Enhancing the Thermal Stability of Avidin

INTRODUCTION OF DISULFIDE BRIDGES BETWEEN SUBUNIT INTERFACES*

Henri R. NordlundDagger , Olli H. LaitinenDagger , Sanna T. H. UotilaDagger , Thomas Nyholm§, Vesa P. HytönenDagger , J. Peter Slotte§, and Markku S. KulomaaDagger

From the Dagger  Department of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland and § Department of Biochemistry and Pharmacy, Åbo Akademi University, P. O. Box 66, FIN-20521 Turku, Finland

Received for publication, October 19, 2002, and in revised form, November 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native right-arrow denatured transition midpoints (Tm) of 98.6 and 94.7 °C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower Tm in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chicken avidin and bacterial streptavidin are widely utilized proteins in many life science applications ranging from purification techniques to modern diagnostics and targeted drug delivery. This methodology, known as (strept)avidin-biotin technology, relies on the extremely tight and specific affinity (Kd sime  10-15 M) between (strept)avidin and biotin (1, 2).

Avidin and streptavidin are exceptionally stable proteins that consist of homotetrameric antiparallel beta -barrels (3-5), which upon biotin binding become even more stable. Transition midpoints of heat denaturation (Tm),1 analyzed by differential scanning calorimetry (DSC), have shown that avidin is more stable than streptavidin in both the absence and presence of biotin (6). A possible reason for this finding may be the intramonomeric disulfide bridge found in each avidin monomer (3, 4, 7). Wild-type (wt) avidin has a high isoelectric point (pI sime  10.5) and is glycosylated, properties that may cause unspecific binding in some applications (7, 8). It has been shown that these unwanted properties can be abolished without markedly affecting the tight biotin binding affinity or the stability characteristics of avidin (9, 10).

The avidin (and streptavidin) tetramer is actually a dimer of two dimers. The monomers that form the tetramer (Fig. 1) interact with each other in a symmetrical manner and form the three types of monomer-monomer interactions described in detail by Livnah et al. (3). The buried surface area between monomers 1-4 (and the equivalent 2-3) is the largest, whereas the interface area between monomers 1-3 (and 2-4) is the smallest. The buried surface area between monomers 1-2 (and 3-4) is also relatively small, but this interface is important because tryptophan 110 in avidin (Trp-120 in streptavidin) from subunit 1 participates in biotin binding at the binding-site of subunit 2, forming the function-related monomer-monomer interface.

A streptavidin mutant with enhanced stability characteristics when compared with those of the wt streptavidin has been reported previously (11, 12). The improved stability was achieved by the addition of two intermonomeric disulfide bridges to the streptavidin tetramer, one between monomers 1-3 and the other between monomers 2-4, by changing histidine residue 127 to cysteine. In avidin these interfaces are similar, and isoleucine residue 117 of avidin is analogous to histidine residue 127 of streptavidin.

In the present study, we improved the stability of avidin through the addition of intermonomeric disulfide bridges by the same strategy and changed isoleucine residue 117 to cysteine. In addition, we produced two supplementary mutants in which even more intermonomeric disulfide bridges were introduced. A cysteineless avidin version was also constructed in which the intramonomeric disulfide bridges of the wt avidin were removed. The stability characteristics of these mutants were studied and compared with those of avidin and streptavidin.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenesis of Avidin cDNA-- Mutations were designed by using the sequence and structure information obtained from analyses with the programs GCG (Genetics Computer Group, Madison, Wisconsin), EMBOSS (European Molecular Biology Open Software Suite), WHAT IF (13), and InsightII (Molecular Simulations Inc., San Diego, CA). Genetic engineering of the coding sequence of avidin was performed by megaprimer (14) and QuikChange (Stratagene) methods using oligonucleotide primers containing the desired mutations.

Production and Purification of Mutant Avidins-- All of the mutants were produced by a baculovirus expression system (Bac-To-BacTM, Invitrogen) in the infected insect cells and purified by affinity chromatography on 2-iminobiotin-agarose as described previously in detail by Airenne et al. (15) and Laitinen et al. (16). The wt avidin was purified from chicken egg white. Using a Vibra cellTM sonicator, the egg white was sonicated for 3 min on ice at power setting 8 and 50% duty cycle with a 1-min break between bursts. After sonication the sample was diluted with two volumes of PBS and centrifuged for 20 min at 20,000 × g, 4 °C. The soluble fraction was purified further by affinity chromatography on 2-iminobiotin-agarose as reported previously (17). Protein samples were concentrated and subjected to a change of buffers with Centricon YM-3 filters (Millipore, catalog no. 4202). The SDS-PAGE analysis was performed with a sample buffer without beta -mercaptoethanol.

Biotin Binding Assays-- Reversibility of biotin binding was measured for avidin and the mutants with an IAsys optical biosensor as reported previously (18). Protein samples were allowed to bind to a biotin-aminosilane cuvette in PBS containing 1 M NaCl. After equilibrium was reached, biotin-containing buffer was added, and protein dissociation was monitored. The affinity of the proteins toward 2-iminobiotin was also determined with an IAsys optical biosensor as described earlier (9). The biotin binding activity of the mutants Avd-ci and Avd-ccci after heat treatment was studied with a microtiter plate assay. Protein samples, at a concentration of 5 µg/ml in PBS, were heated for various time periods at 99.9 °C and then chilled on ice. The samples were transferred to a Nunc Maxisorp plate and incubated at 37 °C for 2 h. The wells were washed three times with PBS-Tween (0.05% v/v). The wells were then blocked with PBS containing 1% bovine serum albumin at 37 °C for 30 min and washed again with PBS-Tween. Biotinylated alkaline phosphatase (Sigma) in PBS, 1% bovine serum albumin was added and incubated at 37 °C for 1 h. Once again, the wells were washed with PBS-Tween and the p-nitrophenyl phosphate substrate in DEA buffer (1 mg/ml) was applied to the wells. Absorbance at 405 nm was measured after incubation for 45 min.

Differential Scanning Calorimetry-- To study the thermostability of the avidins, a Nano II differential scanning calorimeter (Calorimetric Science Corporation, Provo, UT) was used. The protein sample concentration was 0.032 mM (given as the monomer concentration), and the biotin-containing samples had a biotin:avidin (monomer) molar ratio of 3:1. The reference cell was filled with the same buffer in which the sample proteins were dissolved (100 mM sodium phosphate buffer pH 7.4). The thermograms were recorded as a function of temperature, between 25 and 130 °C, at a temperature scan rate of 55.6 °C/h. The base lines were subtracted, and the Tm values of the samples were calculated using proprietary software provided by the manufacturer of the instrument.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design of the Mutants-- The avidin mutant Avd-nc (C4A, C83Y) was constructed to obtain information about the importance of the intrinsic disulfide bridges to the overall stability of the avidin tetramer. According to the sequence alignment with streptavidin (3), the cysteine residues were substituted with the same residues that streptavidin bears in the analogous positions in its primary structure (Fig. 1, Table I). The avidin mutant Avd-cci (D86C, I106C) also adopted the cysteines using the evolutionary approach (18). This sequence information came from the avidin-like domain of the sea urchin fibropellins (19-22). In the case of Avd-cci, two intermonomeric disulfide bridges, designed to form between monomers 1-4 and 2-3, were introduced, thereby constituting a total of four new disulfide bridges per tetramer (Figs. 1 and 2A). In Avd-cci, cysteine 86 from subunit 1 was presumed to pair with cysteine 106 from the adjacent subunit 4 and vice versa. Identical contacts were assumed to be present on the interface of subunits 2 and 3 as well.


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Fig. 1.   Ribbon representation of avidin tetramer (stereo view) showing the locations of mutated residues (sticks). A, subunit color coding is as follows: green = 1, pink = 2, light blue = 3, black = 4 (numbering according to Livnah (3)). When the side chains of isoleucine 117 in each subunit (indicated in yellow) are substituted with cysteines they form the 1-3 (and 2-4) disulfide bridges in mutants Avd-ci and Avd-ccci. The blue side chain represents isoleucine 106 in each subunit, whereas red highlights aspartate 86; in mutants Avd-cci and Avd-ccci these are substituted with cysteines to form the 1-4 (and 2-3) intermonomeric disulfide bridges. White sticks represent the cysteines that form the intrasubunit disulfide bridges in the wt avidin. In Avd-nc they were substituted with alanine (Cys-4) and tyrosine (Cys-83). B, a close-up view from the bottom left corner of panel A with the same color coding of the subunits.

                              
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Table I
Description of the proteins used in this study
The number of intermonomeric disulfide bridges and the measured affinity toward 2-iminobiotin are indicated.


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Fig. 2.   Formation of the intermonomeric disulfide bridges. A, schematic representation of the mutants Avd-ci, Avd-cci, and Avd-ccci displaying the intermonomeric disulfide bridges. The subunits are numbered according to Livnah et al. (3). B, nonreducing SDS-PAGE analysis of avidin (Avd) and avidin mutants Avd-ci, Avd-cci, and Avd-ccci. Samples were boiled for 15 min in SDS-PAGE sample buffer (without beta -mercaptoethanol), and the gel was stained with Coomassie Brilliant Blue. The wt avidin is found mainly as a monomeric form (the dimeric form is an artifact seen in several avidin preparations (28)). The mutants that have intermonomeric disulfide bridges between two subunit pairs (Avd-ci, Avd-cci) and the mutant that forms a continuous macromolecule (Avd-ccci) formed dimeric and tetrameric structures in the manner expected. The double bands in the mutant samples result from different glycosylation patterns of the recombinant avidins (15). The lower molecular weight bands are mainly nonglycosylated forms, whereas the subunits in the higher molecular weight bands bear sugar moieties. LMW, low molecular weight markers.

Avd-ci (I117C) has an extra cysteine residue in each subunit of the tetramer. It was designed according to the mutational strategy that Stayton and co workers (11) and Sano and co workers (12) used to stabilize the streptavidin tetramer. In Avd-ci, cysteine 117 from subunit 1 faces cysteine 117 from the neighboring subunit 3. Identically, the corresponding subunit interface 2-4 of the wt protein contains two equivalent isoleucine residues, which were replaced by cysteines. Therefore, two intermonomeric disulfide bridges in the avidin tetramer were expected to form between subunits 1-3 and 2-4, intensifying the firm association between dimers 1-4 and 2-3 by the addition of two covalent bonds (3, 4). Finally, to introduce six intermonomeric disulfide bridges into the avidin tetramer, Avd-ccci, a combination of mutants Avd-ci and Avd-cci, was constructed.

Purification and Characterization of the Proteins-- The mutant avidins were produced by a baculovirus expression system (Bac-To-BacTM) in infected insect cells and purified by affinity chromatography on a 2-iminobiotin-agarose column with a single-step protocol. All of the mutants showed excellent purification efficiency, indicating that the mutations had no major effects on the 2-iminobiotin-binding properties. Moreover, the mutants showed irreversible biotin binding properties indistinguishable from that of the wt avidin as measured with an IAsys optical biosensor (data not shown). Affinities toward 2-iminobiotin were determined for the wt avidin and the four mutants (Table I). The results indicated that the mutations had not significantly altered the 2-iminobiotin binding characteristics of the mutants. The formation of the intermonomeric disulfide bridges was studied using SDS-PAGE analysis (Fig. 2B), which confirmed that the cysteines formed pairs in the manner expected. The biotin binding activity after heat treatment of Avd-ci and Avd-ccci was studied using a microtiter plate assay (Fig. 3). The mutants proved to be more stable and they remained active, in the sense of binding biotin, longer than the wt avidin.


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Fig. 3.   Binding activity after heat treatment. The binding of biotinylated alkaline phosphatase by wt avidin and the mutants Avd-ci and Avd-ccci after heat treatment (99.9 °C) for various periods of time was determined by a microtiter plate assay.

Differential Scanning Calorimetry-- To study the thermal stability of the purified avidin mutants, we subjected them to analysis by differential scanning calorimetry. The results are shown in a base line-subtracted form (Fig. 4) and numerically (Table II). As shown by the thermograms, Avd-ci was the most stable protein, because its denaturation Tm value was the highest both in the absence and presence of biotin. Avd-ccci was only slightly less stable than Avd-ci. The other mutants and the wt avidin were equally stable when bound to biotin, whereas Avd-nc and Avd-cci showed the lowest Tm values in the absence of biotin. The heat-induced unfolding of the avidins was an irreversible process, and the unfolding of the proteins was often followed by a more or less sharp decrease in the heat capacity, most probably because of aggregation (data not shown).


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Fig. 4.   Differential scanning calorimetry. Heat-induced unfolding of avidin and the mutants in the absence (A) and presence (B) of biotin in a base line-subtracted form.

                              
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Table II
Thermostability analyses
Heat-induced unfolding of the wt and mutant avidins in the absence or presence of biotin was examined using DSC. Values for Tm are the averages of the results from two different experiments (±S.D.). The value for Delta Tm indicates the difference in Tm compared with that of the wt avidin in the presence or absence of biotin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Avidin and streptavidin are valuable and widely used tools in the life sciences. In addition to their high biotin binding affinity, the robustness and flexibility of the system relies on their extreme stability under various demanding conditions (7). In this study, our aim was to investigate whether it is possible to increase the stability of chicken avidin even further without losing its strong biotin-binding ability. To achieve this goal, two, four, or six intermonomeric disulfide bridges were introduced to avidin. In addition, we wanted to clarify the role of the intramonomeric disulfide bridges in the stability of the avidin tetramer by removing them using site-directed mutagenesis.

According to the DSC results, both the intramonomeric and intermonomeric disulfide bridges affected the heat-induced denaturation of avidin. Removal of the intramonomeric disulfide bridges from avidin in the mutant Avd-nc caused a decrease in its Tm in the absence of biotin (Delta Tm = - 8.9 °C), whereas in the presence of biotin the Tm was virtually the same as that of the wt avidin. This finding underlines the importance of the biotin-induced increase in the stability of avidin. Interestingly, the cysteineless mutant Avd-nc had a slightly higher Tm than streptavidin (Tm = 75.5 °C) (6) as an apo form and significantly higher as a complex with biotin (Tm = 112.2 °C) (6).

The most stable of the proteins was Avd-ci, which has an intermonomeric disulfide bridge in monomer interfaces 1-3 and 2-4. In contrast, the disulfide bridges created in interfaces 1-4 and 2-3 caused a decrease in the Tm of the mutant Avd-cci when compared with the wt avidin or even with the cysteineless mutant Avd-nc. This effect was more prominent in the absence of biotin. These interface mutations are interesting in that the sea urchin fibropellins (21, 22) were used as a template in their design. We called this kind of design an "evolutionary approach." These cysteine residue pairs can stabilize the natural fibropellins, although the arrangement failed to further stabilize avidin. The reason behind this phenomenon may return to the fact that the primary structures of avidin and fibropellins differ significantly from each other. Consequently, they may have different three-dimensional structures, and the cysteine residues introduced into avidin according to fibropellins may not be in the ideal position for formation of a disulfide bridge. The thermal stability of Avd-ccci (six intermolecular disulfide bridges) was interesting, because it was lower than that found in Avd-ci (two intermolecular disulfide bridges) but higher, however, than that of Avd-cci (four intermolecular disulfide bridges) and of the wt avidin (no intermolecular bridges). An explanation for the behavior of Avd-ccci being the sum of its components, Avd-ci and Avd-cci, could be that the mutation behind Avd-cci causes a small change in the quaternary structure, and therefore the cysteines originating from Avd-ci form disulfide bridges with non-optimal bond angles or cause more steric hindrance to the main chain. The barrel itself has to be stable, and if, as may well be in the case of Avd-cci, the disulfide bridges cause some unwanted twisting to the loops, the stability of the barrel might therefore be jeopardized.

In the presence of denaturing agents it may be preferable to use the mutants Avd-ci, Avd-cci, or Avd-ccci instead of the wt avidin. This may be the case particularly for Avd-ccci, because all of its monomers are covalently linked to each other (directly or indirectly). The mutant is capable of remaining as a tetramer even after unfolding, as seen in the SDS-PAGE analysis (Fig. 2B). Therefore it could be beneficial in applications where a leakage of subunits from matrix-coupled avidin tetramers would impede the qualitative and quantitative analysis of molecules. According to the DSC and microtiter plate assays, the avidin mutants Avd-ci and Avd-ccci, which had remarkably high Tm values even in the absence of biotin, could be utilized in PCR protocols, because they can withstand the temperatures used to denature double-stranded DNA. For example, the extraction of 2-iminobiotinylated or biotinylated single-stranded DNA molecules at 95 °C should be possible with Avd-ci- and Avd-ccci-coated particles.

In most applications of (strept)avidin-biotin technology, high-affinity biotin binding is essential. Consequently, all of the mutants introduced in this report are promising candidates for use with the technology, as they all showed irreversible biotin binding and high affinity toward 2-iminobiotin. This result was not surprising, because none of the mutations involved residues that were directly responsible for biotin binding. However, cysteine 106, which substituted isoleucine 106 in mutants Avd-cci and Avd-ccci, is located in the same loop (between beta -strands 7 and 8) as the important biotin-binding residue tryptophan 110. The disulfide bridge formed between Cys-106 of subunit 1 and Cys-86 of subunit 4 may directly or indirectly have an influence on Trp-110 and thereby also affect the biotin binding ability of these mutants.

We have discovered that the streptavidin tetramer is unstable in certain conditions without its bound ligand.2 This could be detrimental in many applications because of the loss of signal along with the analytical molecules. Furthermore, it would presumably shorten the life span of materials if they were to contain covalently linked streptavidin molecules. Chilkoti et al. (11) as well as Reznik and et al. (12) have described a streptavidin mutant, H127C. This mutant has an intermonomeric disulfide bridge between subunits 1 and 3 (2 and 4), which is analogous to the isoleucine 117 to cysteine substitution in our avidin mutant Avd-ci. They report that the resultant mutant is more stable than wild-type streptavidin (12). Avidin, however, is more stable than streptavidin, as judged by DSC analysis (6). Therefore, our prediction is that the 1-3 (and 2-4) intermonomeric disulfide bridges bearing mutant Avd-ci could also have a higher Tm than the corresponding streptavidin mutant.

The production of streptavidin (or its mutants) is usually performed as inclusion bodies in Escherichia coli. Its downstream processing includes laborious and time-consuming denaturation and renaturation procedures followed by the contrived formation of disulfide bridges (as in the case of the H127C mutant) and additional purification steps (11, 12, 23-25). In contrast, the production of avidin mutants, even those with intermonomeric disulfide bridges, yielded soluble proteins in insect cells that were easily purified in a single step by affinity chromatography on a 2-iminobiotin column. Moreover, there are reports describing the production of recombinant avidin in transgenic corn (26, 27). Interestingly, recombinant avidin made in corn is less expensive than avidin purified from chicken egg white (Sigma).

It is possible that avidins with even higher stability than that of Avd-ci or Avd-ccci could be created. More disulfide bridges could probably be introduced to link the interfaces, and the amino and carboxyl termini could be joined together. However, the disulfide bridge strategy used in this study is by no means the only way to stabilize proteins. Avidin could be stabilized further by using other strategies such as shortening, to some extent, the loops connecting the beta -strands and by conversion of the glycines in the loops to prolines. In addition, the introduction of new salt bridges in the interface areas would also be interesting from point of view of stability.

In conclusion, the intramonomeric disulfide bridges of the wt avidin seem to be an important factor in making avidin so thermostable. On the other hand, it is possible to enhance the high thermostability of avidin even further by introducing intermonomeric disulfide bridges. The most thermostable avidin mutants described in this study therefore provide more stable tools for avidin-biotin technology, suitable also for new kinds of applications.

    ACKNOWLEDGEMENTS

We thank Irene Helkala, Pirjo Käpylä, and Jarno Hörhä for excellent technical assistance.

    FOOTNOTES

* This study was supported by grants from the Finnish Cultural Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 358-14-2602272; Fax: 358-14-2602221; E-mail: kulomaa@csc.fi.

Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210721200

2 H. R. Nordlund, O. H. Laitinen, S. T. H. Uotila, K. J. Airenne, E. J. Porkka, N. Kalkkinen, and M. S. Kulomaa, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: Tm, transition midpoint; DSC, differential scanning calorimeter; wt, wild type; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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