Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Structural adaptation of a thermostable biotin-binding protein in a psychrophilic environment

Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application.     

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.     

Bifunctional avidin with covalently modifiable ligand binding site

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.     

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.     

The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins

Bradavidin II is a biotin‐binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X‐ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerization‐independent biotin‐binding protein development.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin-biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin-biotin complex. 3. This reassociation still occurred in 3.5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin-biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin-biotin complex do not readily lose their biotin, even in 3.5m-guanidinium chloride, and that monomeric biotin-binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3.0m and 3.5m.     

Stable,high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K_d = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T_m = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.     

Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein

A gene encoding an avidin-like protein was discovered in the genome of B. japonicum. The gene was cloned to an expression vector and a protein, named bradavidin II, was produced in E. coli. Bradavidin II has an identity of 20-30% and a similarity of 30-40% with previously discovered bradavidin and other avidin-like proteins. It has biochemical characteristics close to those of avidin and streptavidin and binds biotin tightly. In contrast to other tetrameric avidin-like proteins studied to date, bradavidin II has no tryptophan analogous to the W110 in avidin (W120 in streptavidin), thought to be one of the most essential residues for tight biotin-binding. Homology modeling suggests that a proline residue may function analogously to tryptophan in this particular position. Structural elements of bradavidin II such as an interface residue pattern or biotin contact residues could be used as such or transferred to engineered avidin forms to improve or create new tools for biotechnological applications.     

Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins

Two distinct circularly permuted forms of chicken avidin were designed with the aim of constructing a fusion avidin containing two biotin-binding sites in one polypeptide. The old N and C termini of wild-type avidin were connected to each other via a glycine/serine-rich linker, and the new termini were introduced into two different loops. This enabled the creation of the desired fusion construct using a short linker peptide between the two different circularly permuted subunits. The circularly permuted avidins (circularly permuted avidin 5 --> 4 and circularly permuted avidin 6 --> 5) and their fusion, pseudotetrameric dual chain avidin, were biologically active, i.e. showed biotin binding, and also displayed structural characteristics similar to those of wild-type avidin. Dual chain avidin facilitates the development of dual affinity avidins by allowing adjustment of the ligand-binding properties in half of the binding sites independent of the other half. In addition, the subunit fusion strategy described in this study can be used, where applicable, to modify oligomeric proteins in general.     

Relationship between biotin-binding proteins from chicken plasma and egg yolk.

The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.     

Brave new (strept)avidins in biotechnology

Avidin and streptavidin are widely used in (strept)avidin-biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)avidins--such as simple amino acid substitutions to reduce biotin binding and alter physico-chemical characters--have recently developed into more sophisticated changes, including chimeric (strept)avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine and nanotechnology.     

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.     

Biochemical and Biological Characterization of a New Oxidized Avidin with Enhanced Tissue Binding Properties

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff''s base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected ¹¹¹In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.     

Zebavidin - An Avidin-Like Protein from Zebrafish

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.     

Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

In order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1-3 interface, and in combination with a histidine conversion in the 1-2 interface (Trp110). The single replacements Met96His and Val115His in the 1-3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1-2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein-ligand complex formation were pH-sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin-biotin technology.     

Limited proteolysis of native proteins: the interaction between avidin and proteinase K.

Avidin is a tetramer of 16-kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between beta-strands 3 and 4, involving amino acids 38-43. Specifically, sites of proteolysis are at Thr 40-Ser 41 and Asn 42-Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4'-hydroxyazobenzene-2-benzoic acid (HABA), a chromogenic reporter that can occupy the biotin-binding site. Saturation of the biotin-binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site. Analysis of the three-dimensional structures of apoavidin and holoavidin reveals that the 3-4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3-4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3-4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry-related molecules. Structural analyses suggest that the "lid" to the biotin-binding site, formed by the 3-4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility.     

DNA family shuffling within the chicken avidin protein family - A shortcut to more powerful protein tools

Avidins represent an interesting group of proteins showing high structural similarity and ligand-binding properties but low similarity in primary structure. In this study, we show that it is possible to create functional chimeric proteins from the avidin protein family when applying DNA family shuffling to the genes of the avidin protein family: avidin, avidin related gene 2 and biotin-binding protein A. The novel chimeric proteins were selected by phage display biopanning against biotin, and the selected enriched proteins were characterized, displaying diverse features distinct from the parental genes, including binding to cysteine.