BACKGROUND
avidins are high-affinity biotin-binding proteins found in the eggs of oviparous vertebrates including bird, reptilian and amphibian species  <cit> .
in addition to its production in the oviduct and secretion to egg white, avidin is expressed in several other tissues of the chicken during injury and inflammation  <cit> .
avidins analogous to those found in the earliest diverging tetrapod species have been isolated from a few bacterial species: streptomyces avidinii  bradyrhizobium japonicum  and rhizobium etli .
avidins are homotetrameric proteins, the only known exception being rhizavidin, which is a homodimer  <cit> .
the avidin subunits consist of eight anti-parallel β-strands, which form a β-barrel structure that has a biotin-binding pocket at the open end of the barrel.
avidins interact extraordinarily tightly with a water-soluble vitamin, d-biotin .
this exceptional strength of interaction has not only been utilized in various biochemical and biophysical applications  <cit> , but has also led to the production of a number of genetically modified forms of avidin and streptavidin  <cit> .
the biotin-binding modes of avidins, and the amino acids involved in biotin binding are highly conserved among different species even though the similarity between the primary sequences of avidins is relatively low.
in addition to biotin binding, comparisons of avidins, either extracted directly from natural sources  <cit>  or produced as recombinant proteins  <cit> , have revealed many differences in their physicochemical properties, e.g., stability and immunogenicity.
consequently, the detailed characterization of novel avidins has provided valuable information that could be exploited in the development of novel molecular tools and devices.
for example, a chimeric avidin containing structural parts from both chicken avidin and avidin related protein  <dig> , has been found to be more stable than either one of the native forms  <cit> .
although avidins are expressed in several different species, only a few avidins have been thoroughly characterized.
experimentally determined three-dimensional  structures are available e.g.
for streptavidin  <cit> , chicken avidin  <cit> , avidin related protein  <dig>   <cit> , avr <dig>  <cit>  and biotin-binding protein a  <cit> .
these structures have proved that the overall fold of the avidins is the same, but that the subtle structural differences explain the observed differences in the functional characteristics of avidins.
here we report, to our knowledge, the first biochemical and structural characterization of an amphibian avidin - xenavidin - a frog avidin from xenopus tropicalis.
being the only diploid species in the xenopus genus with a small genome  and short generation time, x. tropicalis has proven invaluable, e.g., for understanding the mechanisms of vertebrate embryonic development and for functional genomics .
the 56% amino acid sequence identity shared between avidin and xenavidin is relatively high and suggests that avidin and xenavidin have similar biochemical and structural properties.
however, like for other avidins such as streptavidin, bradavidin and rhizavidin, in comparison with chicken avidin, xenavidin has it own unique features determined by the subtle changes found in its sequence and structure.
thus, the characterization of xenavidin gives insights into the biochemical and structural determinants of a novel member of the avidin protein family, a family of extraordinary biotin-binders with many prospects for exploitation in biotechnology and nanotechnology.
RESULTS
cloning, expression and purification
two dna sequences encoding an avidin-like protein from a frog were originally identified by searching an est sequence database  <cit> ; after discovering the avidin-like gene from xenopus tropicalis, the avidin gene for xenopus laevis, another frog species, was also identified.
here, we focus on the protein from xenopus tropicalis, which we named xenavidin.
the cdna of xenavidin was cloned into the pfastbac <dig> baculovirus expression system entry vector, and recombinant xenavidin was successfully produced in sf <dig> insect cells using the bac-to-bac® baculovirus expression system .
the protein was purified with 2-iminobiotin sepharosetm  affinity chromatography.
about 2- <dig> mg of pure protein was typically obtained per one litre of culture media.
attempts to produce xenavidin in a bacterial expression system in e. coli  <cit>  were unsuccessful.
x-ray structure
xenavidin overexpressed in insect cells was crystallized with and without d-biotin  added to the crystallization solution.
the x-ray structures of the xenavidin-btn complex  and the unliganded xenavidin protein  were solved to  <dig>  Å resolution.
the structures crystallized in a rhombohedral space group and had a dimer in their asymmetric unit .
in general, both of the xenavidin structures were highly similar to the known structures of avidin and streptavidin: the 3d structure of xenavidin is homotetrameric, each subunit consisting of an eight-stranded β-barrel .
the amino acids lining the biotin-binding pocket are structurally conserved between xenavidin and chicken avidin.
there were, however, differences in the loop architecture between these proteins, particularly in the l <dig> -loop and the l <dig> -loop.
the unique l <dig> -loop design in different avidins is a key feature regulating the binding affinity of avidins  <cit> , but the role of the variable l <dig> -loop is not clear though it is known not to be directly involved in ligand binding.
moreover, there were some differences in the interface architecture of xenavidin in comparison to chicken avidin.
for example, out of the three residues at the 1- <dig> subunit interface, two  are conserved between avidin and xenavidin, whereas i <dig> in avidin is substituted by alanine in xenavidin, probably contributing to the reduced stability of xenavidin .
the surface properties of avidin, xenavidin and streptavidin vary, too .
athe numbers in parenthesis refer to the highest resolution bin.
bobserved r-factor from xds  <cit> .
cfrom refmac  <dig>  <cit>  using tls  <cit>  & restrained refinement.
the subunits of the dimer found in the asymmetric units of the xenavidin crystals are labelled a and d to match the numbering system generally used to describe the biological unit of chicken avidin .
in the unliganded structure of xenavidin, there was no clear electron density for biotin in subunit a. however, weak electron density was evident for the ligand at the binding site in subunit d; thus, a biotin molecule was built into subunit d of the unliganded xenavidin structure despite the presence of weak electron density around the ligand .
one possible explanation for the varying electron densities at the biotin-binding sites could be sequential binding, which is supported by some previous studies suggesting cooperativity between binding sites of avidin  <cit> .
however, some other studies do not support cooperativity between binding sites  <cit> .
in the btn-complex structure, d-biotin was clearly present in both of the biotin-binding sites of the asymmetric unit.
in the x-ray structures of xenavidin there was no clear evidence that either of the two potential n-glycosylation sites of each polypeptide, n <dig> or n <dig> , would in fact be glycosylated.
however, the presence or absence of glycosylation at n <dig> cannot be reliably established since n <dig> is within the l <dig> -loop and the side-chain atoms of this residue have high b-factors and hence poor electron density.
the structures of xenavidin clearly showed that n <dig> did not have sugar units attached to its side chain.
in xenavidin, three amino acids residues out of  <dig> lining the biotin-binding pocket are different from those in the binding pocket of avidin: the equivalent residues for the non-conserved s <dig>  l <dig> and w <dig> found in xenavidin in avidin are t <dig>  a <dig> and f <dig> .
s40/t <dig> and w78/f <dig> interact with biotin through hydrophobic effect and van der waals interactions, whereas l41/a <dig> are involved in hydrogen bonding via the residue's main-chain nitrogen atom.
in addition, the main chain of t <dig> in xenavidin is not close enough to form a hydrogen bond with biotin , whereas in avidin a hydrogen bond between the main-chain nitrogen atom of the equivalent residue t <dig> is evident .
these subtle differences in the architecture of the biotin-binding sites of xenavidin and avidin may contribute, at least partially, to the different biotin-binding properties that were observed for these two proteins .
a water molecule was detected in the unliganded structure of xenavidin at the bottom of the ligand-binding site bridging the carboxamide group of n <dig> and biotin ; the conformation of n <dig> in the unliganded structure differs from that in the structure of the xenavidin-biotin complex.
to our knowledge, the only other known avidin structures harbouring a water molecule at the corresponding location, referred to as 'site 1' by hyre et al.
<cit> , are two mutant structures of streptavidin: the d128a mutant structure  and the s45a/d128a double mutant structure .
a structural water at site  <dig> has so far not been observed in native avidin structures.
a conserved water molecule, the water molecule at site  <dig> , located at the entrance of the water channel, is present in both of the xenavidin structures.
according to hyre et al.
<cit> , biotin dissociation from streptavidin is initiated by penetration of water molecules into these sites.
the presence of structural water molecules at site  <dig> and/or  <dig> in the xenavidin structures suggests a role for these water molecules in the biotin association/dissociation events of xenavidin; the biotin-binding site of xenavidin may be more vulnerable to potential 'attack' by water molecules than it is in chicken avidin.
this hypothesis is supported by our experimental results, which showed an increased dissociation rate of biotin from xenavidin compared to avidin .
biochemical and physicochemical characterization
the quality and molecular size of the isolated xenavidin protein was analyzed using 15% sds-page and size exclusion chromatography.
in the latter analysis, xenavidin appeared mainly as a single peak and had a size of approximately  <dig> kda suggesting a tetrameric quaternary structure .
recall that xenavidin has two asparagine residues, n <dig> and n <dig> , identified as potential glycosylation sites according to the program netnglyc  <dig>  .
the theoretical molecular weight of xenavidin, calculated on the basis of the primary amino acid sequence, is only  <dig> kda, which suggests that n <dig> might indeed be glycosylated in the insect cells.
sds-page analysis resulted in multiple bands where, in addition to the band corresponding to the nonglycosylated form, two higher molecular weight forms of the monomeric protein were detectable.
these higher molecular weight forms most probably correspond to glycosylated species with a varying number and type of sugar units .
it is well known that proteins overexpressed using the baculovirus expression system may have heterogeneous glycosylation patterns, as reviewed by davies  <cit> .
furthermore, we have also observed this phenomenon in the avr-proteins, which may have multiple glycosylation sites per polypeptide chain  <cit> .
further evidence for the presence of heterogeneously glycosylated xenavidin was obtained by treating the purified protein with the deglycosylation enzymes endo hf and pngase, which caused the disappearance of the higher molecular weight forms of xenavidin in sds-page analysis .
moreover, based on the x-ray analysis of xenavidin , the crystallized proteins may have carbohydrates attached to one of the two potential glycosylation sites.
taken together, our data support a structure in which xenavidin is glycosylated at n43; interestingly, the glycosylation site in xenavidin is located at a position equivalent to that glycosylated in avr <dig>   <cit> .
avidin also has one glycosylation site  but there is no equivalent glycosylation site in xenavidin.
athe analyzed proteins were most probably glycosylated; sugar moieties comprise at least 10% of the mass of avidin  <cit> .
btheoretical values were calculated from the primary structures.
like almost all of the other avidin-like proteins studied so far, xenavidin also tends to form oligomers of tetramers, which could be seen as a minor peak, with an apparent molecular weight of  <dig> kda, before the main peak in the gel filtration chromatogram .
these oligomers could not be detected in an sds-page-based thermostability assay , where xenavidin appeared only as a tetrameric form in the presence of biotin to up to 65°c, and even at 90°c , but dissociated into monomers in the absence of biotin already at room temperature .
xenavidin has a very similar theoretical isoelectric point  to avidin ; values for both proteins were calculated using the compute pi/mw tool  <cit> .
hence, xenavidin and avidin are positively charged at neutral ph, whereas streptavidin  has a negative net charge at the same ph.
sera from human cancer patients  <cit>  who were exposed to avidin and streptavidin were used to probe the cross reactivity among xenavidin, chicken avidin and streptavidin.
in this analysis xenavidin did not react significantly with the avidin/streptavidin immunized sera.
moreover, polyclonal antibodies against avidin and streptavidin did not recognize xenavidin.
taken together, xenavidin appears not to be immunologically cross reactive with avidin .
the complete dataset is reported in helppolainen et al.
<cit> .
protein stability
the thermal stability of xenavidin was determined with a sds-page thermostability assay , and the analysis was performed as described by bayer et al.
<cit> .
without biotin, the tetrameric form of xenavidin was unstable and the protein was monomeric already at room temperature .
when biotin was added, the stability of the tetrameric form of the xenavidin was significantly increased - the transition temperature of dissociation of the tetramer was raised to 75°c.
therefore, in the absence of biotin, the tetrameric quaternary structure of xenavidin is clearly more vulnerable to the denaturing effect of sds than is the structure of avidin  <cit> , whereas in the presence of biotin, xenavidin and avidin have relatively similar stabilities, thus suggesting high affinity to biotin.
the stability of xenavidin against proteinase k was tested in the presence and absence of biotin.
as previously shown, avidin was cleaved in the absence of biotin after extended incubation with proteinase k to fragments of  <dig> kda and below, but the presence of biotin protected avidin from proteolytic cleavage  <cit> .
similar behaviour was observed in the case of xenavidin.
in the absence of biotin, xenavidin was cleaved by proteinase k into small fragments, which could not be seen using gel analysis, whereas adding biotin to the reaction mixture prevented cleavage .
ligand binding properties
the dissociation rate of d-biotin from xenavidin samples was measured at several different temperatures .
xenavidin showed a faster dissociation rate compared to avidin, but a slower rate when compared to either streptavidin or bradavidin .
if a similar biotin-association rate constant were assumed for all the proteins, xenavidin would have a streptavidin-like affinity for biotin .
the dissociation of biotin from xenavidin was also measured by fluorescence spectroscopy using fluorescently labelled bf560-biotin  <cit> .
xenavidin released this biotin conjugate much faster than did avidin - again suggesting a weaker affinity for biotin .
it is important to keep in mind that avidins may differ in the way in which they bind different biotin forms, including free biotin  and conjugated biotin   <cit> .
for example, it has been shown that streptavidin binds bf560-biotin more tightly than avidin, whereas avidin binds free biotin better than streptavidin  <cit> .
in our opinion, the fluorescent biotin assay reflects more the conditions found in life science applications, where biotin is typically conjugated to other molecules, whereas the free biotin assay mimics more closely the conditions in vivo.
the determination of the kinetics of association of free biotin would give an even deeper picture of the ligand-binding properties of avidins and would be interesting to do in future experiments.
athe dissociation rate was too slow to be measured accurately at 25°c.
the ligand-binding characteristics of xenavidin were also studied using a surface plasmon resonance approach using an optical biosensor instrument .
in this analysis, xenavidin showed ~10-fold lower affinity  to the 2-iminobiotin functionalized optical biosensor chip compared to chicken avidin .
both a slower association rate and a faster dissociation rate were observed for xenavidin compared to chicken avidin .
discussion
although thoroughly studied in terms of structural, biochemical and biophysical characteristics, the biological functions of avidins are not fully understood.
here, we expand the research on avidins by reporting xenavidin, the first avidin-like protein from a frog, which was identified as an expressed sequence tag  from the xenopus tropicalis genome project.
avidins appear to be conserved among egg-laying vertebrates  <cit>  and are thought to have a role as defence proteins against microbial infections  <cit> .
bird eggs contain egg yolk, the compartment for embryonic development, and the egg white that surrounds the egg yolk provides both nutrients and protection for the embryo.
in frogs, the equivalent to egg white is called egg jelly  <cit> .
the avidin content of egg jelly may have an important role in the embryonic development of frogs, a hypothesis that is also supported by recent studies within natural bird populations, in which the concentration of avidin in egg white appears to be linked to hatching success .
in this light, it seems likely that avidins are found in all frog and bird proteomes.
it would be interesting to see whether some of the many fish species also carry avidin in their proteome; a search of sequence databases revealed that the zebrafish , for example, may carry a gene encoding an avidin-like protein .
the first avidin from fungi, tamavidin, has recently been reported, too  <cit> .
avidin and streptavidin have been used for pre-targeting immunotherapy and imaging  <cit> .
although the folds of these proteins are similar, and both bind biotin tightly, they differ in their pharmacokinetic characteristics  <cit> .
the glycosylation of avidin, for example, was previously observed to affect its uptake by tumours and accumulation in the liver.
on the other hand, the basic pi of avidin is responsible for its accumulation in the kidneys  <cit> .
therefore, avidin cannot be efficiently used in drug delivery and imaging owing to non-specific binding to extraneous material  <cit> .
in comparison to avidin, streptavidin is known to have a longer half-life in plasma, but it accumulates mainly in the kidneys  <cit> .
since xenavidin has unique pharmacokinetic properties, for example its glycosylation pattern and electropotential surface properties , it could be a valuable substitute for avidin or streptavidin in biomedical applications.
the x-ray structures of xenavidin show that xenavidin has a β-barrel fold like the other known structures of avidins.
the residues around the biotin-binding pocket are also well conserved, but some differences were still found - four residues of xenavidin  had altered interactions with biotin when compared to the equivalent residues of avidin , the alterations affecting hydrogen bonding via the main chain, hydrophobic effect or van der waals interactions .
s <dig>  l <dig> and t <dig> are located within the l <dig> -loop, which is known to be important for determining the ligand-binding specificity of avidins  <cit> .
although t <dig> in avidin is comparable to t <dig> in xenavidin, the interaction with biotin is formed in a different way.
together with the unique subunit interface design, these, even subtle structural changes may be responsible for the observed, unique ligand-binding properties of xenavidin.
moreover, in the unliganded xenavidin structure, a water molecule was found at a site referred to as site  <dig>  <cit> , located at the deep end of the biotin-binding pocket .
in the biotin-complex structure of xenavidin, this water molecule was missing, even though a conserved water molecule at the entrance of the water channel was observed in both the unliganded protein structure and the biotin-complex structure .
thus, the unliganded structure of xenavidin may represent the first example of an intermediate structure of the avidin family, with a structural water molecule located deep within the biotin-binding pocket and lending support to the water-bridged model of biotin association/dissociation  <cit> .
moreover, freitag et al.
<cit>  and hyre et al.
<cit>  have shown that d <dig> in streptavidin has an important role in controlling the movement of water molecules in this area.
interestingly, n <dig>  the residue analogous to d <dig> of streptavidin and n <dig> of avidin, has an altered conformation in unliganded xenavidin compared to n <dig> of the biotin-complex structure of xenavidin, suggesting a potentially important role for this residue in the water-mediated association/dissociation mechanism of avidins in general.
CONCLUSIONS
we have carried out a structural and functional analysis of xenavidin, a novel avidin from xenopus tropicalis.
our results show that xenavidin is a well-conserved member of the avidin protein family having a tetrameric structure, n-glycosylation and extremely tight biotin-binding capability that is mediated via numerous non-covalent interactions including hydrogen bonds, hydrophobic interactions and van der waals contacts.
however, biochemical and structural features unique for xenavidin were also discovered.
our results may facilitate the development of novel and advanced avidins for biotechnological and nanotechnological applications.
overall, this study deepens our understanding of the structure-function relationship of avidins.

 avidins are high-affinity biotin-binding proteins found in the eggs of oviparous vertebrates including bird, reptilian and amphibian species. in addition to its production in the oviduct and secretion to egg white, avidin is expressed in several other tissues of the chicken during injury and inflammation. avidins analogous to those found in the earliest diverging tetrapod species have been isolated from a few bacterial species: streptomyces avidinii bradyrhizobium japonicum and rhizobium etli. avidins are homotetrameric proteins, the only known exception being rhizavidin, which is a homodimer. the avidin subunits consist of eight anti-parallel β-strands, which form a β-barrel structure that has a biotin-binding pocket at the open end of the barrel. avidins interact extraordinarily tightly with a water-soluble vitamin, d-biotin. this exceptional strength of interaction has not only been utilized in various biochemical and biophysical applications, but has also led to the production of a number of genetically modified forms of avidin and streptavidin. the biotin-binding modes of avidins, and the amino acids involved in biotin binding are highly conserved among different species even though the similarity between the primary sequences of avidins is relatively low. in addition to biotin binding, comparisons of avidins, either extracted directly from natural sources or produced as recombinant proteins, have revealed many differences in their physicochemical properties, e.g., stability and immunogenicity. consequently, the detailed characterization of novel avidins has provided valuable information that could be exploited in the development of novel molecular tools and devices. for example, a chimeric avidin containing structural parts from both chicken avidin and avidin related protein, has been found to be more stable than either one of the native forms. although avidins are expressed in several different species, only a few avidins have been thoroughly characterized. experimentally determined three-dimensional structures are available e.g. for streptavidin, chicken avidin, avidin related protein, avr and biotin-binding protein a. these structures have proved that the overall fold of the avidins is the same, but that the subtle structural differences explain the observed differences in the functional characteristics of avidins. here we report, to our knowledge, the first biochemical and structural characterization of an amphibian avidin - xenavidin - a frog avidin from xenopus tropicalis. being the only diploid species in the xenopus genus with a small genome and short generation time, x. tropicalis has proven invaluable, e.g., for understanding the mechanisms of vertebrate embryonic development and for functional genomics. the 56% amino acid sequence identity shared between avidin and xenavidin is relatively high and suggests that avidin and xenavidin have similar biochemical and structural properties. however, like for other avidins such as streptavidin, bradavidin and rhizavidin, in comparison with chicken avidin, xenavidin has it own unique features determined by the subtle changes found in its sequence and structure. thus, the characterization of xenavidin gives insights into the biochemical and structural determinants of a novel member of the avidin protein family, a family of extraordinary biotin-binders with many prospects for exploitation in biotechnology and nanotechnology.  cloning, expression and purification two dna sequences encoding an avidin-like protein from a frog were originally identified by searching an est sequence database; after discovering the avidin-like gene from xenopus tropicalis, the avidin gene for xenopus laevis, another frog species, was also identified. here, we focus on the protein from xenopus tropicalis, which we named xenavidin. the cdna of xenavidin was cloned into the pfastbac baculovirus expression system entry vector, and recombinant xenavidin was successfully produced in sf insect cells using the bac-to-bac® baculovirus expression system. the protein was purified with 2-iminobiotin sepharosetm affinity chromatography. about 2- mg of pure protein was typically obtained per one litre of culture media. attempts to produce xenavidin in a bacterial expression system in e. coli were unsuccessful. x-ray structure xenavidin overexpressed in insect cells was crystallized with and without d-biotin added to the crystallization solution. the x-ray structures of the xenavidin-btn complex and the unliganded xenavidin protein were solved to Å resolution. the structures crystallized in a rhombohedral space group and had a dimer in their asymmetric unit. in general, both of the xenavidin structures were highly similar to the known structures of avidin and streptavidin: the 3d structure of xenavidin is homotetrameric, each subunit consisting of an eight-stranded β-barrel. the amino acids lining the biotin-binding pocket are structurally conserved between xenavidin and chicken avidin. there were, however, differences in the loop architecture between these proteins, particularly in the l -loop and the l -loop. the unique l -loop design in different avidins is a key feature regulating the binding affinity of avidins, but the role of the variable l -loop is not clear though it is known not to be directly involved in ligand binding. moreover, there were some differences in the interface architecture of xenavidin in comparison to chicken avidin. for example, out of the three residues at the 1- subunit interface, two are conserved between avidin and xenavidin, whereas i in avidin is substituted by alanine in xenavidin, probably contributing to the reduced stability of xenavidin. the surface properties of avidin, xenavidin and streptavidin vary, too. athe numbers in parenthesis refer to the highest resolution bin. bobserved r-factor from xds. cfrom refmac using tls & restrained refinement. the subunits of the dimer found in the asymmetric units of the xenavidin crystals are labelled a and d to match the numbering system generally used to describe the biological unit of chicken avidin. in the unliganded structure of xenavidin, there was no clear electron density for biotin in subunit a. however, weak electron density was evident for the ligand at the binding site in subunit d; thus, a biotin molecule was built into subunit d of the unliganded xenavidin structure despite the presence of weak electron density around the ligand. one possible explanation for the varying electron densities at the biotin-binding sites could be sequential binding, which is supported by some previous studies suggesting cooperativity between binding sites of avidin. however, some other studies do not support cooperativity between binding sites. in the btn-complex structure, d-biotin was clearly present in both of the biotin-binding sites of the asymmetric unit. in the x-ray structures of xenavidin there was no clear evidence that either of the two potential n-glycosylation sites of each polypeptide, n or n, would in fact be glycosylated. however, the presence or absence of glycosylation at n cannot be reliably established since n is within the l -loop and the side-chain atoms of this residue have high b-factors and hence poor electron density. the structures of xenavidin clearly showed that n did not have sugar units attached to its side chain. in xenavidin, three amino acids residues out of lining the biotin-binding pocket are different from those in the binding pocket of avidin: the equivalent residues for the non-conserved s l and w found in xenavidin in avidin are t a and f. s40/t and w78/f interact with biotin through hydrophobic effect and van der waals interactions, whereas l41/a are involved in hydrogen bonding via the residue's main-chain nitrogen atom. in addition, the main chain of t in xenavidin is not close enough to form a hydrogen bond with biotin, whereas in avidin a hydrogen bond between the main-chain nitrogen atom of the equivalent residue t is evident. these subtle differences in the architecture of the biotin-binding sites of xenavidin and avidin may contribute, at least partially, to the different biotin-binding properties that were observed for these two proteins. a water molecule was detected in the unliganded structure of xenavidin at the bottom of the ligand-binding site bridging the carboxamide group of n and biotin; the conformation of n in the unliganded structure differs from that in the structure of the xenavidin-biotin complex. to our knowledge, the only other known avidin structures harbouring a water molecule at the corresponding location, referred to as'site 1' by hyre et al., are two mutant structures of streptavidin: the d128a mutant structure and the s45a/d128a double mutant structure. a structural water at site has so far not been observed in native avidin structures. a conserved water molecule, the water molecule at site, located at the entrance of the water channel, is present in both of the xenavidin structures. according to hyre et al., biotin dissociation from streptavidin is initiated by penetration of water molecules into these sites. the presence of structural water molecules at site and/or in the xenavidin structures suggests a role for these water molecules in the biotin association/dissociation events of xenavidin; the biotin-binding site of xenavidin may be more vulnerable to potential'attack' by water molecules than it is in chicken avidin. this hypothesis is supported by our experimental results, which showed an increased dissociation rate of biotin from xenavidin compared to avidin. biochemical and physicochemical characterization the quality and molecular size of the isolated xenavidin protein was analyzed using 15% sds-page and size exclusion chromatography. in the latter analysis, xenavidin appeared mainly as a single peak and had a size of approximately kda suggesting a tetrameric quaternary structure. recall that xenavidin has two asparagine residues, n and n, identified as potential glycosylation sites according to the program netnglyc. the theoretical molecular weight of xenavidin, calculated on the basis of the primary amino acid sequence, is only kda, which suggests that n might indeed be glycosylated in the insect cells. sds-page analysis resulted in multiple bands where, in addition to the band corresponding to the nonglycosylated form, two higher molecular weight forms of the monomeric protein were detectable. these higher molecular weight forms most probably correspond to glycosylated species with a varying number and type of sugar units. it is well known that proteins overexpressed using the baculovirus expression system may have heterogeneous glycosylation patterns, as reviewed by davies. furthermore, we have also observed this phenomenon in the avr-proteins, which may have multiple glycosylation sites per polypeptide chain. further evidence for the presence of heterogeneously glycosylated xenavidin was obtained by treating the purified protein with the deglycosylation enzymes endo hf and pngase, which caused the disappearance of the higher molecular weight forms of xenavidin in sds-page analysis. moreover, based on the x-ray analysis of xenavidin, the crystallized proteins may have carbohydrates attached to one of the two potential glycosylation sites. taken together, our data support a structure in which xenavidin is glycosylated at n43; interestingly, the glycosylation site in xenavidin is located at a position equivalent to that glycosylated in avr. avidin also has one glycosylation site but there is no equivalent glycosylation site in xenavidin. athe analyzed proteins were most probably glycosylated; sugar moieties comprise at least 10% of the mass of avidin. btheoretical values were calculated from the primary structures. like almost all of the other avidin-like proteins studied so far, xenavidin also tends to form oligomers of tetramers, which could be seen as a minor peak, with an apparent molecular weight of kda, before the main peak in the gel filtration chromatogram. these oligomers could not be detected in an sds-page-based thermostability assay, where xenavidin appeared only as a tetrameric form in the presence of biotin to up to 65°c, and even at 90°c, but dissociated into monomers in the absence of biotin already at room temperature. xenavidin has a very similar theoretical isoelectric point to avidin; values for both proteins were calculated using the compute pi/mw tool. hence, xenavidin and avidin are positively charged at neutral ph, whereas streptavidin has a negative net charge at the same ph. sera from human cancer patients who were exposed to avidin and streptavidin were used to probe the cross reactivity among xenavidin, chicken avidin and streptavidin. in this analysis xenavidin did not react significantly with the avidin/streptavidin immunized sera. moreover, polyclonal antibodies against avidin and streptavidin did not recognize xenavidin. taken together, xenavidin appears not to be immunologically cross reactive with avidin. the complete dataset is reported in helppolainen et al.. protein stability the thermal stability of xenavidin was determined with a sds-page thermostability assay, and the analysis was performed as described by bayer et al.. without biotin, the tetrameric form of xenavidin was unstable and the protein was monomeric already at room temperature. when biotin was added, the stability of the tetrameric form of the xenavidin was significantly increased - the transition temperature of dissociation of the tetramer was raised to 75°c. therefore, in the absence of biotin, the tetrameric quaternary structure of xenavidin is clearly more vulnerable to the denaturing effect of sds than is the structure of avidin, whereas in the presence of biotin, xenavidin and avidin have relatively similar stabilities, thus suggesting high affinity to biotin. the stability of xenavidin against proteinase k was tested in the presence and absence of biotin. as previously shown, avidin was cleaved in the absence of biotin after extended incubation with proteinase k to fragments of kda and below, but the presence of biotin protected avidin from proteolytic cleavage. similar behaviour was observed in the case of xenavidin. in the absence of biotin, xenavidin was cleaved by proteinase k into small fragments, which could not be seen using gel analysis, whereas adding biotin to the reaction mixture prevented cleavage. ligand binding properties the dissociation rate of d-biotin from xenavidin samples was measured at several different temperatures. xenavidin showed a faster dissociation rate compared to avidin, but a slower rate when compared to either streptavidin or bradavidin. if a similar biotin-association rate constant were assumed for all the proteins, xenavidin would have a streptavidin-like affinity for biotin. the dissociation of biotin from xenavidin was also measured by fluorescence spectroscopy using fluorescently labelled bf560-biotin. xenavidin released this biotin conjugate much faster than did avidin - again suggesting a weaker affinity for biotin. it is important to keep in mind that avidins may differ in the way in which they bind different biotin forms, including free biotin and conjugated biotin. for example, it has been shown that streptavidin binds bf560-biotin more tightly than avidin, whereas avidin binds free biotin better than streptavidin. in our opinion, the fluorescent biotin assay reflects more the conditions found in life science applications, where biotin is typically conjugated to other molecules, whereas the free biotin assay mimics more closely the conditions in vivo. the determination of the kinetics of association of free biotin would give an even deeper picture of the ligand-binding properties of avidins and would be interesting to do in future experiments. athe dissociation rate was too slow to be measured accurately at 25°c. the ligand-binding characteristics of xenavidin were also studied using a surface plasmon resonance approach using an optical biosensor instrument. in this analysis, xenavidin showed ~10-fold lower affinity to the 2-iminobiotin functionalized optical biosensor chip compared to chicken avidin. both a slower association rate and a faster dissociation rate were observed for xenavidin compared to chicken avidin. discussion although thoroughly studied in terms of structural, biochemical and biophysical characteristics, the biological functions of avidins are not fully understood. here, we expand the research on avidins by reporting xenavidin, the first avidin-like protein from a frog, which was identified as an expressed sequence tag from the xenopus tropicalis genome project. avidins appear to be conserved among egg-laying vertebrates and are thought to have a role as defence proteins against microbial infections. bird eggs contain egg yolk, the compartment for embryonic development, and the egg white that surrounds the egg yolk provides both nutrients and protection for the embryo. in frogs, the equivalent to egg white is called egg jelly. the avidin content of egg jelly may have an important role in the embryonic development of frogs, a hypothesis that is also supported by recent studies within natural bird populations, in which the concentration of avidin in egg white appears to be linked to hatching success. in this light, it seems likely that avidins are found in all frog and bird proteomes. it would be interesting to see whether some of the many fish species also carry avidin in their proteome; a search of sequence databases revealed that the zebrafish, for example, may carry a gene encoding an avidin-like protein. the first avidin from fungi, tamavidin, has recently been reported, too. avidin and streptavidin have been used for pre-targeting immunotherapy and imaging. although the folds of these proteins are similar, and both bind biotin tightly, they differ in their pharmacokinetic characteristics. the glycosylation of avidin, for example, was previously observed to affect its uptake by tumours and accumulation in the liver. on the other hand, the basic pi of avidin is responsible for its accumulation in the kidneys. therefore, avidin cannot be efficiently used in drug delivery and imaging owing to non-specific binding to extraneous material. in comparison to avidin, streptavidin is known to have a longer half-life in plasma, but it accumulates mainly in the kidneys. since xenavidin has unique pharmacokinetic properties, for example its glycosylation pattern and electropotential surface properties, it could be a valuable substitute for avidin or streptavidin in biomedical applications. the x-ray structures of xenavidin show that xenavidin has a β-barrel fold like the other known structures of avidins. the residues around the biotin-binding pocket are also well conserved, but some differences were still found - four residues of xenavidin had altered interactions with biotin when compared to the equivalent residues of avidin, the alterations affecting hydrogen bonding via the main chain, hydrophobic effect or van der waals interactions. s l and t are located within the l -loop, which is known to be important for determining the ligand-binding specificity of avidins. although t in avidin is comparable to t in xenavidin, the interaction with biotin is formed in a different way. together with the unique subunit interface design, these, even subtle structural changes may be responsible for the observed, unique ligand-binding properties of xenavidin. moreover, in the unliganded xenavidin structure, a water molecule was found at a site referred to as site, located at the deep end of the biotin-binding pocket. in the biotin-complex structure of xenavidin, this water molecule was missing, even though a conserved water molecule at the entrance of the water channel was observed in both the unliganded protein structure and the biotin-complex structure. thus, the unliganded structure of xenavidin may represent the first example of an intermediate structure of the avidin family, with a structural water molecule located deep within the biotin-binding pocket and lending support to the water-bridged model of biotin association/dissociation. moreover, freitag et al. and hyre et al. have shown that d in streptavidin has an important role in controlling the movement of water molecules in this area. interestingly, n the residue analogous to d of streptavidin and n of avidin, has an altered conformation in unliganded xenavidin compared to n of the biotin-complex structure of xenavidin, suggesting a potentially important role for this residue in the water-mediated association/dissociation mechanism of avidins in general.  we have carried out a structural and functional analysis of xenavidin, a novel avidin from xenopus tropicalis. our results show that xenavidin is a well-conserved member of the avidin protein family having a tetrameric structure, n-glycosylation and extremely tight biotin-binding capability that is mediated via numerous non-covalent interactions including hydrogen bonds, hydrophobic interactions and van der waals contacts. however, biochemical and structural features unique for xenavidin were also discovered. our results may facilitate the development of novel and advanced avidins for biotechnological and nanotechnological applications. overall, this study deepens our understanding of the structure-function relationship of avidins. 
xenavidin was identified from an est sequence database for xenopus tropicalis and produced in insect cells using a baculovirus expression system. biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog. avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. the location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin. the recombinant xenavidin was found to be homotetrameric based on gel filtration analysis.
 avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.  xenavidin was identified from an est sequence database for xenopus tropicalis and produced in insect cells using a baculovirus expression system. the recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. x-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. the location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin.  the novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. this information may facilitate the discovery of novel tools for biotechnology. 