From the Department of Biological and Environmental
Science and the ¶ Department of Chemistry, P. O. Box 35, FIN-40014 University of Jyväskylä, Finland, the
A. I. Virtanen Institute, University of Kuopio, FIN-70211
Kuopio, Finland, the ** Department of Biological Chemistry,
The Institute of Life Sciences, The Wolfson Centre for Applied
Structural Biology, The Hebrew University of Jerusalem, Givat Ram,
Jerusaleum 91904, Israel, and the
Department of Biological Chemistry, The
Weizmann Institute of Science, 76100 Rehovot, Israel
Received for publication, June 12, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Homotetrameric chicken avidin that binds four
molecules of biotin was converted to a monomeric form (monoavidin) by
mutations of two interface residues: tryptophan 110 in the 1 Chicken avidin and bacterial streptavidin are structurally
analogous proteins, which are well known for the uniquely tenacious affinity they exhibit toward their ligand biotin. This affinity is the
firmest protein-ligand interaction known in nature
(Kd ~10 The scientific merit of mutation studies of avidin and streptavidin has
been 2-fold. On the one hand, mutations of designated residues have
provided important structural and functional information regarding the
native protein. On the other, the mutant proteins themselves can be of
applicative value for use in avidin-biotin technology, since they
frequently exhibit special and sometimes surprising binding properties
or physicochemical characteristics. In this context, the affinity of
the native biotin-binding protein may be too high for some
applications. In some cases, for example, a reversible binding might be
desired, e.g. for affinity chromatography where the facile
release of biotinylated material from an avidin column is required.
Several reports have demonstrated clearly that in most cases the strong
affinity of avidin or streptavidin for biotin is dependent upon the
tetrameric architecture of the protein. Indeed, long before the
structures of avidin (3, 4) and streptavidin (5, 6) were known, it was
recognized that the avidin monomer shows a highly reduced affinity
constant, which can be recovered upon conversion of the monomer to the
tetrameric state (7). The crystal structures of avidin and streptavidin
have revealed that the major reason for this common property of the two
biotin-binding proteins is the cross-subunit insertion of a conserved
tryptophan residue (Trp-110 in avidin and Trp-120 in streptavidin) from
one monomer into the binding site of its neighbor. Mutation of this tryptophan in either protein has resulted in both a reduction of
binding affinity toward biotin and a reduction in the stability of the
tetramer (8-10).
The quaternary structure of avidin and streptavidin has long been
viewed as a dimer of dimers (11) and the transition from tetramer to
dimer to monomer has always been intriguing. To study the tetrameric
architecture further, we have previously probed the interface
interactions in avidin by site-directed mutagenesis. In one study, we
mutated the critical 1 In the present communication, we describe a monomeric avidin mutant
(monoavidin), in which Trp-110 was converted to lysine and Asn-54 was
mutated to alanine. The resultant mutant binds biotin specifically in a
reversible manner and remains in the monomeric state even upon binding
to biotin. The resultant monomeric avidin may have applicative
advantages over previously described native and mutated forms of the
tetrameric and dimeric avidins and streptavidins for affinity-based
separations of biotinylated biomolecules.
Construction of Recombinant Baculoviruses--
A modified
(Invitrogen) donor BacToBacTM Baculovirus Expression System
plasmid containing the W110K mutation (8) of avidin cDNA was
further mutagenized by adding an N54A substitution by the mega-primer
method (13). Subcloning of the PCR-amplified insert was performed as
earlier reported by Laitinen et al. (12). A recombinant
baculovirus capable of producing the N54A+W110K double mutant in avidin
was constructed according to the instructions of the BacToBacTM
Baculovirus expression system kit.
Production and Purification of Mutant Avidin--
Mutant avidin
was produced in Baculovirus-infected insect cells as described
previously (14). Purification on a biotin-agarose column was performed
from cell extracts grown in biotin-free medium according to Laitinen
et al. (8).
Biotin-binding Assays--
Reversibility of biotin binding was
determined by competitive binding to biotinylated biosensor surfaces,
and the determination of binding constants for the mutant avidin to
biotin were performed using an IAsys optical biosensor as reported
earlier (8). Fluorescence spectroscopy measurements were performed
using a PerkinElmer LS55 luminescence spectrometer. The excitation
wavelength of 280 nm was used, and the emission spectra was obtained
from 320 to 420 nm using 2.5 nm excitation and 17 nm emission
slitwidths. An average of four scans with scan at speed 240 nm/min was
used to calculate the final result. The protein concentration in this
analysis was 20 nM, and the biotin was added in small
volumes to final concentrations from 0.02 nM to 13 mM. The protein sample was thoroughly mixed in a cuvette
with a micropipette and incubated for 5 min covered from UV light
before scanning. The measurements were performed at 23 °C in
phosphate-buffered saline containing 1 M NaCl. The measured spectra were integrated, and the intensities were plotted against biotin concentration. Binding curve (quenching (%) = a[Btn]/(Kd + [Btn]) + c),
where a and c are scaling factors, was fitted to this data using the least square method.
Structure Analyses--
The molecular mass of the mutant was
calculated from the known amino acid composition using the GCG package
program Peptidesort (Genetic Computer Group, Madison, WI). SDS-PAGE,
immunoblot analyses, and assays for protease sensitivity were performed
according to Laitinen et al. (8). For stability analysis,
protein samples were combined with sample buffer and incubated at
selected temperatures for 20 min before being subjected to SDS-PAGE
(15). The quaternary status of avidin and the mutant was determined by
fast-protein liquid chromatography, performed on a Superdex 200 HR
10/30 (Amersham Biosciences) using a Shimadzu SCL-10A VP system
controller with RF-10A xl fluorescence detector and SPD-M 10A VP diode
array detector. Samples (sodium phosphate buffer, 0.65 M
NaCl, pH 7.2) were applied, and chromatography was carried out at a
flow rate of 0.5 ml/min.
ELISA1
Analyses--
The immunological properties of the mutant were compared
with avidin by an indirect ELISA analysis as previously described in
Laitinen et al. (16). ELISAs were performed using two
monoclonal avidin antibodies (produced at the Institute of Medical
Technology, University of Tampere, Tampere, Finland) and a polyclonal
rabbit avidin antibody (produced at the Laboratory Animal Center,
University of Oulu, Oulu, Finland) as primary antibodies.
Construction, Production, and Purification of the Avidin
Mutant--
The W110K+N54A avidin mutant was produced in biotin-free
insect cell medium as described previously (14). The mutant was purified efficiently on biotin agarose and eluted with mild acid treatment (8) to yield virtually pure mutant protein (not shown). The
location of residues W110K and N54A in the avidin tetramer is shown in
Fig. 1.
Biotin-binding Analyses--
Reversibility of biotin binding was
studied using an IAsys apparatus. The binding of the vitamin by the
W110K+N54A mutant was found to be completely reversible (Fig.
2A). The dissociation constant
of mutant for the immobilized biotin was determined at ~1-3 × 10 Structural Analyses--
The tendency of the W110K+N54A double
mutant to form higher order quaternary structures (dimers, tetramers,
oligomers) was studied by SDS-PAGE based assay (15). The mutant
migrated as a monomer even at room temperature, both in the absence and
presence of biotin (not shown). The quaternary structure status of the mutant was also determined in solution by fast-protein liquid chromatography assay. The calculated value for the monomeric peptide sequence is 14.2 kDa (~15.7 kDa including the oligosaccharide side
chain). This value fits well with the estimated molecular weights both
in the absence or presence of biotin (15.2 and 17.5 kDa, respectively),
indicating a monomer in solution in both cases. Biotin-free and
biotin-bound forms of the native protein were tetrameric (Fig.
3). Henceforth, the W110K+N54A mutant was
termed monoavidin.
The resistance of monoavidin to proteolytic cleavage was studied by
proteinase K treatment and compared with the wild-type avidin. In the
absence of biotin, monoavidin was degraded within minutes, whereas the
presence of the ligand had a short-lived protective effect on the
mutant against the proteolytic action of proteinase K. Complete
proteolysis took place within an hour, whereupon the protein was
digested into small peptides. Native avidin was clearly more stable,
both in the absence and especially in the presence of biotin (Fig.
4). As reported previously (18), the
native protein was cleaved into two long peptides.
Immunochemical Analyses--
Monoavidin was compared
immunologically to native avidin by ELISA using a polyclonal and two
monoclonal anti-avidin preparations. The polyclonal rabbit sera
recognized monoavidin clearly weaker than native avidin and the
monoclonal antibodies failed to recognize the mutant (Table
II).
The linkage of the tetrameric architecture to the strong affinity
of the avidin/streptavidin-biotin complex was first evident from
biochemical studies and then confirmed by the determination of the
crystal structures (3-6). In view of these findings, we and others
have employed site-directed mutagenesis of avidin and/or streptavidin
(8, 9, 12, 19-23), both to better understand this intimate
structure-function relationship and to design mutated forms of the
proteins that would exhibit interesting physicochemical or binding
properties. In this context, it has been one of our expressed goals to
prepare an active, reversible biotin-binding monomer of one or both
proteins. Indeed, in recent work (8), we succeeded in producing a
dimeric form of avidin and streptavidin that bound biotin reversibly.
In another work, we developed monomeric forms of avidin that
fortuitously assembled into tetramers upon binding biotin (12). The
question outstanding was whether a combination of the two types of
mutant would lead to a stable, reversible biotin-binding monomer.
The dimeric mutants of avidin and streptavidin were produced by
rational design (8), which involved a radical point mutation of a
single biotin-binding residue (i.e. Trp-110 in avidin or the
conserved Trp-120 in streptavidin) to a lysine in both cases. The
rationale for this particular "irrational" mutation was inspired by
comparing the sequences of avidin and streptavidin to those of the sea
urchin fibropellins (24). One of the domains of the latter proteins is
remarkably similar to avidin and streptavidin, including most, but not
all, of the biotin-binding residues. In this case, the designated
binding site tryptophan in the avidins is replaced by a lysine in the
fibropellins, hence our decision to effect the Trp The monomeric avidin mutants were also produced by rational design upon
categorizing the molecular forces involved in maintaining the other two
interfaces of the avidin
tetramer.2 Using this
approach, all three residues involved in the 1 In the present work, combined single point mutations of two
critical 1 Monomeric avidin columns have been described in the past for isolation
of biotinylated materials. The immobilized monomers were produced by
treating native egg-white avidin columns with strong chemical
denaturants (7, 25, 26). However, the avidin monomers tend to form
tetramers spontaneously, despite the fact that the immobilized monomers
are covalently bound to the resin (7). Once formed, the tetramers again
bind biotin irreversibly. To preserve the reversibility of such
columns, they are commonly stored in the presence of denaturant, but
the long term utility of the column is thus compromised. The stable
monoavidin described in the present communication provides a more
effective and advantageous component for such columns. In addition,
strategies for producing avidin-based fusion proteins would benefit
from the use of monomeric avidin as a fusion partner, compared with
using avidin tetramers. If a fusion partner (like avidin or
streptavidin) has an intrinsic propensity toward oligomerization, the
mature fusion protein would likely form higher order aggregates with
undesirable physicochemical properties that would interfere with their
intended function. It is not surprising that, to date, the most
successful (strept)avidin-containing fusion proteins have included
relatively small proteins as fusion partners, such as hevein fused to
avidin (27, 28) or antibody Fab fragments fused to streptavidin
(29).
The reversible biotin-binding characteristics of monoavidin are also
advantageous for isolation of monoavidin-containing fusion proteins. To
date, the most popular affinity purification protocols for avidin have
utilized 2-iminobiotin as an affinity matrix. The relevant protocols
include a binding step under conditions of high pH (>10), whereas the
elution step is accomplished by lowering the pH to 4 or even lower
values (30). Many proteins, however, are labile at high or low pH
values, and purification procedures that include such conditions may
lead to inactive fusion products. Consequently, the reversible biotin-
binding properties of monoavidin are amenable to protocols that require
physiological binding conditions and mild elution with biotin.
Pérez-Luna et al. (31) have recently
studied the wild-type and two tetrameric streptavidin mutants by
surface plasmon resonance optical biosensor. They observed that
dissociation rates for these streptavidins did not follow the simple
first-order kinetics in their experimentation. The reasons for this
behavior were proposed to be nonspecific interactions of the bound
proteins for non-evenly organized surface, cooperativity of binding to
a dense biotin surface caused by multiple binding sites in tetrameric
proteins, and/or interactions between adsorbed proteins and tethering
effects of the ligand. In contrast, first-order association and
dissociation kinetics were obtained for monoavidin using IAsys in the
present study. Together with the complete reversibility of biotin
binding, the data suggest that the latter phenomena did not
significantly affect the observed interactions. Indeed, the lack of
cooperativity would not be surprising due to the monovalency of the
studied molecule. The character of the surface used for these
experiments can also have a strong impact on the kinetic parameters.
Edwards et al. (32) have compared the binding kinetics of
human serum albumin to an antibody immobilized either to a dextran
matrix or to an aminosilane surface. They found that when the dextran matrix was used the binding phenomena followed second-order kinetics due to steric hindrance between the binding matrix and the analyte, whereas kinetics obtained by using the aminosilane surface followed first-order kinetics. In this context, the binding analyses were performed on a planar aminosilane surface in the present study.
Kohanski and Lane (25) have previously described association and
dissociation values for column-immobilized monomeric avidins (generated
from egg white avidin by incubation with chaotropic agents) using
radiolabeled biotin. The resultant on and off rates as well as the
derived dissociation constant were remarkably similar to those obtained
in the present work for monoavidin. In addition comparable affinity
constant was determined for monoavidin under soluble conditions using
fluorescence spectrophotometry. Finally, similar association and
dissociation rates have recently been reported for monomeric
streptavidin mutants (23).
In conclusion, the current study demonstrates how the use of the
rational mutagenesis can be used to disrupt the extraordinary stability
of the avidin tetramer in producing functional monomers by changing
only two structurally important amino acids. The resultant monoavidin
is appropriate as a reversible biotin-binding component of affinity
columns and fusion proteins for use in developing novel applications of
the (strept) avidin-biotin system.
2
interface was mutated to lysine and asparagine 54 in the 1
4
interface was converted to alanine. The affinity for biotin binding of
the mutant decreased from Kd ~10
15
M of the wild-type tetramer to Kd
~10
7 M, which was studied by an optical
biosensor IAsys and by a fluorescence spectroscopical method in
solution. The binding was completely reversible. Conversion of the
tetramer to a monomer results in increased sensitivity to proteinase K
digestion. The antigenic properties of the mutated protein were
changed, such that monoavidin was only partially recognized by a
polyclonal antibody whereas two different monoclonal antibodies
entirely failed to recognize the avidin monomer. This new monomeric
avidin, which binds biotin reversibly, may be useful for applications
both in vitro and in vivo. It may also shed
light on the effect of intersubunit interactions on the binding of ligands.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
15 M), and, under
physiological conditions, the binding can essentially be considered
irreversible (1). This tight interaction has been utilized during the
past three decades for various applications in the life sciences to
purify, probe, and target various materials both in vitro
and in vivo (2).
2 interface residue, Trp-110 of avidin and
the conserved Trp-120 of streptavidin, to lysine, which led to the
production of dimeric avidin and streptavidin mutants that bound biotin
reversibly (8). In another study, we mutated the critical 1
4
interface residue, asparagine 54, to alanine, which also led to
destabilization of tetramer (12). One of the surprising outcomes of
that study was the production of avidin mutants that, in the absence of
biotin, appeared as monomers in solution, whereas upon binding the
vitamin the monomers formed stable tetramers. It was thus of interest
to investigate whether the combination of these two types of mutation
might lead to a stable avidin monomer.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ribbon diagram of the avidin tetramer.
The location of the mutated residues between subunit interfaces
1 2 and 1
4 (and 3
4 and 2
3) are shown as
sticks. The subunits are color-coded as follows: subunit 1, blue; subunit 2, green; subunit 3, dark
red; subunit 4, gray. Tryptophans 110 of
each subunit are indicated in yellow (Trp-110 of subunit 2 is located at the left side of the figure). On its
right is Trp-110 of subunit 4, and at the upper
part of the figure Trp-110 residues of subunits 1 and 3 are
situated. Asparagines 54 are labeled in red. The Asn-54
residues of subunits 1 and 4 are located on the left side of
the figure and the corresponding asparagines of subunits 2 and 3 on the
right. The diagram was created using InsightII (Molecular
simulatios Inc., San Diego, CA).
7 M in IAsys analyses (Fig. 2B,
Table I). The binding and dissociation curves were consistent with simple first-order kinetics. The resultant kinetics data were in accord with self-consistency tests described by
Schuck and Minton (17) whereby dissociation constants calculated from
equilibrium response data were similar to those obtained directly from
the binding curves (Table I). The binding was also examined under
soluble conditions by fluorescence spectroscopy monitoring the
quenching of the intrinsic fluorescence due to biotin binding to
avidin. Total decrease of about 45% in protein intrinsic fluorescence
intensity was observed when monoavidin sample was titrated with biotin
(Fig. 2C). The fitted binding curve
(r2 = 0.98) gave dissociation constant of
~7.6 × 10
8 M.
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Fig. 2.
Biotin-binding characteristics of the
monoavidin. A, reversibility of biotin binding by
monoavidin (concentration 1.5 × 10 6 M)
and apparent irreversibility by avidin (concentration 3.2 × 10
7 M). B, interaction analysis of
monoavidin with the biotin cuvette. Various concentrations of
monoavidin were added to the biotin-coated cuvette, and the interaction
was measured by IAsys. The equilibrium response
(Req) is plotted versus protein
concentration. The Kd of the protein is equal to the
concentration at Rmax/2. C, quenching
of the intrinsic fluorescence of monoavidin plotted as a function of
biotin concentration. Binding curve gave half-maximal quenching at
biotin concentration 7.6 × 10
8 M
(r2 = 0.98). The triangle indicates
the value for quenching at 13 mM concentration of
biotin.
IAsys biosensor data for binding of biotin to monoavidin
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Fig. 3.
Gel filtration profiles of the W110K+N54A
mutant, in the presence or absence of free biotin. Human
immunoglobulin G (IgG, 158 kDa), avidin (63 kDa), ovalbumin (44 kDa),
and B12 vitamin (B-12, 1.35 kDa) were used as molecular
mass markers to calibrate the column. The logarithm of molecular
mass was plotted versus
Ve/Vo
(Ve = elution time, Vo = void volume). Molecular mass for monoavidin (D27) was
calculated according to the slope of the curve (15.2 kDA in the absence
and 17.5 kDa in the presence of biotin), indicating a monomer both in
the presence and absence of the ligand. On this basis, the mutant was
referred to as monoavidin.
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Fig. 4.
Proteinase K assay of monoavidin and native
avidin. A, monoavidin treated with proteinase K in the
presence of biotin. Samples were extracted at the indicated time
intervals. Untreated monoavidin (MA) was included as a
control. B, monoavidin in the absence of biotin. The same
analysis was performed for native avidin in the presence (C)
and in the absence (D) of excess biotin. Proteinase K
treatment of the native avidin resulted in only two defined peptides,
representing a single cleavage site. In contrast, monoavidin was
completely digested by proteinase K into small peptides.
Molecular mass markers (L) were 21.5 and 14.4 kDa
(Bio-Rad).
ELISA analysis of avidin and monoavidin using anti-avidin polyclonal
(rabbit) and two different monoclonal antibody preparations
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Lys mutations
of avidin and streptavidin. Since the same tryptophan also plays a
crucial role in the 1
2 monomer-monomer interface, this radical
type of point mutation also generates dimeric forms of the two proteins.
3 interface were
converted to alanines (12). The resultant 1
3 mutant still formed
a tetramer, but its stability properties were compromised. In contrast
to the relatively weak 1
3 intermonomer interaction, the
1
4 interface exhibits an extensive hydrogen bonding network.
Nevertheless, one of the residues involved in this interface (Asn-54)
contributes numerous hydrogen bonding interactions with several
residues of the neighboring monomer. The combination of the single
Asn-54
Ala mutation with those of the 1
3 interface was
sufficient to disrupt the stable avidin tetramer into monomer (12).
Such monomers, however, re-assembled into stable tetramers upon biotin binding.
2 and 1
4 interface residues generated a stable avidin monomer. These data infer that once these two interfaces are
disrupted, the relatively weak 1
3 interface is insufficient to
maintain a viable avidin dimer. Disruption of the 1
2 and 1
4 intermonomer interactions apparently causes changes in the tertiary structure of the avidin monomer, since the monomer is more
labile than the tetramer to protease K treatment. In the conditions
where proteinase K cleaves wild-type avidin at only one position (18),
monoavidin was completely digested into small peptides. Moreover,
immuochemical analysis using polyclonal anti-avidin antibodies revealed
a reduction of cross-reactivity with monoavidin, and monoclonal
antibodies (elicited against the native avidin tetramer) failed to
react with the avidin monomer.
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ACKNOWLEDGEMENTS |
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We thank Irene Helkala and Anne Ettala, Satu Pekkala, Piia Karisola, Jarno Hörhä, and Tikva Kulik for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Academy of Finland and the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem, Israel).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.
§ These authors contributed equally to this work.
§§ To whom correspondence should be addressed: Dept. of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland. Tel.: 358-14-2602272; Fax: 358-14-2602221; E-mail: kulomaa@csc.fi.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M205844200
2 O. Livnah and E. A. Bayer, unpublished results.
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ABBREVIATIONS |
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The abbreviation used is: ELISA, enzyme-linked immunosorbent assay.
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