Preparation and characterization of geodin A bc-crystallin-type protein from a sponge Concetta Giancola 1 , Elio Pizzo 2 , Antimo Di Maro 3 , Maria Vittoria Cubellis 2 and Giuseppe D’Alessio 2 1 Department of Chemistry, University ‘Federico II’ of Naples, Italy 2 Department of Biological Chemistry, University ‘Federico II’ of Naples, Italy 3 Department of Life Sciences, Second University of Naples, Italy Vertebrate crystallins are proteins that last an entire life- time, tightly packed in the eye lens which they provide with the appropriate refractive index essential for vision. There are three families of crystallins: the a-crystallins, complex multimers made up of small proteins which perform also chaperon-like functions; and the b- and c-crystallins, which together constitute a superfamily of homologous proteins including monomeric c-type and oligomeric b-type proteins [1]. bc-Crystallins have been extensively studied as models of molecular evolution [2–5] and for their structural features [6,7]. The 3D structures of many members of the superfamily have been determined by X-ray crystallography [8–11], and NMR [12,13]. They indicate that the smallest structural unit in the bc-crystallin superfamily is a b-stranded Greek key motif, with two such motifs making up a domain; two domains connected by a peptide linker constitute a c-crystallin-type monomer, or a subunit of a b-crystallin-type oligomer. A long evolutionary history can be traced for bc-crystallins, back to single-domain homologues from moulds [14], and bacteria [12], and a two-domain homologue from amphibians [15]. Recently, a bc-crys- tallin-type gene from a sponge, Geodia cydonium, has been identified [16] and cloned [17]. The finding that this is an intron-less gene [17] (as compared with ver- tebrate bc-crystallin genes, all of which are endowed with several introns) and the very early divergence of porifera ) the most primitive metazoans ) support the idea that the protein encoded by this gene, called geo- din, is the most ancient member of the metazoan bc-crystallin-type superfamily. Thus, it appeared to be of interest to inspect the structural features of this pro- tein, to verify the hypothesis that the same structural Keywords geodin; bc-crystallins; metazoans; calorimetry; protein stability Correspondence G. D’Alessio, Department of Biochemistry, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy Fax: +39 081 5521217 Tel: +39 081 2534731 E-mail: dalessio@unina.it (Received 4 November 2004, revised 30 November 2004, accepted 20 December 2004) doi:10.1111/j.1742-4658.2004.04536.x Geodin is a protein encoded by a sponge gene homologous to genes from the bc-crystallins superfamily. The interest for this crystallin-type protein stems from the phylogenesis of porifera, commonly called sponges, the earliest divergence event in the history of metazoans. Here we report the preparation of geodin as a recombinant protein from Escherichia coli, its characterization through physico-chemical analyses, and a model of its 3D structure based on homology modelling. Geodin is a monomeric protein of about 18 kDa, with an all-beta structure, as all other crystallins in the superfamily, but more prone to unfold in the presence of chemical denatu- rants, when compared with other homologues from the superfamily. Its thermal unfolding, studied by far- and near-CD, and by calorimetry, is des- cribed by a two-state model. Geodin appears to be structurally similar in many respects to the bacterial protein S crystallin, with which it also shares a significant, albeit more modest stabilizing effect exerted by calcium ions. These results suggest that the crystallin-type structural scaffold, employed in the evolution of bacteria and moulds, was successfully recruited very early in the evolution of metazoa. Abbreviations DSC, differential scanning microcalorimetry; GuHCl, guanidine hydrochloride; TFA, trifluoroacetic acid. FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1023 scaffold was used in the evolution of bacteria and moulds, and then very early recruited in the metazo- ans. Here we report the expression and purification of recombinant geodin and its thermodynamic and spect- roscopic characterization, and propose a putative structure of the protein, as derived through homology modelling. Results and Discussion Preparation of recombinant geodin As described in Experimental procedures, E. coli cells transformed with the cDNA encoding geodin, the putative bc-crystallin-type protein from the sponge G. cydonium (UNIPROT accession number: O18426), were lysed by sonication followed by centrifugation. By SDS ⁄ PAGE, both the resulting supernatant and pellet were found to contain a protein of  18 kDa, expected for geodin on the basis of the amino acid sequence encoded by the available gene sequence (Fig. 1). Thus, both fractions were investigated: the soluble fraction (S preparation) and the pelleted fraction, after solubilization and renaturation (see Methods), called IB preparation. The S and IB preparations were fract- ionated in parallel by size exclusion chromatography followed by ion exchange chromatography. The chro- matography runs, described in the Experimental proce- dures, are illustrated in Fig. 2. When the proteins purified from the S or IB prep- arations, respectively, were analysed by SDS ⁄ PAGE, they were both found to contain a single protein with the molecular size of geodin (Fig. 1). Identical results were obtained when the two preparations 100 kDa 66 kDa 14 kDa 8 kDa 18 kDa M1 2 34 Fig. 1. SDS ⁄ PAGE of protein preparations from the lysate of E. coli cells transformed with geodin encoding cDNA. Lane 1, IB prepar- ation from inclusion bodies; lane 2, S preparation from the lysate soluble fraction; lane 3, geodin purified from the IB preparation; lane 4, geodin purified from the S preparation. A B C D Fig. 2. Gel filtration on Superdex G-75 of the proteins from the S preparation (A) and the IB preparation (B) from the lysate of E. coli cells transformed with geodin encoding cDNA. The inserts show the results of SDS ⁄ PAGE separations of individual fractions from each column as indicated by fraction numbers. Fractions 48 through 56 were pooled and dialysed. Each pool was then chroma- tographed on a cation exchange column (Resource-S) as illustrated in C (for the S preparation) and D (for the IB preparation). Geodin, a sponge bc-crystallin-type protein C. Giancola et al. 1024 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS were subjected to RP-HPLC on a C4 column as des- cribed in Methods. Also by this procedure, the pro- tein, isolated from the S or IB preparation, was found to be homogeneous and eluted in a single, symmetrical peak from the HPLC column (data not shown). The two proteins eluted from the HPLC column, analysed by MALDI-TOF MS, were found to have the following molecular masses: 17 788, protein puri- fied from S preparation; 17 785 protein purified from IB preparation. These values compare very satisfactor- ily with the geodin mass value calculated from its amino acid sequence (17 781 kDa). The amino acid sequence determination carried out on the proteins purified from either the S or IB prepa- rations gave the following N-terminal sequence (one- letter code), identical for both protein preparations: NH 2 –STAKVTLVTSGGSSQDFT–, which is exactly the sequence deduced for the N terminus of the protein encoded by the geod gene from G. cydonium. These results indicated that: (a) the same protein was contained in both the S and IB protein prepara- tions, as derived from the soluble and insoluble frac- tions, respectively, of the E. coli lysate; (b) this protein was geodin, the expression product of the geod gene from the sponge G. cydonium. Thus, a single form of geodin was expressed by E. coli under the conditions described above. The finding that the expressed recombinant geodin was distributed between cytosol and inclusion bodies of transformed E. coli cells can be explained by considering that the E. coli synthetic machinery allowed the production of free, soluble geodin up to a solubility limit, beyond which the excess protein was sequestered in inclusion bodies. Stability against urea and guanidinium chloride Figure 3 shows the CD spectra of geodin in far-UV and near-UV at T ¼ 20 °C and pH 5.0 and 7.0, respectively. At both pH values, the far-UV CD spec- trum of the protein exhibits a strong positive band at 217 nm, which indicates a well defined b-conformation in solution. This suggests that geodin is an all-b pro- tein. The different protonation state apparently does not affect the secondary structure, as no drastic chan- ges are observed in the far-UV signal (Fig. 3). In the near-UV region the effects of protonation are signifi- cant in the protein tertiary structure with a higher exposure of chromophors at pH 5.0. As for vertebrate crystallins ) such as mammalian cB and bB2 crystallins ) geodin tends to aggregate, especially at higher temperatures and concentrations. However, because at relatively high concentrations geodin was more soluble at pH 5.0 than at pH 7.0, a pH value closer to geodin pI value of 7.9, as calculated for from its amino acid sequence, we chose to study the thermodynamic properties of the protein at pH 5. Geodin stability against chemical denaturants was investigated by measuring the molar ellipticity at 217 nm, and the shift in fluorescence maximum wave- length, as a function of urea or guanidine (GuHCl) concentration. As shown in Fig. 4, all denaturation curves have monophasic, sigmoidal shapes with single midpoints of denaturation determined at 3.5 m and 1.2 m for urea- and GuHCl-induced denaturation, respectively. When the denatured protein solutions were dialysed, their far-UV CD spectra were found to be identical to that of the native protein. This indicates that the unfolding of geodin as induced by chemical denaturants is reversible. Fig. 3. Far-UV (A) and near-UV (B) CD spec- tra of geodin at pH 5.0 (solid lines) and pH 7.0 (dashed lines). C. Giancola et al. Geodin, a sponge bc-crystallin-type protein FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1025 Geodin appears to be very sensitive to chemical denaturation, in fact more sensitive than a typical crystallin-type protein of the vertebrate c family (Table 1). In particular, with guanidinium ⁄ HCl as a denaturant, the midpoint of denaturation determined for geodin is lower than those determined for other monomeric crystallin-type proteins, but very close to that determined for spherulin 3a. The latter crystal- lin-type protein is a homodimeric protein in which two single-domain protomers associate into the topo- logical equivalent of a monomeric crystallin. Thus, the finding of a similar dependence from chemical denaturation between geodin and spherulin 3a might be suggestive of weaker inter-domain interactions in geodin, weaker than those at the interfaces of monomeric crystallins, but comparable with those in a noncovalent two-domain homologue such as spherulin 3a. As for the denaturation of geodin in urea, as com- pared with other monomeric crystallin-type proteins, our findings (Table 1) suggest that the stability of geo- din in urea resembles that of protein S rather than that of a mammalian crystallin such as cS-crystallin, a qualitative, indirect indication of a closer structural relationship between geodin and protein S. Geodin also displays a single denaturation midpoint, detectable at pH 5, but with no aggregation at any urea concen- tration (Fig. 4, Table 1). Fig. 4. (A, B) Urea and (C, D) GuHCl-induced transitions of geodin at pH 5.0. The transi- tions were monitored by the shift in the wavelength corresponding to the maximum of the fluorescence spectrum (A and C), and by far-UV CD at 217 nm (B and D). Geodin, a sponge bc-crystallin-type protein C. Giancola et al. 1026 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS Stability against temperature When the thermal denaturation of geodin was investi- gated by the dichroic absorption in far- and near-UV (Fig. 5), at 217 nm and 280 nm, respectively, the curves showed cooperative transitions in agreement with a two-state model. For both, the midpoint tem- perature was found to be 61 °C, which indicates a sim- ultaneous collapse of secondary and tertiary structures. The calorimetric profile obtained by differential scan- ning microcalorimetry (Fig. 6), was not influenced by protein concentration, although the endothermic peak was followed by an exothermic process that coincides with aggregation and precipitation, which lead to an irreversible overall denaturation process. A description of an irreversible protein denaturation is in the model proposed by Lumry and Eyring [18]: N $ U $ F where N is the native protein, U is the reversibly unfolded protein and F is the final, irreversibly dena- tured protein. Starting from the Lumry–Heyring model, Sanchez-Ruiz [19] has shown that when the transition is calorimetrically irreversible, but the irre- versible step takes place with a significant rate at a temperature even slightly above those corresponding to the transition, the equilibrium thermodynamic analysis is permissible. In this case the unfolding process: N ! K U is described by the van’t Hoff equation: @ ln K @T  P ¼ DH 0 RT 2 ð1Þ where K is the equilibrium constant and DH 0 is the enthalpy that determines the variation of K with the absolute temperature. The integrated form can be writ- ten as: K ¼ exp  DH 0 ðTÞ R   1 T  1 T m  ð2Þ At each temperature value, the enthalpy of this ther- modynamic system can be described as: Table 1. Physico-chemical data for geodin and other two-domain crystallin-type proteins. Values of T m , DH 0 and DG 0 , determined by DSC, are from reference [39] for protein S, and reference [26] for spherulin 3a and human-cS. Data for denaturation with chemical denaturants were obtained by CD spectroscopy for protein S [22], spherulin 3a and human-cS [25]. c 1 ⁄ 2 GuCl ( M) c 1 ⁄ 2 urea ( M) T m (°C) DH 0 (kJÆmol )1 ) DG 0 (293 K) (kJÆmol )1 ) Geodin 1.2 a 3.5 a 61.0 c 532 41 1.2 b 3.6 b 60.5 d +Ca 2+ 65.0 c,d 570 48 Human-cS 2.6 8.0 75 750 84 Protein S 1.7 3.7 52 ⁄ 64 e 399 ⁄ 263 29 ⁄ 16 +Ca 2+ 1.9 4.8 64 ⁄ 65 e 454 ⁄ 332 39 ⁄ 25 Spherulin 3a 1.1 53.3 523 81 +Ca 2+ 2.5 68.7 1020 137 a Data from near-UV CD spectroscopy. b Data from fluorescence spectroscopy. c Data from near- and far-UV CD spectroscopy. d Data from DSC. e Values for first ⁄ second transition. Fig. 5. Temperature-induced unfolding transitions at pH 5.0 monit- ored by far-UV CD (A) and near-UV CD (B). C. Giancola et al. Geodin, a sponge bc-crystallin-type protein FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1027 HðTÞ¼f N H N þ f D H D ¼ H N þ f D ðH D  H N Þ ¼ H N þ f D DH 0 ðT m Þð3Þ where f N and f D are the fractions of molecules in the native and denatured states, respectively. H N and H D are the corresponding enthalpies of native and dena- tured states. Choosing the native state as reference state, the fol- lowing equation for the excess enthalpy is obtained: < DH 0 ðTÞ > ¼ HðTÞH N ¼ f D DH 0 ðT m Þ ¼½K=ð1 þ KÞ  DH 0 ðT m Þð4Þ which, derived with respect to the temperature and based on Eqn (1), leads to: < DC 0 P ðTÞ > ¼ ½DH 0 ðT m Þ 2 RT 2 ½K=ð1 þ KÞ 2  þ DC 0 P ðT m Þ½K=ð1 þ KÞ ð5Þ This equation allows the simulation of a calorimetric curve for a two-state transition [20,21]. When the experimental curve of geodin denaturation and the DSC profile predicted by Eqn (5) were juxtaposed, a satisfactory agreement was found (Fig. 6). This con- firms that the equilibrium thermodynamic treatment can be applied to this case, and the Gibbs’ energy value can be calculated. In Table 1 we compare the thermodynamic parame- ters of geodin with those of two-domain crystallin-type proteins for which values have been determined for most physico-chemical parameters, such as monomeric bacterial protein S and mammalian human-cS, and spherulin, which is a two-domain noncovalent dimer. The latter two proteins, as well as geodin, display a single transition in the denaturation process. Protein S instead undergoes a two-step denaturation, but the main transition is centred at 64.4 °C, closer to that of geodin (61 °C) when compared with the T m values of 75 °C and 53 ° C determined for human-cS and spheru- lin, respectively. Furthermore, the values for DG°,asa parameter of thermodynamic stability, are of about 80 kJÆmol )1 for human-c S and spherulin 3a, and of 41 and 45 kJÆmol )1 for geodin and protein S, respectively. The value of 45 kJÆmol )1 for protein S is the value cal- culated for the overall unfolding process. Thus, the conclusion can be proposed that, based on thermody- namic behaviour, geodin is a crystallin-type protein closer to protein S than to a mammalian (human-cS) or mould (spherulin 3a) crystallin. Stability of geodin in the presence of calcium ions Several two-domain members of the bc-crystallin superfamily are stabilized by calcium ions, such as pro- tein S [22], spherulin 3a [14,23,24], and c-crystallin [25]. To investigate the effect of Ca 2+ on geodin stabil- ity, DSC and CD measurements were performed in parallel at pH 5.0 in ammonium acetate buffers con- taining either 1 mm CaCl 2 or 1 mm Na 2 EDTA. The near-UV spectrum, shown in Fig. 7A, indicates that upon CaCl 2 addition the positive band at 280 nm increases, revealing perturbation in the exposure of some aromatic residue(s). Also the far-UV dichroic spectrum is affected, as shown in Fig. 7B, with a less intense minimum at 217 nm and a shoulder at about 230 nm in the pres- ence of Ca 2+ . The latter findings, which suggest a reas- sessment in the secondary structural order of the protein upon calcium binding, are in contrast with the results obtained in the case of homologous protein S, for which calcium binding affects only the protein ter- tiary structure [26]. The CD melting profile determined for geodin at 217 nm in the presence of Ca 2+ was found to be sig- moidal as that obtained in the absence of calcium ions, but the midpoint denaturation was increased to 65 °C (Fig. 7C). A perfectly coincident value was obtained by calorimetric measurements in the presence of cal- cium ions (Table 1). The higher melting temperature (increased by 4–5 °C) and higher denaturation enthalpy (Table 1) indicate that calcium ions provide an additional stabilization to geodin. In fact, the calcu- lated DG 0 at 293 K was found to be 48 kJÆmol )1 , com- pared to 41 kJÆmol )1 calculated from measurements in the absence of Ca 2+ ions. Fig. 6. Experimental (solid line) and simulated (dotted line) calori- metric curves of geodin at pH 5.0. Geodin, a sponge bc-crystallin-type protein C. Giancola et al. 1028 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS A structural model for geodin We first searched for homologues of geodin in Hom- strad (http://www-cryst.bioc.cam.ac.uk/data/align), a curated database [27] of structure-based alignments for homologous protein families, which makes use of FuGUE (http://www-cryst.bioc.cam.ac.uk/fugue/ prfsearch.html [28]. The first hit found by fugue (z score ¼ 20, average sequence length ¼ 174) correspon- ded to the family of bc-crystallins. The second hit (z score ¼ 13.5, sequence length ¼ 101) corresponded to spherulin 3a, a homodimer in which each chain con- tains a single crystallin-type domain [24]. The same two hits with statistically significant z scores were obtained when the sequence of geodin was divided into two halves and each half was used independently as a query sequence. Therefore, it is reasonable to assume that geodin (sequence length ¼ 163) has two tandemly repeated domains, each with a crystallin-type fold. We submitted the geodin sequence to a threading server (http://www.sbg.bio.ic.ac.k/~3dpssm), and obtained the same fold. Geodin was then aligned with a structure-based available alignment of bc-crystallins with experiment- ally solved structures, stored in Homstrad and decor- ated with joy [29]. This makes visible structural features, e.g. buried residues (in upper case), exposed residues (lower case) (Fig. 8). The alignment of geodin to this pre-existent alignment of crystallins was carried out using fugue and was manually modified in the N-terminal portion. The improvement produced was tested by comparative model validation using anolea [30,31] and prosaii [32]. Buried residues belonging to b-strands and facing the intradomain hydrophobic core of each domain, as well as other buried residues and positive-u glycine residues were found to be mostly conserved in geodin, which indicated the good quality of the alignment. It should be noted that the sequence of protein S shows the insertion of a valine at position 50, where the sequences of the other homologues show a glycine. The alternative to this insertion in the Homstrad align- ment would have required both an insertion plus a preceding gap. In the alignment by fugue of geodin a glycine could align with the glycines of the other homologues, and only a gap was required. Recombinant geodin is monomeric, as are c-crystal- lins and protein S. c-Crystallins have a symmetric inter-domain interface made up by the second motifs of both domains whereas protein S has a different interface, made up by the second motif of the first domain and the first motif of the second domain. The inter-domain interface in c-crystallin (PDB code 4gcr) is quite extended, made up of hydrophobic interactions between the triad M43 ⁄ F56 ⁄ I81 in the N-terminal domain and the other triad V132 ⁄ L145 ⁄ V170 in the C-terminal domain, and Q54–F145 and F56–Q143 hydrogen bonds. These positions, marked by * in the alignment (shown in Fig. 8), do not appear to be com- pletely conserved in geodin, as some of the hydro- phobic residues involved in c-crystallin are replaced by nonhydrophobic residues. Fig. 7. The effects of Ca ions on geodin structure. Near-UV (A) and far-UV (B) CD spectra of geodin at pH 5.0 in the presence of Na 2 EDTA (dotted line) or 1 m M CaCl 2 (solid line). In (C) the temperature unfolding transition of geodin in the presence of Ca 2+ (1 mM, solid line) is compared to that recorded in the absence of Ca 2+ (1 mM Na 2 EDTA, dotted line). C. Giancola et al. Geodin, a sponge bc-crystallin-type protein FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1029 The inter-domain interface in protein S (PDB code 1 prs) is instead less extended, made up of the V65 ⁄ A67 ⁄ Y121 triad, and a N68–D107 hydrogen bond [13]. These positions, marked in the alignment by #, are not conserved in geodin. Therefore, neither alignment could explain how domains are assembled in geodin. To infer the relative orientation of domains in geodin, two independent models were built with modeller. As templates we used, respectively, c-crystallins (PDB codes 4gcr, 1elp,1a45,1a5d) for a ‘mod-gamma’, and protein S (PDB code 1 prs) for a ‘mod-prs’. anolea [30,31] and prosaii [32] were run on the models and, as a control, on the template structures. Even if the energy calcula- ted for c-crystallins is much lower than that calculated for protein S, anolea indicated that the energy of the model based on 1 prs is comparable with the energy of the model based on c-crystallins, while prosaii indica- ted that the model based on 1 prs is much better than that based on c-crystallins. Figure 9 shows that in mod-prs, the model built with protein S as a template, the inter-domain Fig. 8. Alignment of geodin amino acid sequence with the sequences of homologous bc-crystallins. The sequences of bovine (PDB codes 1elp, 1a45, 4gcr, 2bb2) and murine (1a5d) crystallins, and of spore coat protein S from Myxococcus xanthus (1 prs) are decorated as follows: blue for b-strands; red for a-helices, brown for 3–10 helices. Buried residues are in uppercase letters; residues with a positive u angle, in ital- ics; hydrogen bonds to main-chain amides in bold; hydrogen bonds to main-chain carbonyls are underlined. Buried residues belonging to b-strands and facing the intra-domain hydrophobic core of each domain, other buried residues, and positive-u glycine residues conserved in geodin, are highlighted in green, blue and yellow, respectively. The inter-domain interface residues in 4gcr are marked by asterisks. The inter-domain interface residues in 1 prs are marked by #. Geodin, a sponge bc-crystallin-type protein C. Giancola et al. 1030 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS interface is hydrophobic, as made up of W57, I58, L101, P102 and P106 (shown as yellow as ball-and- stick residues). Furthermore, correctly intercalated resi- dues were found, both positively charged (K59 and R107; blue ball-and-stick residues), and negatively charged residues (D61 and D100; red ball-and-stick residues, Fig. 9). These charged residues, located at the surface of the two domains, could also contribute with hydrogen and ⁄ or ionic bonds to the stability of geodin inter-domain interface. As these interactions were not used as restraints with modeller, they further validate the model built on protein S, and shown in Fig. 9. On the other hand, the model built using c-crystallins as templates did not possess a hydrophobic interface or any other interactions capable of stabilizing mono- meric geodin. Since the key question at hand was to predict the relative orientation of domains, we submitted the coordinates of the two models, mod-prs and mod-gamma, to a protein–protein interaction server, http://www.biochem.ucl.ac.uk/bsm/PP/server [33]. The results, summarized in Table 2, indicated that the interface in mod-prs is larger, more planar, although less circular than that in mod-gamma. The interacting surfaces in mod-prs are more complementary, as proved by a lower gap volume and gap volume index (gap volume ⁄ interface ASA), with a balanced number of hydrophobic residues and, interestingly, a higher number of interdomain hydrogen bonds, and salt brid- ges. In conclusion, the analysis confirms the results reported above: although mod-prs and mod-gamma represent alternative ways to assemble the two domains of geodin, measurement of several descriptive parameters of the interfaces and visual inspection both suggest that in geodin, as in protein S, the inter- domain interface is made up by the second motif in the first domain and the first motif of the second domain. As it has been found that geodin is stabilized by Ca 2+ (see above), the 3D model of geodin with protein S as a template was analysed to verify whether it could accommodate a Ca 2+ binding site. Protein S has two binding sites for Ca 2+ , one per domain. They are formed by residues in the folded hairpin of the first Greek key motif and by residues in the loop connect- ing the penultimate and ultimate strands in the second Greek key motif of each domain. The site with the highest affinity for Ca 2+ in protein S is in the N-ter- minal domain and is defined by residues E10 and E71. It can be proposed that T10, S11 and E62, located in hydrophilic loops in the corresponding region of geo- din, could play the same role. This tentative Ca 2+ binding site was thus modelled (green ball-and-stick residues) in the geodin structure shown in Fig. 9. Another presumable but weaker binding site could be identified in the C-terminal domain of geodin, defined by Asn93 and Asn145. Spherulin 3A, a single-domain mould protein with crystallin fold, has two distinct Ca 2+ binding sites per domain, one site is located between the folded hairpin of the first Greek key motif and the loop between the last two strands of the second Greek key motif [13]. This site corresponds to the sites identified in protein S and modelled in geodin. Conclusions The results of the physico-chemical studies reported above, and those from homology modelling, lead to Fig. 9. A model of geodin built using the structure of protein S (1 prs) as a template. Inter-domain interface residues are shown as yellow ball-and-stick residues when they are hydrophobic; in red when they are negatively charged; in blue when they are positively charged. Residues proposed to describe the binding site for Ca 2+ are in green ball-and-stick notation. The structure was drawn using MOLSCRIPT [37] and rendered using RASTER3D [38]. Table 2. Domain–domain interface analysis of alternative models of geodin domain assembly. Mod-prs domain-1 Mod-prs domain-2 Mod-gamma domain-1 Mod-gamma domain)2 Interface ASA 643.29 741.38 415.18 404.38 % Interface ASA 13.62 14.73 8.92 8.29 Planarity 2.14 2.13 1.02 1.28 Length ⁄ breadth 0.49 0.54 0.74 0.71 % Polar atoms in interface 38.09 43.61 32.79 64.11 % Non–polar atoms in interface 61.90 56.30 67.20 35.80 Hydrogen bonds 3 3 1 1 Salt bridges 1 1 0 0 Gap volume 1644.62 1644.62 2927.67 2927.67 Gap volume index 1.19 1.19 3.57 3.57 C. Giancola et al. Geodin, a sponge bc-crystallin-type protein FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1031 the first structural description of geodin, and validate its identification as a homologue of the bc-crystallin superfamily. Geodin is a monomeric, two-domain protein, made up of b strands apparently folded in Greek-key motifs, just as its mammalian, amphibian and bacterial homologues. Of particular interest is that its source is a sponge, G. cydonium, which makes geo- din the most primitive metazoan bc-crystallin studied so far. By both fluorescence and CD spectroscopic analyses, geodin is found to be, with respect to other bc-crystal- lins, more readily but reversibly unfolded by chemical denaturants, with a single midpoint of denaturation, both in urea and GuHCl. These features are very sim- ilar to those found for protein S [22]. Consistently, our conclusion from homology modelling strongly suggests that the closest structural homologue to geodin is bac- terial protein S. Geodin thermal denaturation, studied both by near- and far-UV CD and calorimetry, shows that geodin unfolds with a typical cooperative transition in agree- ment with a two-state model, with a simultaneous col- lapse of secondary and tertiary structures. This is difficult to explain, given the larger structural differ- ences in primary structure and 3D architecture between the two geodin domains, as they result from the proposed sequence alignment and model, com- pared with the more similar sequence and architecture of the two domains in the other monomeric crystallins studied so far. It should be considered that, besides the structural differences among the proteins under com- parison, different pH values were adopted in the experiments. The similarity in structural properties between geodin and protein S extends to the stabilization exerted by calcium ions on both crystallin-type proteins, although the removal of calcium affects in geodin both secon- dary and tertiary structure, whereas for protein S only the tertiary structure is affected. Furthermore, the effect of calcium on geodin stability is less conspicuous, with an increase in DG 0 of denaturation of 7 kJÆmol )1 , compared to that reported for protein S (19 kJÆmol )1 ). This outcome is due mainly to the higher DH 0 contribu- tion for protein S denaturation in the presence of Ca 2+ , in turn apparently due to strengthened inter- domain interactions. As from the evolutionary point of view, it is of interest that the crystallin-type structural organization, already evolved in bacteria and moulds, was readily recruited for porifera, the earliest metazoans. Further- more, it is interesting that in the model as derived for geodin the inter-domain interfaces appear to be stabil- ized by hydrophobic interactions, but also by a number of polar interactions, such as H-bonds and salt linkages. These contacts can be interpreted, as it has been proposed for vertebrate bc-crystallins [5], as remi- niscent of the polar, solvent exposed surfaces in the putative single-domain ancestor(s) that evolved to associate into two-domain geodin. Experimental procedures Cloning and expression of geodin Cloning into the pET22b (+) vector (Novagen, Madison, WI, USA) of the DNA segment encoding geodin, a puta- tive bc-crystallin-type protein from G. cydonium, was carried out as described previously [17]. The plasmid was used to transform E. coli strain BL21 (DE3) (from AMS Biotechnology). For over-expression of geodin, the bacterial cultures were grown at 37 °CtoD 600 ¼ 1, then induced by addition of 0.1 m isopropyl-1-thio-d-galactopyranoside. After overnight growth at room temperature, cells were pel- leted by centrifugation, and lysed by sonication. An Ultra- sonic sonicator (Heat System Ultrasonic) was used at 20 kHz, with 30-s impulses, each followed by a 30-s rest period, for a total time of 15 min. A protein with the molecular size of geodin was detected by SDS ⁄ PAGE as a soluble protein in the lysate superna- tant (henceforth termed S preparation), but also in the insoluble material (Fig. 1). Thus, the insoluble material, washed twice in 50 mm Tris ⁄ HCl pH 8 containing 20 mm Na 2 EDTA, and once in 100 mm Tris ⁄ HCl pH 8 containing 1mm Na 2 EDTA, was solubilized by denaturation with 6 m GuHCl in 25 mm sodium phosphate pH 7 (buffer P). Rena- turation followed, obtained through extensive dialysis against the same buffer. This yielded another preparation containing a protein with the mobility of geodin, henceforth termed IB preparation, as presumably it consists of proteins sequestered in inclusion bodies. Both preparations S and IB were fractionated by gel fil- tration, carried out on a column of Superdex G-75 (Amer- sham Biosciences) equilibrated with buffer P containing 0.3 m sodium chloride. Figure 2 illustrates that the fraction- ation of both S and IB preparations yielded three main UV absorbing fractions. Only in the middle peak from either S or IB preparations SDS ⁄ PAGE runs revealed the presence of a protein band with the molecular size expected for geo- din. The corresponding fractions (Fig. 2A,B, insets) were pooled and dialysed against 50 mm ammonium acetate buf- fer pH 5. The Superdex G-75 fraction pools obtained from the S and IB preparations were each loaded onto a cation exchange Resource-S column (Amersham Biosciences) equilibrated in 50 mm ammonium acetate pH 5, and eluted with a 60-min linear gradient from 50 to 300 mm ammo- nium acetate. As shown in Fig. 2C most of the protein Geodin, a sponge bc-crystallin-type protein C. Giancola et al. 1032 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS [...]... eluted at the same concentration of ammonium acetate as for the S preparation, again containing a protein with the molecular size of geodin (Fig 1, lane 3) Analytical methods Sequence analyses were performed by automated Edman degradation as previously reported [34] Samples were cleaned and concentrated prior to analysis through absorption on a ProSorb cartridge (Applera Italia, Monza, Italy) MALDI-TOF...C Giancola et al material from the gel filtration step did not bind to the cation exchanger, and a single, symmetrical peak was eluted from the column at about 100 mm ammonium acetate This contained a protein with the molecular size of geodin (Fig 1, lane 4) Figure 2D illustrates the same fractionation by cation exchange chromatography of the IB fraction In the latter case, only a single peak was detected,... to an IBM PC computer for automatic data collection and analysis using previously described software [36] The excess heat capacity function was obtained after baseline subtraction, assuming that the baseline is given by the linear temperature dependence of the native state heat capacity [20] A buffer vs buffer scan was subtracted from each peak The denaturation enthalpies, DH0(Tm), were obtained... Giancola C & Graziano G (1993) THESEUS: a new software package for the handling and analysis of thermal denaturation data of biological macromolecules J Thermal Anal 38, 2779–2790 37 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallog 24, 946–950 38 Merrit EA & Bacon DJ (1997) Raster 3D: Photorealistic Molecular Graphics Methods Enzymol... Parente A, Verde C, Malorni A, Montecucchi P, Aniello F & Geraci G (1993) Amino-acid sequence of the cooperative dimeric myoglobin from the radular muscles of the marine gastropod Nassa mutabilis Biochim Biophys Acta 1162, 1–9 35 Laemmli U.K & Favre M (1973) Maturation of the head of bacteriophage T4 I DNA packaging events J Mol Biol 80, 575–599 36 Barone G, Del Vecchio P, Fessas D, Giancola C & Graziano... Publishers, Amsterdam, the Netherlands Wenk M & Mayr EM (1998) Myxococcus xanthus spore coat protein S, a stress-induced member of the beta gamma-crystallin superfamily, gains stability from binding of calcium ions Eur J Biochem 255, 604–610 Kretschmar M, Mayr EM & Jaenicke R (1999) Kinetic and thermodynamic stabilization of the beta gammacrystallin homolog spherulin 3a from Physarum polycephalum by calcium... C, Mayr EM, Jaenicke R & Holak TA (1997) Ca2+-loaded spherulin 3a from Physarum polycephalum adopts the prototype gamma-crystallin fold in aqueous solution J Mol Biol 271, 645–655 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS C Giancola et al 25 Rajini B, Shridas P, Sundari CS, Muralidhar D, Chandani S, Thomas F & Sharma Y (2001) Calcium binding properties of gamma-crystallin: calcium ion binds at the... droplet of the resulting mixture (0.5– 2 lL) was placed on the mass spectrometer’s sample target and dried at room temperature for loading The accuracy of the instrument is about 0.1% for single determinations Proteins were analysed by SDS ⁄ PAGE on 15% polyacrylamide gels as described by Laemmli and Favre [35], and stained with Coomassie blue HPLC was carried out on C4 columns (Vydac) equilibrated in... Kretschmar M, Mayr EM & Jaenicke R (1999) Homodimeric spherulin 3a: a single-domain member of the beta gamma-crystallin superfamily Biol Chem 380, 89– 94 Wistow G, Jaworski C & Rao PV (1995) A non-lens member of the beta gamma-crystallin superfamily in a vertebrate, the amphibian Cynops Exp Eye Res 61, 637–639 Krasko A, Muller IM & Muller WE (1997) Evolutionary relationships of the metazoan beta gamma-crystallins,... by integrating the area under the heat capacity vs temperature curves Tm was the temperature corresponding to the maximum of each DSC peak 0 DCP (Tm), was the value of the excess heat capacity function at Tm The entropy changes, DS0(Tm), were determined by integrating the curve obtained by dividing the heat capacity curve by the absolute temperature The denaturation enthalpies, entropies and Gibbs’ . Preparation and characterization of geodin A bc-crystallin-type protein from a sponge Concetta Giancola 1 , Elio Pizzo 2 , Antimo Di Maro 3 , Maria Vittoria Cubellis 2 and Giuseppe D’Alessio 2 1. that of protein S rather than that of a mammalian crystallin such as cS-crystallin, a qualitative, indirect indication of a closer structural relationship between geodin and protein S. Geodin also. 575–599. 36 Barone G, Del Vecchio P, Fessas D, Giancola C & Graziano G (1993) THESEUS: a new software package for the handling and analysis of thermal denaturation data of biological macromolecules.