(Received for publication, May 20, 1996, and in revised form, December 2, 1996)
From the Departamento de Química
Biológica, Centro de Quimica Biologica de Cordoba (CIQUIBIC),
Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba, 5000 Córdoba, Argentina, the
§ Departamento de Bioquímica, Universidad del
País Vasco, E-48080 Bilbao, Spain, and the ¶ Departments
of Biomedical Engineering and Pharmacology, Boston University,
Boston, Massachusetts 02215
The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 °C in unliganded protein to 112 °C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka ~1015 reproduce the behavior observed, but the estimated values of Tm are 15-20 °C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions.
The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.
The biological function of many proteins is triggered and modulated by the binding of ligands. For this reason, an understanding of the mechanism of protein-ligand interactions is essential for a detailed knowledge of protein function at the molecular level. Ligand binding, in most cases, involves the formation of noncovalent bonds at specific interacting surfaces between the protein and the ligand. The binding of a ligand can be accompanied by conformational changes at the protein site that sometimes are propagated throughout the entire protein. It is desirable to have a way to monitor these structural changes to understand any new properties acquired by the complex. The high affinity of the biotin-streptavidin binding not only offers useful bioanalytical advantages (1), but it also makes this system an attractive model for studying protein-ligand interactions (2-5). The biotin·STV1 association constant of about 1015 is the highest known in biochemistry.
In the present work we explore protein thermostability by heating STV in the absence of ligand or under conditions of partial or full ligand saturation. A dramatic increase in the Tm of protein denaturation, from 75 °C in absence of biotin to 112 °C at full ligand saturation, was revealed using differential scanning calorimetry (DSC). An analysis of the cooperativity of the denaturation was made on the basis of a reversible non-two-state model of protein unfolding to gain understanding of the system that unfolds differently if biotin is present.
Conformational changes were characterized by FT-IR and fluorescence spectroscopy. The large changes in the thermostability of STV·biotin complex as compared with the free protein can be explained by two simultaneous processes. One is related to the binding energy; the other is related to structural changes induced by the binding of biotin to STV.
All reagents used were of analytical grade. STV was from Life Technologies, Inc. Biotin was from Sigma.
CalorimetryProtein denaturation was monitored with a
MicroCal MC-2D scanning calorimeter with digital data acquisition and
analyzed by means of the software provided by the manufacturer. The
calorimetric data were analyzed assuming a reversible non-two-state
model of denaturation (6). A possible justification for the
applicability of reversible thermodynamics to apparently irreversible
processes have been discussed previously (6-9) for the case where
reversible unfolding is followed by a rate-limited irreversible step.
This model was used after checking that no endotherms were found after complete denaturation and that similar thermograms, and results were
obtained either: (i) at lower scan rates (26.8 degrees·h1 and 9.2 degrees·h
1) than the
one usually employed (55 degrees·h
1) or (ii) at
different protein concentrations (from 0.020 to 0.080 mM)
or (iii) by fitting the whole curve using only the first 50% of the
experimental data of the thermogram. The calorimetric enthalpy (
H) is determined only by the area under the transition
peak of the thermogram, while the van't Hoff enthalpy
(
HVH) depends on the shape of the transition
peak. The sharper the transition, the larger is the
HVH,
independently of
H. The
H refers to heat change/mol,
while
HVH is the heat change per unfolding
unit (7). Thus, the ratio
H/
HVH can be
thought of as the number of cooperative units/mol of STV monomer. To
interpret the cooperative unit value for oligomeric proteins as a
quantitative evidence for coupling of the denaturation process between
the monomers, the calculations should be done on the basis of protein
monomer concentration.
Biotin and STV were dissolved in 100 mM phosphate buffer,
pH 7.3. The monomer protein concentration was 0.066 mM in
all experiments and was determined at 280 nm using an extinction
coefficient 0.1% = 3.4 (10). The molar ratios of
biotin:STV (mole of biotin/mol of STV monomer) used were: 0, 0.25, 0.5, 0.75, and 2. Samples were exhaustively degassed before injection into
the calorimeter cell (volume: 1.2388 ml) to prevent air bubble
formation at the high temperatures reached (near 120 °C). A nitrogen
pressure of 2 atmospheres was applied to both cells. The reference cell
was filled with a matching buffer identical to that used with the sample. Samples containing biotin:STV at different molar ratios were
mixed and incubated at room temperature for at least 10 min, in a final
volume of 1.5 ml, before being degassed and injected into the
calorimeter cell. Some representative DSC experiments were performed
with the homogeneous pure core STV provided by Apcel Ltd., Berkshire,
United Kingdom, with identical results to those found for the
heterogeneous STV from Life Technologies, Inc.
The thermal denaturation of a protein that binds a ligand at a single binding site is predicted to behave at subsaturating levels of ligand, in an unimodal or bimodal manner, depending on the value of the association constant (11). This behavior arises solely from the coupling of the binding to the denaturing process. The model predicts the thermogram of a monomeric protein that binds to a ligand with no changes in the protein conformation or in the association constant as a function of temperature. Under these conditions the transition temperature Tm (temperature at half-completion), in the presence of ligand, changes according to Ref. 11,
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(Eq. 1) |
Infrared spectra were recorded in a Nicolet Magna 550 spectrometer equipped with an MCT detector. Samples for IR were made up by dissolving the lyophilized protein in 10 mM Hepes, pH or pD (H2O- or D2O-based buffers) 7.4 and placed in a thermostatted cell equipped with CaF2 windows. A path length of 50 µm was used for D2O measurements and 6 µm for H2O solutions. A total of 1000 scans were accumulated for each spectrum, using a shuttle device. Thermal stability studies were carried out by heating the samples in steps of about 3 °C, in the temperature interval 30-85 °C. After every heating step the sample was left to stabilize for 5 min and the spectrum recorded. Solvent subtraction, deconvolution, band position determination, and curve fitting of the amide I band were performed as reported previously (12). Briefly, band component positions are obtained from deconvolution and derivatization; initial heights are set to 90% in the center and the edges and 70% of the original heights in the other components. Bandwidth estimates are obtained from derivative spectra and the Gaussian fraction is set to 90%. The decomposition method assumes that the absorption coefficient is the same for all the components (12). The results obtained after iterations may not be unique, so restrictions must be applied (13). The most important points are that the band position cannot differ from the initial guesses by more than the distance between data points and that the width of the bands should be less than half of the amide I bandwidth. The combined use of several spectra, recorded at different temperatures below denaturation, makes the solution practically unique (12).
Fluorescence SpectroscopyCorrected steady-state emission spectra and fluorescence phase lifetime measurements were performed with a phase modulation fluorimeter, SLM-Aminco model 4800C, equipped with a xenon arc lamp and thermostatted cell holder. The excitation wavelength was 295 nm in all fluorescence experiments, using a band-pass filter of 8 nm. Fluorescence intensity was corrected for inner filter effects (14) and dilution. All fluorescence experiments were carried out in triplicate, and the temperature was 25 °C (controlled by a circulating bath). Data were analyzed using the SLM 4800 C software package.
The fluorescence phase shifts for phase lifetime determinations were
measured at 30 MHz. Fluorescence was observed using a 310-nm cut-off
filter to eliminate scattered radiation from the light source. In the
reference cell a solution of p-terphenyl in ethanol ( = 1.05 ns) was used to correct for possible color effects (14). The
standard deviations of the phase lifetime were below 2%. Quenching
experiments performed with acrylamide were done at a final
concentration of 4.2 µM STV in 100 mM
phosphate buffer, pH 7.3. Acrylamide was added by successive pipetting
of small aliquots from a stock solution (4.8 M) into the
cuvette.
SDS-polyacrylamide gel electrophoresis analyses were performed in a Bio-Rad Miniprotean II using a 13% acrylamide gel according to the method described by Schägger and von Jagow (15). The cathodic buffer was 0.1 M Tris, 0.1 M Tricine, 0.1% (w/v) SDS, pH 8.25; the anodic buffer was 0.2 M Tris-HCl, pH 8.45; the sample buffer was 0.125 M Tris-HCl pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.01% (v/v) bromphenol blue. Each sample, containing 35 µg of STV or biotin:STV at different molar ratios was heated for 5 min at 95 °C, cooled down, then mixed with the sample buffer and incubated at room temperature for 10 min before application to the gel. For protein staining, the gel was previously washed in 20% methanol, 10% acetic acid for at least 30 min, then incubated for 30 min with gentle shaking in a mechanical shaker with 0.15% Coomassie Brilliant Blue R-250 (Sigma), dissolved in 50% methanol, 10% acetic acid. For destaining, the gel was left overnight in the same solution but without Coomassie Brilliant Blue R-250.
The effect of ligand on the thermally induced
denaturation of STV was studied at different biotin:STV molar ratios.
The results appears to be a biphasic or a monophasic process depending
on the biotin concentration (the data in Fig. 1 and
Table I are based on STV monomer concentration). A
monophasic pattern is obtained only either in the absence of ligand or
under fully saturating conditions. Biphasic behavior is observed at
subsaturating biotin:STV 0.25 or 0.5 molar ratios; this is on average
one or two molecules of biotin per tetramer, respectively (Fig. 1). The
thermogram of free STV shows a single, symmetric peak with a
Tm centered at 75.5 °C (Fig. 1 and Table I).
Under biotin-saturating conditions, the Tm of
STV is considerably increased and centered at 112.2 °C (Fig. 1).
Denaturation of ligand-stabilized STV is marked by a higher excess heat
capacity peak, reaching a value close to three times the one obtained
for STV, and by an increase in the calorimetric enthalpy (Fig. 1 and
Table I). Concomitantly an increase in the cooperativity of the
denaturation process, given by a cooperative unit lower than 1, is also
observed (Table I). Each single thermogram peak of unliganded or fully
liganded STV can be deconvoluted into two transition components (not
shown). This can be attributed to the two major different species of
STV present in the samples studied (16). In Table I we show
calorimetric data for the overall denaturation process, which is an
average over the properties of these two species.
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Subsaturating levels of a high affinity ligand have effects on thermally induced protein denaturation that arise from the coupling of protein denaturation and ligand binding equilibria. In the model developed by Shrake and Ross (11), at subsaturating ligand levels a biphasic thermogram can be obtained, since the ratio of the free ligand concentration to the native protein is continuously changing as the denaturation proceeds. The midpoint denaturation temperature increases proportionally with the free ligand concentration and the value of the association constant Ka (see Equation 1 under "Experimental Procedures"). The model assumes that the Ka and the calorimetric enthalpy are independent of the temperature (11). The inset in Fig. 1 shows the simulated thermograms for a hypothetical STV-like protein with a calorimetric denaturation enthalpy of 150 Kcal/mol, a Tm of 75 °C, and a Ka = 1015 for an initial fractional saturation of 0.05, 0.25, 0.50, 0.75, 0.99, and 0.999. At intermediate levels of ligand saturation, the bimodality is evident and, at full saturation, the biphasic behavior disappears, and the Tm is increased to near 96 °C.
The thermostability induced by high affinity ligand binding, taking into account only the coupling of protein denaturation process to the binding equilibrium, explains only partially the experimental thermograms observed for STV in presence of biotin (compare the Fig. 1 with its inset). The experimental midpoint denaturation temperature values for the STV·biotin complex are higher than the theoretical calorimetric parameters expected for such an interaction. Also, at 0.75 fractional saturation the theoretical thermogram predicts that a considerable fraction of the protein will denature at a lower temperature (inset in Fig. 1). However, the experimental thermogram at a similar biotin:STV molar ratio shows just a single peak at 110 °C.
The observed changes in the cooperativity of thermally-induced unfolding provides calorimetric evidence that there is an increment in the strength of the intermolecular association of the subunits induced by biotin binding. The ratio of the calorimetric enthalpy (the area under the transition peak) to the van't Hoff enthalpy (calculated from the shape of the transition peak) indicates whether protein denaturation represents a simple or a complex cooperative system (7). Since all the calorimetric data shown in Table I were calculated on a basis of STV monomer concentration, the ratios are referred to the numbers of unfolding units of the system. In the absence of biotin, STV denatures with a cooperative unit value closer to 2 than 1 (Table I), indicating probably more than one domain in the STV monomer that would unfold independently. At 0.50 biotin:STV molar ratio the cooperative unit is about 0.5 in the higher transition component, indicating that the dimer undergoes a single coupled transition. As the molar ratio increases the intermolecular interaction among the subunits is larger, and a cooperative unit value higher than 1 is no longer observed. Even when there is only one molecule of biotin per tetramer (0.25 biotin:STV molar ratio), the unliganded subunits are affected by the biotin bound subunit (see Table I and Fig. 1).
The effect of biotin on the stability of different oligomeric forms of
STV after heating was investigated. Samples of STV at different biotin
molar ratios were subjected to heating (95 °C, 5 min) and analyzed
by SDS-polyacrylamide gel electrophoresis (Fig. 2).
Non-heated STV remains as a ~60-kDa tetramer, and in the absence of
biotin the treatment produces mainly protein bands of ~14 and ~30
kDa, corresponding to the monomer and dimer forms of the protein,
respectively. A progressive increase in the biotin:STV molar ratios
increases the proportion of the tetramer, and at a 0.75 biotin:STV
molar ratio, most of the protein is in the tetrameric form, in
agreement with the DSC results. The electrophoretic observations support strongly the idea that a structural cooperativity is built up
upon biotin binding to STV. Sano and Cantor (17) reported previously
that the exchange of free and radioactive biotin in biotin-liganded STV
is hard to achieve even in the presence of high concentrations of free
ligand. This could indicate that STV would not release the biotin prior
to denaturation, instead the STV·biotin complex might undergo the
unfolding transition. The idea of an increment in the intermolecular
communication between the STV subunits upon biotin binding was
suggested by previously the electrophoretic behavior of the complex in
presence of 6 M urea (18). This idea is further supported
by site-directed mutagenesis of tryptophan 120, which indicates that
this residue, which contacts bound biotin, is strongly involved in
intersubunit contacts (17).
Secondary Structure of Streptavidin by FT-IR Spectroscopy
The
secondary structure of STV has been studied by means of infrared
spectroscopy. The amide I band envelope from samples in H2O
and D2O buffers was decomposed into its constituents. Fig. 3 shows the spectrum of STV in the absence
(A) or presence (B) of biotin in a
D2O buffer. Table II presents the band
positions and the relative area of the components shown in Fig. 3.
Seven bands are seen that arise from conformations of the peptide
backbone. These are located at around 1692, 1681, 1671, 1641, 1633, and 1629 cm1. The assignment of the amide I component bands
to conformational structures is not yet straightforward because of the
complexity of the interactions involved (19). Still, some of the bands can be unambiguously assigned (13). In STV, a high content of
-sheet
is expected because the maximum of the amide I band is around 1630 cm
1, which is coherent with the previous x-ray studies
(5, 20). Also, a high frequency band around 1680 cm
1 is
produced by antiparallel
-sheet. The band at 1641 cm
1
is due to unordered structure, and bands between 1660 and 1690 cm
1 arise from
-turns.
-Helix usually gives rise to
a band around 1650 cm
1; however, bands originating from
turns, with dihedral angles comparable with
-helix, have also been
described at this frequency (21, 22). Binding of biotin to STV does not
change the
-sheet structure content; that remains at 40%, but the
two bands at 1641 and 1651 cm
1 in STV alone appear as a
single band at 1647 cm
1 after biotin binding (Table
II).
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The integrity of STV conformation with temperature was also studied by
FT-IR (Fig. 4). In our hands, this methodology permits heating up to 85 °C only. STV in the presence of saturating biotin concentrations does not change its FT-IR profile significantly in the
temperature range tested (Fig. 4A), while free STV undergoes thermal denaturation with a half-midpoint temperature of 72-73 °C (Fig. 5). This agrees with the Tm
value observed by DSC. The denaturation process followed by FT-IR is
revealed by broadening of the amide I region due to the emergence of
bands at 1620 and 1680 cm1, both associated with
denatured conformations (23). The increase in regular structures
associated with a raise in thermal denaturation temperature has also
been observed in concanavalin A, a protein with a high
-sheet
content upon metal binding (24).
Fluorescence Experiments
STV has six tryptophans (16). Four
of these tryptophan residues are clustered in the hydrophobic binding
site in close contact with biotin. The binding pocket is located in a
shared interface contact domain, since one of the tryptophans, residue
120, belongs to the adjacent subunit (5, 17, 20). As expected, biotin binding leads to considerable changes in STV fluorescence. When biotin
binds to STV, a progressive blue shift and a decrease in fluorescence
intensity are observed. Under saturation conditions a blue shift of
about 5 nm (from 330 to 325 nm) together with a 25% decrease of the
fluorescence intensity are observed (not shown) in agreement with the
data reported by Kurzban et al. (25). Control experiments
performed by mixing N-acetyltryptophanamide, used as a model
tryptophan-containing protein, and biotin do not reveal appreciable
changes in N-acetyltryptophanamide fluorescence. These
results indicate that neither dynamic nor static tryptophan quenching
are the direct result of the presence of biotin (data not shown). Thus,
changes observed in max and fluorescence intensity when
biotin binds to STV are likely to arise mainly from conformational changes of the protein and not directly from a quenching effect of the
ligand.
To further explore the influence of biotin binding on the fluorescence
of tryptophan in STV, we performed quenching experiments with
acrylamide in the presence and absence of ligand. We also measured the
phase lifetime of STV and the STV·biotin complex. The major
contribution to the total fluorescence emission intensity, when STV is
excited at 295 nm, is provided by the four tryptophans at the binding
site of each subunit. The accessibility of these amino acids to the
water-soluble, neutral quencher acrylamide was evaluated from
steady-state fluorescence data by using the modified form of the
Stern-Volmer equation: Fo/F = eVapp[Q]
(1 + KSV(app) [Q]); where
F and Fo are, respectively, the
fluorescence intensities in the presence and in the absence of quencher
at concentration [Q]. KSV(app) and
Vapp are the apparent dynamic and static
contributions to the total quenching, respectively. These parameters
were obtained from the best fit of the experimental data. In the case
of a multitryptophan protein, the Stern-Volmer parameters are a crude
estimate of the average exposure of the fluorescent residues in the
protein (26). In unliganded STV, the quenching takes place without a
shift in the max of the fluorescence emission spectrum
(not shown). This suggests that all forms of tryptophan have similar
exposure to the quencher. In contrast, the conformational changes in
STV that accompanies biotin binding make these tryptophans practically
inaccessible to acrylamide. Consequently, higher values of
KSV(app) and Vapp were found for STV
alone compared than for biotin:STV at saturating conditions. The
results are summarized in Table III.
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We also measured the fluorescence phase lifetime of STV and STV:biotin in the absence and in the presence of 0.15 M acrylamide. In the absence of acrylamide, the phase lifetime of STV is reduced by almost 50% when biotin binds, reflecting the conformational change that occurs upon ligand binding. In the presence of acrylamide, a reduction of about 30% in phase lifetime of STV was found, whereas for the STV·biotin complex the phase lifetime is practically unchanged (Table III). These results are in agreement with the values obtained for the Stern-Volmer constants, suggesting that the solvent exposure of the tryptophans is strongly reduced in the presence of biotin.
Previous x-ray studies of a truncated form of STV have shown a
-barrel involving around 70% of the residues of this form (5, 20).
This barrel involves the
-strands and the connecting turns, besides
other turns and two flexible loops have been described. Our FT-IR
studies on the unliganded protein also show antiparallel
-sheet as
the major structure. In the presence of biotin, no changes in the
-sheet conformation are produced, confirming the previous x-ray data
(20). The most striking feature of the infrared spectrum as compared
with the x-ray structure is the presence of a band at 1651 cm
1 that could be related in principle to
-helix
structure. Note that our samples consist primarily of full-length STV.
Additionally, as demonstrated by Krimm and Bandekar (21, 22), bands
around 1653 cm
1 can also arise from
-turns that have
dihedral angles similar to a turn of the helix. In other
-sheet
proteins, such as concanavalin A, a band around 1651 cm
1
has also been described, even if no structured
-helices have been
found in the x-ray structure (24). Thus, this band can arise either
from helical segments that would be in the full-length species of our
sample (about 60%), or more probably they correspond to turns with a
helix-like geometry. The presence of biotin produces a significant
change in the infrared spectrum. This is appreciable even in the crude
spectrum (Fig. 3), it corresponds to an increase in the intensity at
1647 cm
1 at the expense of the bands at 1651 and 1641 cm
1 (Table II). With the help of the H2O
spectrum (data not shown), where this shift is not observed, it can be
concluded that the component at 1641 cm
1, associated with
unordered structure, has decreased. Thus biotin binding leads to a more
structured protein. In fact, the x-ray results showed that two loops
that cannot be defined in the unliganded STV are defined in the
presence of biotin (20). Also, from the infrared results it can be
suggested that the residues not included in the truncated form are
disordered, and after biotin binding they become structured. This
assumption is sustained by the large increase in denaturation
temperature observed after biotin binding. Fluorescence quenching
experiments are also in keeping with the higher structural order
observed upon biotin binding, since a drastic decrease of the aqueous
quencher accessibility to the tryptophan residues is found for liganded
as compared with free STV (Table III). A difference of around 40 °C
in the denaturation temperature should imply a protein reorganization
similar to that observed in concanavalin A after demetallization (24),
where no changes in the
-sheet structure are produced, but
variations in the protein flexible segments are seen.
It is well known that ligand binding gives an apparent increment in the
thermal stability of a protein due only to the coupling of binding with
unfolding (11). So, it is clear that not only thermodynamic factors are
influencing the experimental STV stability upon thermally induced
unfolding (Fig. 1). The increase in structural order seen by previous
authors in x-ray crystals (5, 20) and also confirmed by FT-IR in a more
dynamic aqueous solution condition is perhaps the main reason for the
increment from 75 to 112 °C in the thermal stability of the
STV·biotin complex. The calorimetric data also indicate an increase
in the cooperativity of the unfolding process. The sharpening and the
3-fold increment in the excess heat capacity changes the calorimetric
cooperative unit from 1.7 in unliganded STV to 0.5 at full ligand
saturation (Table I). A cooperative unit in calorimetry is a
thermodynamic concept that may be related to a structural domain and
represents a region of a protein with a thermodynamically stable
structure (7). The structural changes in STV upon biotin binding as
observed by several techniques include Trp-120 from one subunit in the dimer moving across the surface binding site of the second interacting subunit (20). At full ligand saturation, the increased packing due to
the interdigitation of the region that contains Trp-120 apparently
leads the dimers to undergo a coupled single transition upon heating.
In unliganded STV the interaction of the Trp-120 domain in the binding
cleft might not be strong enough, and this would imply the possibility
of observing some intermediate state in the transition ascribable to
one of the cooperative units (or units of unfolding). In a
non-cooperative model of the biotin-STV interaction (see below) and at
subsaturating conditions, a considerable portion of the unliganded STV
monomer is in close contact with the neighboring liganded monomer. So,
it is probable that, at subsaturating conditions, part of free STV
monomer melts at a lower temperature, and a second domain (including
the loop containing Trp-120) melts cooperatively with the liganded
neighboring STV. This may explain the lower calorimetric enthalpy of
the low transition component found at subsaturating conditions (Table
I). At full saturation, the molecular interdigitation between the STV
monomers is at maximum, and the system undergoes denaturation with
maximum calorimetric enthalpy (Table I). Replacement of Trp-120 by Phe reduces substantially the affinity constant to approximately
108 M1, indicating that the
contact made by Trp-120 to biotin has a considerable contribution to
the tightness of biotin binding to STV (17). So, it can be expected
that the Phe-120 mutant would behave with a different cooperativity of
unfolding upon heating than the native protein.
The association constant, Ka for the binding of biotin to STV is among the highest for any known non-covalent structure (2, 17). Even with this huge Ka it has been suggested previously that biotin induces binding cooperativity (17, 18, 27). Usually the term cooperativity in binding is used to explain changes in the affinity (with Ka in micromolar-nanomolar range) of an oligomeric protein in the presence of increasing amounts of bound ligand. The typical textbook example is oxygen binding to tetrameric hemoglobin, in which one molecule of oxygen increases the affinity of the second and so forth. It is very difficult to imagine (and experimentally difficult to evaluate) whether the cooperativity that appears to occur with biotin binding to STV works in a similar way than that observed for the interaction of oxygen with hemoglobin, even when the quaternary structural changes seen in STV are consistent with the possibility of cooperativity.
A model in which the conformational communication between dimers in the
tetrameric structure seen in the present work and in previous papers,
induced by the interaction of a first ligand, influences the affinity
of the remaining ligands giving an overall macroscopic
Ka of biotin (~1015) is not in
agreement with the calorimetric data. Instead, biotin appears to induce
structural cooperativity into the subunits without substantial changes
in the binding affinity. According to the conventional picture of
cooperative binding, at subsaturating conditions of 0.25 biotin:STV
monomer, the fully liganded tetrameric and unliganded species should
predominate in the population of the species (option A in
Fig. 6). If this were the case, and assuming that STV
melts with the biotin attached, the calorimetric profile should be
biphasic with a main transition centered at 75 °C (STV tetramer
without biotin) and a transition centered at 112 °C (fully liganded
STV tetramer). However, the actual experiments shown an intermediate
calorimetric profile that depends on the occupancy (biotin:STV molar
ratios, see Fig. 1), so option B, pictured in Fig. 6, is
more consistent with the data. Evidence for non-cooperativity in biotin
binding to STV has been recently reported by Jones and Kurzban (28) by
using equilibrium binding and separation of the present species by
anion exchange chromatography. Binding appears to occur with
cooperative structural changes without affecting the binding affinity
of the overall process. The STV-biotin system may be thought as
an extreme case of Koshland-Nemethy-Filmer sequential model of binding
(29).