of 5 x Stop Solution (0.5% SDS, 40

[ - 32 P

 32 ]ATP), increasing amounts of Hel112 gel filtration peak fraction corresponding to the monomer[START_REF] Brosh | Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity[END_REF][START_REF] Ozsoy | Analysis of helicase activity and substrate specificity of Drosophila RECQ5[END_REF][START_REF] Cheok | The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA[END_REF] 50, 100 and 200 ng) in helicase buffer. Samples were incubated for 10 min at 60 °C. Then ATP was added at 5 mM into each sample, incubation was continued for additional 15 min at 60 °C and stopped with 2.5 µl of 5 x Stop Solution. Samples were subjected to gel electrophoresis as described for the DNA helicase assay.ATPase assay -The ATPase reaction mixture (10 l) contained 100 M [ -32 P]ATP (0.5-1 Ci) and the indicated amounts of Hel112 (gel filtration peak fractions corresponding to the monomeric or dimeric form of the protein). Incubation was performed for 30 min at 60°C in a heated-top PCR machine to prevent evaporation and stopped in ice. A 1-l sample of each mixture was spotted onto a polyethyleneimine-cellulose thin layer plate (Merck) and run in 0.5 M LiCl and 1 M formic acid. The [ -32 P]ATP hydrolysed to [ 32 P]orthophosphate was quantified using a phosphorimager.The rate of ATP hydrolysis was determined in the linear range of reaction time and protein concentration dependence. The amount of spontaneously hydrolysed ATP was determined using blank reactions without enzyme and subtracted from the reaction rate values calculated as above. Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  stable, samples of the peak fractions (fraction # 65 corresponding to the dimer and fraction # 74 corresponding to the monomer) were subjected to analytical gel filtration chromatography on a Superdex 200 column (GE Healthcare Life Sciences; unpublished observations). They both eluted as single peaks corresponding to molecular globular species of 65 and 140 kDa suggesting that both the monomeric or the dimeric form are stable in solution. When the gel filtration analyses were carried out in different salt conditions (0.5 M and 1 M NaCl), the stability of the dimeric form was affected and it was converted into the monomeric one (data not shown). In addition, a sample of purified dimer was subjected to a prolonged incubation with -mercaptoethanol (at 5 mM) and, then, analysed by gel filtration. Treatment with this sulphydryl-reducing agent did not affect the oligomeric state of Hel112 revealing that disulphide bonds are not required for the formation of the protein dimeric assembly. Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

3 '

 3 -tail DNA are the Hel112 favourite substrates, while no activity was detected on bubble or bluntended DNA. Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society Both monomeric and dimeric forms of Hel112 possess ATPase activity -The ATPase activity of the two Hel112 oligomeric forms was analysed, as shown in Fig. 5. The amount of [ 32 P]

  oligonucleotide were incubated with increasing amounts Hel112 monomer (from 5 to 200 ng) for 10 min at 60 °C in the absence of ATP. Then, ATP was added to the reaction mixtures and the Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society samples were incubated for additional 15 min. As shown in Fig 6F a slower migrating product was

  Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

[ 32 ]Fig. 1 .

 321 Fig. 1. Oligomeric state of Hel112. (A) The Hel112 pool from the Heparin Sepharose chromatography was run on a Superdex 200 HR 16/60 gel filtration column, as described in Experimental. A 20-µl sample of each fraction (1.0 ml) was loaded on a SDS polyacrylamide gel which was stained with Coomassie Blue. Arrows indicate the position of two protein markers used to calibrate the column (bovine serum albumin, BSA; and aldolase, Ald). (B) Samples of the gel filtration fraction # 65 and # 74 were fractionated on a glycerol gradient as described in Experimental. Aliquots of each fraction were analysed by SDS-PAGE followed by Coomassie Blue staining. Arrows indicate the position of protein markers fractionated on a parallel gradient. (C) A plot of the S values versus the position in the gradient for each marker protein is reported. S values for the protein in the peak fraction # 65 and # 74 were estimated to be 8.5 and 5.7, respectively.

Fig. 2 .

 2 Fig. 2. Catalytic activities of Hel112 dimeric and monomeric form. Enzymatic assays were carried out using the Hel112 dimeric and monomeric form. Increasing amounts of each protein (1.4, 7, 14, 70, 140 and 280 ng) were used either in DNA helicase (A-B) or in DNA pairing (D-E) assays. The labelled single-stranded DNA was quantified using a phosphorimager system and plotted versus the amount of protein used. The labelled single-stranded DNA present in the blank reaction was considered as 0 or 100 % in the plots shown in C and F, respectively. Plots obtained for the DNA helicase (C) and DNA pairing (F) assays are shown. Symbols used are as follows: diamonds, monomeric Hel112; circles, dimeric Hel112. ssDNA stands for single-stranded DNA. The enzymatic assays were performed at least in triplicate and the results were averaged. The error bars of the graphs are the standard error of the mean.

Fig. 3 .

 3 Fig. 3. DNA helicase directionality and ATP-hydrolysis dependency. (A) Increasing amounts of monomeric Hel112 (100, 200, 400 and 800 ng) were used in DNA unwinding assays with 3'-mis or 5'-mis DNA molecules as the substrates. (B) To check the DNA helicase dependency on ATPhydrolysis, increasing amounts of monomeric Hel112 (100, 200, 400 and 800 ng) were assayed in the presence or absence of ATP on the same 3'-mis DNA substrate. Dots on the oligonucleotides represents the radio-labelled phosphate group at the 5'-end.

Fig. 4 .

 4 Fig. 4. DNA helicase activity of Hel112 on various DNA substrates. (A) Increasing amounts of Hel112 in the monomeric form (25, 50, 100, 200, 400 and 800 ng) were used to analyse the DNA unwinding activity on various DNA molecules, as described under Experimental. (B) The displaced oligonucleotide was quantified by a phosphorimager system and plotted against the amount of protein used. Symbols used are: squares, 3'-mis; diamonds, fork; circles, bubble; triangles, double-

Fig. 5 .

 5 Fig. 5. ATPase activity of Hel112. ATPase assays were performed, as described in Experimental. (A) Increasing amounts (5, 10, 25, 50, 100 and 250 ng) of Hel112 monomer (triangles) or dimer (squares) were used. The orthophosphate released during the ATP hydrolysis was plotted versus the amount of protein used. The enzymatic assays were performed at least in triplicate and the results were averaged. The error bars of the graphs are the standard error of the mean.

Fig. 6 .

 6 Fig. 6. DNA annealing activity of Hel112. (A) Annealing of partially complementary oligonucleotides to form bubble DNA molecules was carried out in reaction mixtures containing (or not) monomeric Hel112 (100 ng). (B) The percentage of annealed probe was plotted versus the reaction time in the absence (circles) or presence of monomeric Hel 112 (squares). The annealed probe present in the blank reaction was considered as 0 %. The enzymatic assays were performed at least in triplicate and the results were averaged. The error bars of the graphs are the standard error of the mean. (C) Different DNA molecules were used to analyse the substrate specificity of monomeric or dimeric Hel112 in the annealing reaction. (D-E) The annealed probe was plotted versus the amount (0, 10, 25, 50, 100 ng) of Hel112 in the monomeric (D) or dimeric (E) form. The annealed probe present in the blank reaction was considered as 0 %. Symbols used are as follows: circles, double-stranded DNA; squares, fork; triangles; 3'-mis; diamonds, bubble DNA. The enzymatic assays were performed at least in triplicate and the results were averaged. The error bars of the graphs are the standard error of the mean. (F) Increasing amounts of monomeric Hel112 (5, 10, 30, 50, 100, 200 ng) were tested in strand exchange assays as described in the text. TSJ stands for three-strand junction.

Fig. 7 .Table 1 .

 71 Fig. 7. DNA binding activity of Hel112. The ability of Hel112 to bind different 32 P-labeled synthetic oligonucleotides was analysed by gel shift assays as described under Experimental. (A) Increasing amounts of Hel112 (50, 100, 200 and 400 ng) were used in the presence of the indicated DNA molecules. For each DNA ligand used a plot is reported of the shifted DNA versus the amount of monomeric (B) or dimeric (C) protein used. Symbols used are as follows: open squares, bubble; filled squares, fork; filled triangles, double-stranded DNA; open triangles, singlestranded DNA; circles, 3'-mis. The enzymatic assays were performed at least in triplicate and the results were averaged. The error bars of the graphs are the standard error of the mean.

Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society a

  linear gradient of NaCl in buffer (from 0.1 to 1 M NaCl). Fractions containing the recombinant Hel112 were pooled and concentrated. The sample was then loaded onto a Superdex 200 16/60 coulmn (GE Healthcare Life Sciences) equilibrated in buffer A containing NaCl at 200 mM. The column was developed at a flow rate of 0.3 ml/min. 1-ml fractions were collected. A 20-µl sample of each fraction was analysed by SDS-PAGE. Fractions containing the Hel112 protein were stored at 4 °C for subsequent biochemical analyses. The column was calibrated by running a set of gel

filtration markers that included: tyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa) and bovine serum albumin (BSA, 69 kDa).

Glycerol gradient sedimentation -

Glycerol gradient ultracentrifugation was performed by applying the protein sample (60 µg of fraction # 74 and 30 µg of fraction # 65 in 150 µl) to a 8 ml preformed 20-40 % glycerol gradient in buffer containing 25 mM Tris-HCl pH 8.0, 2.5 mM MgCl2, 200 mM NaCl. A parallel gradient was used to fractionate a mixture of standard proteins which included: catalase (232 kDa, 11.3 S), aldolase (158 kDa, 7.3 S), bovine serum albumin (67 kDa, 4.6 S) and ovalbumin (43 kDa, 3.5 S). After centrifugation at 38,000 rpm for 18 h in a Beckman SW41 rotor at 10 °C, fractions (200 ml) were collected from the bottom of the tube and analysed by SDS-PAGE followed by Coomassie Blue Staining of the gel.

Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGMENTS

This work was partially supported by grants from Agenzia Spaziale Italiana (Project MoMa N°1 /014/06/0) and from ATIBB-BioTekNet Centro Regionale di Competenza in Biotecnologie Industriali. Biochemical Journal Immediate Publication. Published on 24 Jul 2007 as manuscript BJ20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society