Báo cáo khoa học: Preparation and characterization of geodin A bc-crystallin-type protein from a sponge pot

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:
(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 ( a
curated database [27] of structure-based alignments for
homologous protein families, which makes use of
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
(.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,
[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
material from the gel filtration step did not bind to the cat-
ion 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, 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 MS was performed using a Microbe-
Lynx
TM
System instrument (Micromass Corporation, xxx,
xxx). Lyophilized geodin was dissolved ( 1 lgÆlL
)1
)in
0.1% (v ⁄ v) trifluoroacetic acid (TFA). Prior to the acquisi-
tion of spectra, 2 lL geodin solution were mixed with 2 lL
satured a -cyano-4-hydroxycinnamic acid matrix solution
[10 lgÆlL
)1
in 50 : 50 (v ⁄ v) ethanol ⁄ water containing 0.1%
(v ⁄ v) TFA], and a 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% poly-
acrylamide gels as described by Laemmli and Favre [35],
and stained with Coomassie blue.
HPLC was carried out on C4 columns (Vydac) equili-
brated in 95% solution A [0.1% (v ⁄ v) TFA] ⁄ 5% solution B
[acetonitrile containing 0.1% (v ⁄ v) TFA]. The column was
eluted with a gradient in which the concentration of solu-
tion B was raised to 60% in 1 h.
Fluorescence analyses
Intrinsic protein fluorescence was recorded using a Jasco
FP-750 spectrofluorimeter equipped with thermostat-con-
trolled cell holders and a circulating water bath. The pro-

tein concentration ranged from 0.1 to 0.2 mgÆml
)1
. The
excitation wavelength was set at 280 nm, and the emission
measured between 300 and 450 nm. The spectra were recor-
ded at 20 °C with a 1-cm cell and a 5-nm emission slit
width, and corrected for background signal. The GuHCl-
or urea-induced transition curves at 20 °C were obtained
by recording the shift in fluorescence maximum wavelength
as function of denaturant concentration. The maximum
emission wavelength of geodin, recorded at 327 nm, was
shifted to 350 nm in the presence of denaturants. Measure-
ments were performed after an overnight incubation of
samples at 4 °C.
CD spectroscopy
CD spectra were obtained on a JASCO 715 CD spectro-
photometer equipped with a programmable, thermoelectri-
cally controlled cell holder (JASCO PTC-348). The
wavelength was varied from 200 to 340 nm. Near-UV and
far-UV spectra were recorded at 20 °C using a square
quartz cell with a 1 cm or 0.1 cm pathlength, respectively.
Protein concentration was 0.2 or 0.7 mgÆml
)1
for far- or
near-UV measurements. Molar ellipticity per mean residue
[h] in deg cm
2
Ædmol
)1
was calculated from the equation

[h] ¼ 100Æ[h]
obs
l
)1
ÆC where [h]
obs
is the ellipticity measured
in degrees, l is the pathlength of the cell in cm and C is the
protein concentration referred to the mean residue molecu-
lar weight. CD spectra were recorded with a response of
8 s at 2.0 nm bandwidth with 10 nmÆmin
)1
scan rates. The
buffer spectrum was subtracted to each sample spectrum.
Each reported spectrum is an average of at least three
scans. The GuHCl- or urea-induced transition curves at
20 °C were obtained by recording the CD signal at 217 nm
for each independent sample. Measurements were per-
formed after an overnight incubation of the samples at
4 °C. Thermal unfolding curves were recorded in tempera-
ture mode at 217 nm, from 20 to 90 °C, with a scan rate
of 1.0 °CÆmin
)1
.
Differential scanning calorimetry (DSC)
DSC measurements were performed on a second generation
Setaram Micro-DSC (Caluire, France). The instrument was
interfaced to an IBM PC computer for automatic data col-
lection and analysis using previously described software
[36]. The excess heat capacity function <DC

P
> was
obtained after baseline subtraction, assuming that the base-
line 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,
DH
0
(T
m
), were obtained by integrating the area under the
heat capacity vs. temperature curves. T
m
was the tempera-
ture corresponding to the maximum of each DSC peak.
DC
0
P
(T
m
), was the value of the excess heat capacity function
at T
m
. The entropy changes, DS
0
(T
m
), were determined by
integrating the curve obtained by dividing the heat capacity
curve by the absolute temperature. The denaturation

enthalpies, entropies and Gibbs’ energies in function of tem-
perature were calculated according to the classical relations:
DH
0
ðTÞ¼DH
0
ðT
m
ÞþDC
0
P
ðT
m
ÞðT  T
m
Þð6Þ
DS
0
ðTÞ¼
DH
0
ðT
m
Þ
T
m
þ DC
0
P
ðT

m
Þln
T
T
m
ð7Þ
DG
0
ðTÞ¼DH
0
ðTÞTDS
0
ðTÞð8Þ
Gibbs energies of denaturation, at the temperature of
293 K, were determined combining Eqns (6),(7),(8):
C. Giancola et al. Geodin, a sponge bc-crystallin-type protein
FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1033
DG
0
ð293Þ¼DH
0
ðT
m
Þ
T
m
 293
T
m


 DC
0
P
ðT
m
ÞðT
m
 293Þ
þ 293  DC
0
P
ðT
m
Þln
T
m
293

ð9Þ
The thermodynamic parameters in Table 1 represent means
from heating curves of several experiments. The reported
errors for thermodynamic parameters are the deviation of
the mean from multiple determinations.
Acknowledgements
This work was supported by the Ministry of University
and Research. The authors thank I. Duro for skillful
rendering of the figures in the manuscript.
References
1 Augusteyn RC & Stevens A (1998) Macromolecular
structure of the eye lens. Prog Polym Sci 23, 375–

413.
2 Lubsen NH, Aarts HJ & Schoenmakers JG (1988) The
evolution of lenticular proteins: the beta- and gamma-
crystallin super gene family. Prog Biophys Mol Biol 51,
47–76.
3 Slingsby C & Clout NJ (1999) Structure of the crystal-
lins. Eye 13 (3b), 395–402.
4 Wistow GJ & Piatigorsky J (1988) Lens crystallins: the
evolution and expression of proteins for a highly specia-
lized tissue. Ann Rev Biochem 57, 479–504.
5 D’Alessio G (2002) The evolution of monomeric and
oligomeric beta gamma-type crystallins. Facts and hypo-
theses. Eur J Biochem 269, 3122–3130.
6 Jaenicke R (1999) Stability and folding of domain pro-
teins. Prog Biophys Mol Biol 71, 155–241.
7 Jaenicke R (2000) Stability and stabilization of globular
proteins in solution. J Biotechnol 79, 193–203.
8 White HE, Driessen HP, Slingsby C, Moss DS &
Lindley PF (1989) Packing interactions in the eye-lens.
Structural analysis, internal symmetry and lattice inter-
actions of bovine gamma IVa-crystallin. J Mol Biol 207,
217–235.
9 Bax B, Lapatto R, Nalini V, Driessen H, Lindley PF,
Mahadevan D, Blundell TL & Slingsby C (1990) X-ray
analysis of beta B2-crystallin and evolution of oligo-
meric lens proteins. Nature 347, 776–780.
10 Blundell T, Lindley P, Miller L, Moss D, Slingsby C,
Tickle I, Turnell B & Wistow G (1981) The molecular
structure and stability of the eye lens: x-ray analysis of
gamma-crystallin II. Nature 289, 771–777.

11 Clout NJ, Kretschmar M, Jaenicke R & Slingsby C
(2001) Crystal structure of the calcium-loaded spherulin
3a dimer sheds light on the evolution of the eye lens
beta gamma-crystallin domain fold. Structure (Camb)
9, 115–124.
12 Ohno A, Tate S, Seeram SS, Hiraga K, Swindells MB,
Oda K & Kainosho M (1998) NMR structure of the
Streptomyces metalloproteinase inhibitor, SMPI, iso-
lated from Streptomyces nigrescens TK-23: another
example of an ancestral beta gamma-crystallin precursor
structure. J Mol Biol 282, 421–433.
13 Bagby S, Harvey TS, Eagle SG, Inouye S & Ikura M
(1994) NMR-derived three-dimensional solution struc-
ture of protein S complexed with calcium. Structure 2,
107–122.
14 Kretschmar M, Mayr EM & Jaenicke R (1999) Homo-
dimeric spherulin 3a: a single-domain member of the
beta gamma-crystallin superfamily. Biol Chem 380, 89–
94.
15 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.
16 Krasko A, Muller IM & Muller WE (1997) Evolution-
ary relationships of the metazoan beta gamma-crystal-
lins, including that from the marine sponge Geodia
cydonium. Proc R Soc Lond B Biol Sci 264, 1077–1084.
17 Di Maro A, Pizzo E, Cubellis MV & D’Alessio G
(2002) An intron-less beta gamma-crystallin-type gene
from the sponge Geodia cydonium. Gene 299, 79–82.

18 Lumry R & Eyring H (1954) Conformation changes of
proteins. J Phys Chem 58, 110–120.
19 Sanchez-Ruiz JM (1992) Theoretical analysis of Lumry-
Eyring models in differential scanning calorimetry. Bio-
phys J 61, 921–935.
20 Freire E & Biltonen RL (1978) Thermodynamics of
transfer ribonucleic acids: the effect of sodium on the
thermal unfolding of yeast tRNAPhe. Biopolymers 17,
1257–1272.
21 Barone G, Del Vecchio P, Fessas D, Giancola C &
Graziano G (1994) Denaturation of Biological Macromo-
lecules: New Programs for the Deconvolution of DSC
Measurements. Kluwer Academic Publishers,
Amsterdam, the Netherlands.
22 Wenk M & Mayr EM (1998) Myxococcus xanthus spore
coat protein S, a stress-induced member of the beta
gamma-crystallin superfamily, gains stability from bind-
ing of calcium ions. Eur J Biochem 255, 604–610.
23 Kretschmar M, Mayr EM & Jaenicke R (1999) Kinetic
and thermodynamic stabilization of the beta gamma-
crystallin homolog spherulin 3a from Physarum
polycephalum by calcium binding. J Mol Biol 289,
701–705.
24 Rosinke B, Renner C, Mayr EM, Jaenicke R & Holak
TA (1997) Ca
2+
-loaded spherulin 3a from Physarum
polycephalum adopts the prototype gamma-crystallin
fold in aqueous solution. J Mol Biol 271, 645–655.
Geodin, a sponge bc-crystallin-type protein C. Giancola et al.

1034 FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS
25 Rajini B, Shridas P, Sundari CS, Muralidhar D, Chan-
dani S, Thomas F & Sharma Y (2001) Calcium binding
properties of gamma-crystallin: calcium ion binds at the
Greek key beta gamma-crystallin fold. J Biol Chem 276,
38464–38471.
26 Jaenicke R & Slingsby C (2001) Lens crystallins and
their microbial homologs: structure, stability, and func-
tion. Crit Rev Biochem Mol Biol 36, 435–499.
27 Mizuguchi K, Deane CM, Blundell TL & Overington
JP (1998) HOMSTRAD: a database of protein structure
alignments for homologous families. Protein Sci 7,
2469–2471.
28 Shi J, Blundell T & Mizuguchi K (2001) FUGUE:
sequence-structure homology recognition using environ-
ment-specific substitution tables and structure-dependent
gap penalties. J Mol Biol 310, 243–257.
29 Mizuguchi K, Deane CM, Blundell TL, Johnson MS &
Overington JP (1998) JOY: protein sequence-structure
representation and analysis. Bioinformatics 14, 617–623.
30 Melo F, Devos D, Depiereux E & Feytmans E (1997)
ANOLEA: a www server to assess protein structures.
Proc Int Conf Intell Syst Mol Biol 5, 187–190.
31 Melo F & Feytmans E (1998) Assessing protein struc-
tures with a non–local atomic interaction energy. J Mol
Biol 277, 1141–1152.
32 Sippl MJ (1993) Recognition of errors in three-dimen-
sional structures of proteins. Proteins 17, 355–362.
33 Jones S & Thornton JM (1996) Principles of protein–
protein interactions. Proc Natl Acad Sci USA 93,

13–20.
34 Parente A, Verde C, Malorni A, Montecucchi P, Aniello
F & Geraci G (1993) Amino-acid sequence of the coop-
erative 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 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 277, 505–524.
39 Wenk M & Jaenicke R (1999) Calorimetric analysis of
the Ca
2+
-binding beta gamma-crystallin homolog pro-
tein S from Myxococcus xanthus : intrinsic stability and
mutual stabilization of domains. J Mol Biol 293, 117–
124.
C. Giancola et al. Geodin, a sponge bc-crystallin-type protein
FEBS Journal 272 (2005) 1023–1035 ª 2005 FEBS 1035