doi:10.1016/j.jmb.2005.05.070
J. Mol. Biol. (2005) 351, 672–682
The Folding Mechanism of a Dimeric b-Barrel Domain
Gonzalo de Prat-Gay1*, Alejandro D. Nadra1
Fernando J. Corrales-Izquierdo2, Leonardo G. Alonso1
Diego U. Ferreiro1 and Yu-Keung Mok2
1
Instituto Leloir, CONICET
and Facultad de Ciencias
Exactas y Naturales
Universidad de Buenos Aires
Patricias Argentinas 435
(1405) Buenos Aires, Argentina
2
Department of Chemistry
Cambridge University, Lensfield
Road, Cambridge CB2 1EW
UK
The dimeric b-barrel domain is an unusual topology, shared only by two
viral origin binding proteins, where secondary, tertiary and quaternary
structure are coupled, and where the dimerization interface is composed of
two four-stranded half-b-barrels. The folding of the DNA binding domain
of the E2 transcriptional regulator from human papillomavirus, strain-16,
takes place through a stable and compact monomeric intermediate, with
31% the stability of the folded dimeric domain. Double jump multiple
wavelength experiments allowed the reconstruction of the fluorescence
spectrum of the monomeric intermediate at 100 milliseconds, indicating
that tryptophan residues, otherwise buried in the folded state, are
accessible to the solvent. Burial of surface area as well as differential
behavior to ionic strength and pH with respect to the native ground state,
plus the impossibility of having over 2500 Å2 of surface area of the halfbarrel exposed to the solvent, indicates that the formation of a non-native
compact tertiary structure precedes the assembly of native quaternary
structure. The monomeric intermediate can dimerize, albeit with a weaker
affinity (w1 mM), to yield a non-native dimeric intermediate, which
rearranges to the native dimer through a parallel folding channel, with a
unimolecular rate-limiting step. Folding pathways from either acid or urea
unfolded states are identical, making the folding model robust. Unfolding
takes place through a major phase accounting for apparently all the
secondary structure change, with identical rate constant to that of the
fluorescence unfolding experiment. In contrast to the folding direction, no
unfolding intermediate was found.
q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: folding; E2; DNA-binding; b-barrel; papillomavirus
Introduction
Protein folding is an intricate biological and
physicochemical problem because of all the theorPresent addresses: Y.- K. Mok, Department of Biological
Sciences, National University of Singapore, Singapore
117543; F. J. Corrales-Izquierdo, Laboratorio de Proteomica, Genomica y Bioinformatica, and Division de
Hepatologia y Terapia Genica, Facultad de Medicina,
Universidad de Navarra, 31008 Pamplona, Spain; D. U.
Ferreiro, Centre for Theoretical Biological Physics and
Department of Chemistry and Biochemistry, University of
California, San Diego, 9500 Gilman Dr, 92093-0378 La
Jolla, CA, USA.
Abbreviation used: TSE, transition state ensemble.
E-mail address of the corresponding author:
[email protected]
etically possible conformations that a protein
molecule can attain, the multiple ways of doing it,
and the chemical composition of the milieu where
the reaction under study takes place, whether
in vivo or in vitro. Studies on protein folding
concentrate to a larger extent on monomeric
proteins undergoing unimolecular reactions aiming
at simplified models and the ability to carry out
molecular simulations of the folding process.
A number of studies on oligomeric proteins have
also been described, but are mostly on large or
multidomain proteins that complicate the dissection of the reaction at the molecular level (see
Jaenicke & Lilie1 ). Often, the folding of the
monomer is followed by association into oligomers,
with some structural adjustments but following
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Folding of a Dimeric b-Barrel
essentially native or hierarchical pathways, or can
take place through multi-channel processes.2
At the interface between small monomers and
large oligomers, we find dimeric proteins that are
small enough to address a molecular dissection of
the process and eventually computational studies,
such as the domain swapping cell-cycle regulatory
protein p13suc1.3 Some of these dimers have the
particularity that their structure is highly intertwined, i.e. the folding of each monomer depends
on the formation of the interface of the dimer, which
gives a characteristic highly concerted folding and
association reaction and coupling between secondary, tertiary and quaternary structure.4,5 Good
models of this are Trp and arc repressors6,7 or the
leucine zipper peptide of the GCN4 activator.8 Since
they are mostly DNA binding proteins or domains
and bind to palindromic sequences, a link between
their folding mechanism and their function, while
unclear, is expected.
The C-terminal domain of the papillomavirus E2
protein was shown to constitute a new family of
dimeric folding topology, as well as a newly
673
described DNA binding motif from the crystal
structure of the bovine E2–DNA complex.9 This
domain is comprised of four antiparallel b-strands
from each monomer forming a dimeric b-barrel at
the interface of the dimer. Two a-helices pack onto
the outside of the barrel, and the major helix
participates in DNA binding (Figure 1(a) and (b)).
The solution structure of the homologous domain
from human papillomavirus strains showed an
identical folding topology,10 and so far this topology
is only shared by the Epstein–Barr EBNA1 origin
binding protein.11 We have recently determined the
solution structure of the HPV-16 E2C12 (Figure 1).
At the equilibrium, the HPV-16 E2 domain
unfolds and dissociates in a highly concerted
manner by urea with no stable intermediates
accumulated.13 On the other hand, its kinetic
folding pathway shows a monomeric intermediate
formed in less than 100 ms, with large fluorescence
and ellipticity changes, increased ANS binding
properties and burial of surface area.14 Because of
the particular folding topology, we proposed that
the intermediate could not be fully native. Further-
Figure 1. Folding topology of the
HPV16 DNA binding domain of
the E2 transcriptional regulator.
(a) Backbone superimposition of
the 20 lowest-energy structures
calculated for HPV-16 E2C dimer.
(b) Strand view of average HPV-16
E2C with Trp residues highlighted.
Figures were produced with SwissPdbViewer.31
674
more, the urea denatured state of E2C was shown to
contain clusters of residual non-native structures.15
Here, we present a characterization of the folding
and unfolding mechanism of this domain with a
unique topology, and we fully characterize the
monomeric intermediate, including its unfolding
cooperativity. We reconstructed its fluorescence
spectrum at 100 milliseconds and, using doublejump experiments, we measured its conformational
stability. We show that the folding pathways from
urea and acid unfolded states are equivalent. We
characterize the unfolding reaction and found that
it does not involve a detectable intermediate.
Independent of whether it is started from acid or
urea unfolded E2C, the folding pathway proceeds
through a monomeric intermediate with non-native
compact tertiary structure preceding the formation
of native quaternary structure.
Results
Effect of ionic strength, pH, and temperature on
the folding rates of E2C
Refolding of acid unfolded E2C was shown to
proceed through two major phases: (i) the formation of a monomeric intermediate with a
unimolecular rate constant of 32 sK1 (k1), and (ii) a
subsequent change with a second-order rate constant of 1.6!105 sK1 MK1 (k 2) involving both
association and folding events, and a most likely
first-order phase of w0.5 sK1 (k3), taking place in
parallel with the association/folding event.14 To
further characterize the nature of the interactions
involved in the refolding phases of the E2C dimeric
domain, we analyzed the effect of ionic strength.
Figure 2(a) shows the effect of sodium chloride on
the rate of formation of the monomeric intermediate, where a large and linear increment is
Folding of a Dimeric b-Barrel
observed, increasing up to sixfold from 0 M to 1.0 M
salt. The slow phases were also affected but in a
rather different, biphasic, manner; from 0 to 0.1 M
salt both the rates were doubled (Figure 2(a)), and
the major phase (k2) increased linearly while the
minor slower phase (k3) remained invariant from
0.2 M to 1.0 M salt. A rate increase could be
explained by screening of unfavorable ionic interactions in the transition state of this intermediate or,
alternatively, the increase in ionic strength in the
milieu might strengthen hydrophobic interactions,
or both effects added together.16 In addition,
preferential hydration effects could account at
least in part for this effect.
The rate of the folding intermediate (k1) is affected
by pH, showing an apparent transition midpoint at
around pH 6.5 (Figure 2(b)), where the rate is
doubled along the pH transition. The rates of the
slower phases (k2 and k3), however, increase by 120
and 180-fold, respectively (Figure 2(b)). As
observed for the stability of the E2C dimer in
equilibrium unfolding experiments,13 the pH transition appears to involve one or more histidine
groups. The pH midpoint of the slow phases is
lower than 5.8, a significant difference with the
intermediate (Figure 2(b), inset).
The monomeric intermediate was shown to form
through a transition state ensemble (TSE) with
significant change in exposure of residues.14 We
carried out a pH jump refolding experiment at
different temperatures to investigate the thermodynamic properties of the TSE for the slow
phases. The observed rates increase with temperature, and we can analyze the data as previously
described.14,17 Both slow phases show a significant
curvature, indicative of a measurable DCp,
explained by change in solvent exposure
(Figure 2(c), legend). Since it is difficult to estimate
the value of the pre-exponential term of the
modified Eyring equation, the absolute values of
Figure 2. Effect of solvent variables on the folding kinetics of E2C. (a) Refolding of acid denatured E2C at different salt
concentrations. Final concentration of E2C was 2 mM, in 50 mM Mes buffer (pH 6.1), and refolding by dilution 1:1, under
the conditions indicated in Materials and Methods. (b) Effect of pH on refolding of acid denatured E2C. The buffers used
were Mes and Mops at the indicated pH. (c) Temperature dependence of the slow folding phases. The experimental
conditions are those described for (a), and data were fitted as described.10 The absolute values of DH and DS are not
reliable, since the pre-exponential of the non-linear Eyring equation term is not known.
Folding of a Dimeric b-Barrel
activation entropies and enthalpies are uncertain,
but both phases display a similar free energy of
activation. The temperature does not affect their
relative amplitudes (not shown). Values for DCp
were K567(G199) cal molK1 degK1 for k2 and
for
k3.
The
K520(G89) cal molK1 degK1
value previously obtained for k 1 was
K385(G31) cal molK1 degK1, corresponding to the
burial of surface in the monomeric intermediate.10
The DCp values for the parallel folding channels are
identical, since they involve a similar amount of
rearrangement from their respective intermediates
to the final folding state.
Similar folding pathways from urea and acid
denatured E2C
We investigated the folding pathway starting
from the urea denatured domain, by monitoring
tryptophan fluorescence upon dilution of E2C
unfolded in 4.0 M urea into folding buffer. The
refolding reaction yielded a monomeric intermediate and slow phases with similar behavior to
the acid unfolded reaction. The rate of formation of
the monomeric intermediate decreased with the
increase in urea concentration on the refolding
buffer, as Figure 3(a) shows. Extrapolation of the
kobs to zero denaturant yields a value of 31 sK1, in
excellent agreement with pH jump refolding.14
The slope of this plot, the m ‡ value, is
0.59 kcal molK1 MK1. The amplitude of the intermediate increases linearly over the protein concentration range and extrapolates to zero, indicating
that a parallel reaction for the formation of the
intermediate, if present, is minimal (Figure 3(a),
inset).
The slow refolding association phases show a
fluorescence decrease similar to the pH jump
refolding experiments, with a second-order rate
constant of 1.5!105 sK1 MK1 (k2) in excellent
675
agreement with the folding pathway from acid
unfolded E2C14 (Figure 3(b)). The observed rate of
this folding/association phase is increased with E2C
concentration displaying a slope of 2.4!105 sK1 MK1
corresponding to (k2), thus validating the secondorder approximation (Figure 3(c) and (b)). The
slowest phase yields a first-order rate of w0.2 sK1
(k3) in excellent agreement with the equivalent
process from acid unfolded E2C.14 The relative
amplitudes undergo an inversion as the protein
concentration increases, stabilizing at 50% each
approximately (not shown), which agrees with the
fact that k2 is bimolecular and k3 unimolecular. As
previously noted, there is a deviation of the observed
rate from linearity at high concentration of E2C,
where a first-order rate becomes limiting, providing a
rough estimate of w1–2 mM KD for the monomer–
monomer interaction.13,14
The effect of urea on the rates of the slow
association/folding phases was investigated; both
showed a decrease as the denaturant was increased,
with observed rates of 0.41 sK1 and 0.05 sK1, for k2
and k3, respectively at zero denaturant (Figure 3(c)).
The sensitivity to urea is very similar in both phases
(identical m‡ values of 1.0 kcal molK1 MK1), slightly
more pronounced than in the intermediate.
The monomeric intermediate unfolds
cooperatively and has a non-native fluorescence
spectrum
The monomeric intermediate showed an increase
in fluorescence, with a subsequent decrease as the
reaction proceeds to the folded state as the slow
association/folding takes place, in both acid or urea
unfolded E2C (Figure 4(a)14). We carried out kinetic
refolding experiments at different wavelengths
from 300 nm to 400 nm, starting the reaction from
the acid denatured E2C domain in order to
minimize possible effects of residual denaturant.
Figure 3. Folding from urea versus acid denatured E2C. (a) Effect of urea on the folding rate (k1) of the monomeric
intermediate from E2C unfolded in 4 M urea. Concentration of E2C was 10 mM using the same conditions as in Figure 2,
except that the dilution was 1:10 in refolding buffer. The inset shows a wider concentration range plotted logarithmically.
(b) Effect of protein concentration on the slow folding rates, k2 and k3, of urea unfolded E2C. The values are indicated in
the text. The amplitudes equilibrate at 50% each, but since it is fluorescence intensity, they do not necessarily measure
absolute populations. (c) Effect of urea on the folding rates of the slow phases, k2 and k3. Conditions are similar to those in
(a).
676
Folding of a Dimeric b-Barrel
By taking fluorescence values at each time for 20
different wavelengths, we reconstructed the spectrum of the intermediate at its maximum amplitude
(80 ms). An increase in the fluorescence intensity at
80 ms is observed, but with otherwise no change in
its wavelength maximum (Figure 4(a) and (b)). This
indicates that the tryptophan residue (or residues)
responsible for the signal remains fairly accessible
to the solvent in the intermediate. The large increase
in the fluorescence quantum yield could be related
to another group in the proximity of the fluorophore, causing quenching effects in the acid
unfolded state. A further association/folding step
leads to the observed blue-shift in the fluorescence
spectrum (Figure 4(c)), concomitant with a decrease
in the intensity, to finally recover the spectral
properties of the folded dimeric domain. This is
consistent with the burial of the tryptophan
residues at the center of the barrel.
The transient nature of the monomeric species
was previously demonstrated in a double jump
refolding-unfolding experiment, were the unfolding amplitude of the phase reached a maximum at
60–80 ms and then disappeared.14 Since the unfolding amplitude is proportional to the amount of
intermediate present, varying the urea concentration in the unfolding (second) mixture would in
theory cause the gradual disappearance of a folded
population of the intermediate, allowing for a
quantitative estimate of its stability. Figure 5
shows the effect of urea on the unfolding amplitudes of the monomeric intermediate in a double
mixing refolding/unfolding experiment. The curve
displays a cooperative transition reaching a plateau
at 2.5 M urea, and a further linear change to 4.0 M
denaturant. Since both the folding and unfolding of
this intermediate are first-order reactions, as the
lack of protein concentration dependence previously showed, we can analyze its stability
by assuming a two-state, concentrationindependent model. By this procedure, we obtain
a DGunf of 3.45(G0.40) kcal molK1, with an m value
of 2.55(G0.32) kcal molK1 MK1 (urea). In similar
conditions of buffer, pH, temperature, and protein
concentration the DGunf of the native folded dimeric
domain was measured to be 11.0 kcal molK1, with
an m value of 3.1 kcal molK1 MK1 (urea).13
Unfolding kinetics of E2C
Figure 4. Evolution of the fluorescence spectrum of the
monomeric intermediate to the folded E2C. (a) The
complete folding time trace of acid unfolded E2C,
followed at two wavelengths as an example (dark dots,
340 nm; light dots, 350 nm). (b) A time trace was followed
at 20 different wavelengths at 5 nm intervals, and the
spectra at selected times reconstructed and shown.
The unfolding kinetics of the dimeric domain was
studied by mixing the folded E2 C-terminal domain
to a final concentration of 4.0 M urea.13,15 The
fluorescence data can be best described by a
double-exponential model (Figure 6(a)). Analysis
of the relative amplitudes indicates a major phase
accounting for 85% of the fluorescence change and a
minor phase of 15%, and the unfolding rate of both
phases remained unchanged up to 10 mM E2 (not
shown). The observed fluorescence change corresponds to the full change upon unfolding; no
changes were observed at very short times or in
the dead time (not shown).
Folding of a Dimeric b-Barrel
Figure 5. Stability of the monomeric intermediate from
a double jump folding-unfolding experiment at different
urea concentrations. Acid unfolded E2C at 10 mM was
refolded by 1:1 dilution into Mes (pH 6.1) buffer and, after
a 100 ms delay, unfolded by 1:1 dilution at the indicated
urea concentrations. The amplitude change is negative
(disappearance of the intermediate) and their absolute
value plotted, against urea. The data were fitted to a
standard two-state unimolecular transition, and the
values obtained indicated in the text.
The observed rate of the major unfolding phase
decreased with urea concentration, and extrapolation of the data to 0 M denaturant gives
the unfolding rate constant in water, ku1, which is
0.009 sK1, corresponding to a half life of 32 seconds
and an m value of 0.26(G0.01) kcal molK1 MK1
(urea) (Figure 6(b)). The rate of the minor phase
(ku2Z0.5 sK1) appears unaffected by urea but the
677
signal is too small for an accurate determination of
changes in rate and amplitude with the denaturant.
The effect of ionic strength in the unfolding
kinetics was investigated by increasing the concentration of NaCl in the unfolding mixture. The major
phase (ku) shows a decrease in the unfolding rate as
the salt concentration is increased (Figure 6(c)). We
had observed a marked effect of NaCl on the
stability of E2C,18 and this will have an effect on the
rate of unfolding. In fact, the amplitude of
unfolding actually decreases due to the stabilizing
effect of the salt on the ground state18 (not shown).
Thus, as the salt concentration is increased, a
smaller fraction of the molecules become unfolded.
However, at 0.15 M salt, the effect on the rate is 90%,
and the amplitude is unchanged. This suggests that
the transition state of unfolding is rather sensitive to
ionic strength, the rate is halved at low NaCl
concentrations at which the rate of folding is
doubled (Figure 6(c)).
Unfolding experiments at different wavelengths
on the larger unfolding phase show gradual change
at all the wavelengths, with no fluorescence
increase beyond native or unfolded species, which
suggests that the major unfolding reaction is
accompanied by a single spectral shift, different
from the folding direction (not shown), suggesting
the absence of a populated unfolding intermediate.
Unfolding of E2C was also followed by circular
dichroism and the ellipticity change of the major
phase goes in parallel with the fluorescence change
and fits to a single-exponential decay with a firstorder rate of 0.018 sK1 in excellent agreement with
the rate of the same phase obtained from fluorescence (0.02 sK1, Figure 7). Since ellipticity traces
tend to be much noisier than fluorescence the
presence of a minor unfolding phase cannot be
determined accurately, assuming it does involve an
ellipticity change. In addition, the dead time of the
CD unfolding is at least five seconds, which
Figure 6. Unfolding kinetics of E2C by urea. (a) Unfolding trace of 10 mM E2C in 4 M urea in Mes (pH 6.1) (see
Materials and Methods), shown as an example. The data were fitted to two exponentials (inset, residuals). The two
observed rates and their amplitudes are independent of the protein concentration (not shown). (b) Urea concentration
dependence of the major unfolding phase (see the text for values). (c) Effect of salt concentration on the major urea
unfolding phase of E2C. Conditions are the same as in (a), except for the added salt. The inset shows the effect of pH,
using the buffer system explained in Figure 2(b).
678
Figure 7. Concomitant loss of secondary and tertiary
structure upon unfolding of E2C. E2C was unfolded by
dilution into 4 M urea, at pH 6.1 and 25 8C (see Materials
and Methods), and the ellipticity change (line) monitored
in a time-course manner, and adjusted to a singleexponential equation. The dead time of the experiment
was five seconds. An experiment in identical conditions
was carried out and the fluorescence (dots) was
monitored, but fitted to two exponentials. The rate
constants are coincident (see the text).
precludes an accurate determination, since the halflife of the minor fast unfolding phase is less than
two seconds. At this stage it is not possible to
completely rule out an intermediate from this
experiment, but a major phase clearly displays a
concomitant disappearance of secondary and
tertiary structures, with no detectable fast changes.
The two slow phases represent parallel folding
channels
The transient nature of the monomeric intermediate was determined from its amplitude of
unfolding in double jump experiments. Two phases
(k2 and k3) are present in the refolding of the E2C
from both acid and urea unfolded states (Mok
et al.14 and Figure 3(b)). Above 5 mM E2C, these
amplitudes correspond roughly to 50% of the
fluorescence change (Figure 3(b)). To further
investigate these phases, we carried out a double
jump experiment, in which the pH unfolded
domain was allowed to refold for times greater
than 100 ms, after the intermediate disappears, and
then unfolded in 4.0 M urea. The unfolding process
is described by two exponential decays, as in
unfolding of fully folded E2C (Figure 6(a), inset).
At short times of refolding delay (100 ms), there are
already two phases, suggesting a fast equilibrium.
The slower unfolding phase (ku1) corresponds to
60% of the amplitude and at longer delay times,
80% (Figure 8). We can use the change in the
amplitude of unfolding as a measure of the folded
species at different delay times (Figure 8). This can
be fitted to an exponential phase and the rate, 1.7 sK1,
Folding of a Dimeric b-Barrel
Figure 8. Two folding channels demonstrated by time
delay double jump folding-unfolding. Acid unfolded E2C
(20 mM) was refolded in 100 mM Mes (pH 6.1), and at the
indicated delay times, after the disappearance of the
monomeric species, the mixture was unfolded to a final
concentration of 4.0 M urea. The fluorescence change was
monitored and the data fitted to a double exponential
equation. The resulting amplitudes, plotted against time,
represent the two parallel folding channels, one (ku2) from
a monomeric intermediate and the other (ku3) from a fast
formed dimeric intermediate.
corresponds to that of the folding reaction through
this channel. Multiplying k2 (2!105 MK1 sK1) times
the protein concentration (2 mM), we obtain a kobs of
0.4 sK1, in very good agreement, considering the
large differences in accuracy between a stoppedflow fluorescence trace and the amplitudes of a
double jump experiment.
Therefore, the two phases occur early in the
refolding process, after the formation of the
monomeric intermediate, and presumably arise
from an isomerization of the latter. From there, the
folding proceeds to the final state by two parallel
channels that are partitioned depending on protein
concentration. As described before, the dissociation
constant for the dimeric intermediate is estimated to
be around 2 mM (Figure 3(b) and Mok et al.15).
Discussion
Here, we completed the folding pathway of the
DNA binding domain of a dimeric b-barrel
transcriptional regulator, HPV E2C, by further
characterization of the monomeric intermediate,
and its unfolding mechanism. E2C is an excellent
model to address problems concerning the folding
of intertwined dimeric proteins in general and a
dissection of their kinetic folding pathways in
particular.
The refolding process from the urea unfolded
E2C occurs through a pathway highly equivalent to
Folding of a Dimeric b-Barrel
that of the acid unfolded state; this means that
the unfolded state ensembles in urea and acid are
very similar, or at least energetically equivalent,
lacking persistent long-range interactions.14,15 The
monomeric intermediate is present in both
reactions, indicating that it is not related to pH
effects on the chromophores and is a reproducible
species. The large fluorescence increase in the
intermediate appears to be due to the release of an
interaction of the chromophore with a quencher in
the unfolded state. By reconstructing a fluorescence
spectrum for this species from kinetic experiments,
we were able to determine that there is not a change
in the maximum, which remains with properties of
an exposed Trp, supporting the idea that the Trp at
the interface are largely exposed in the monomeric
intermediate. The slow association/folding step
includes a shift of the spectra to that of a buried
Trp residue.
The analysis of the relative amplitudes of the
slow refolding/association phases from both high
urea and low pH suggest that there is a dimerization of the intermediate at higher protein
concentrations. This would generate a parallel
folding pathway, to attain the folded dimeric
species through a first-order reaction. The change
in molecularity indicates that above a certain
concentration, the rate-limiting step is unimolecular. From the plot of rate versus E2C concentration,
and the second to first-order transition,15 we can
estimate a dissociation constant between 1 mM and
5 mM for a dimerization of an early species,
approximately 8 kcal molK1 in terms of free
energy, compared to 11 kcal molK1 under similar
conditions at equilibrium.13
The transition state ensemble for the formation of
the intermediate is very sensitive to the ionic
strength, at 1 M salt the rate increased linearly by
sixfold. On the other hand, the slow folding/
association rates are only slightly increased. The
large effect of ionic strength on the formation of the
intermediate could be explained by the strengthening of hydrophobic interactions on its transition
state by an increase in the ionic strength of the
milieu. Alternatively, as experiments on the arc
dimer suggest, the high salt concentration could be
screening unfavorable ionic interactions in the
transition state, contributing to large increases in
rate. The salt dependence of the arc dimer is slightly
less pronounced than the E2C intermediate but
more pronounced compared to the slow association/folding phase of the E2 domain.19 However,
in the arc repressor the transition state leads to the
folded state, since it is a single step process, and
appears to be a single ensemble of structures. The
fact that rates of neither the fast nor the slow phases
are decreased by ionic strength strongly suggests
that the important ionic interactions for the native
dimer, if any, are formed after the transition state of
the slower folding/association step. Since this
transition state is dimeric, it is tempting to suggest
the absence of ionic interactions at the dimer
interface; this is supported by the largely hydro-
679
phobic nature of the interface of the folded state
with some hydrogen bonding added.12 A monomeric protein such as lysozyme shows a large
decrease in the refolding rate of the slowest phase as
the ionic strength is increased, suggesting that in
this case the ionic interactions in the transition state
are favorable.20 Lysozyme also has a fast phase
where the fluorescence change goes beyond that of
the native protein, but different from the E2C
intermediate, the rate is insensitive to ionic
strength.
Previous work showed that the monomeric
intermediate underwent substantial structural
change from an ellipticity change corresponding
to 50% of the total change, increased binding of
ANS, and burial of residues.14 Here, we were able to
reconstruct the fluorescence spectrum of the intermediate, indicative of solvent accessible tryptophan
residues. Moreover, we characterized the stability of
this intermediate towards urea denaturation, and
found that it proceeds through a cooperative twostate process. The intermediate has w31% of the
stability of the folded dimeric domain, indicating
that it is clearly a distinctive thermodynamic entity
with a cooperative behavior. The m value, or
cooperativity parameter is 82% that of the folded
E2C dimer at equilibrium.13 The m value was
shown to correlate very well with the change in
solvent-accessible surface area upon unfolding
(DASA), and this, in turn, with the number of
residues and with changes in DCp.21 This would
mean that there is more burial of surface area in the
formation of the native dimer from unfolded dimer
than in the formation of the intermediate from the
unfolded. In the Trp repressor burst intermediate,
its m value is one-third of the fully folded dimeric
repressor and the stability of this intermediate is
15% that of the folded state, half of the comparative
stability of the monomeric intermediate.22
Unfolding studies showed a major phase, as
judged by the major CD change observed with the
same rate. We determined a ku value of 0.01 sK1 in
the absence of denaturant, of similar magnitude to
the 33-residue leucine zipper peptide derived from
the yeast transcriptional activator GCN4,8 and
tenfold slower than the unfolding of the arc
repressor.23 The Trp repressor seems to unfold
even slower than E2C, and its larger stability to
urea denaturation at equilibrium may contribute to
this slow unfolding rate.6 In contrast to the folding
direction, the unfolding of E2C occurs through a
gradual change in fluorescence spectral properties
with no species with enhanced fluorescence
detected, strongly suggesting an apparent concurrent two-state unfolding and dissociation with
no intermediate being populated.
The bimolecular folding rate constant of E2C is
105 M K1 s K1, substantially lower than the
theoretical rate expected from the collision of two
spheres,24 and much slower than that of Trp or arc
repressors, occurring closer to the diffusion rate
limit.19,25 If the monomeric intermediate was
native-like, the association rate would be much
680
faster, i.e. approaching diffusion control. Nevertheless, it is clear that the intermediate has compact
non-native tertiary structure and represents a
distinct thermodynamic species. This species is
stable and cooperative and must partially unfold
or decompact when proceeding to the dimeric
folded species. However, such rearrangement is
not expected for the formation of the weak dimeric
intermediate I2. However, at this stage, the decompaction of the monomeric intermediate cannot be
studied by direct methods in isolation from all other
reactions.
The Trp repressor showed a burst phase monomeric intermediate and the arc repressor showed no
detectable monomeric intermediate, but its
extremely fast folding rate suggests the fast
formation of a partly folded monomer or an
unfolded monomer with a structure compatible
with native folding.25,26 The GCN4-p1 leucine
zipper domain lies between the Trp repressor and
the E2C domain, in that an intermediate is not
populated but the folding/association rate constant
is two orders of magnitude lower than the near
diffusion rates of Trp and arc repressors.8 However,
it is not clear why, given the ultrafast helix-coil
equilibrium, the pre-formed native helical structure
does not yield a near diffusion limit second-order
folding/association rate.
It is well known that the folding in regions that
are helical in the folded state can occur in the dead
time of most of the standard kinetic techniques.27 It
is difficult, however, to determine whether the
structures formed are full helices or represent a
pre-helix conformation that may act as pre-formed
native-like structures that would accelerate a
concomitant folding and association reaction. It is,
nevertheless, clear that b-sheet formation requires
precise tertiary interactions that will require a larger
conformational search or rearrangement of
collapsed structures. The unusual dimeric interface
present in the half-barrel of a dimeric b-barrel
domain such as E2C appears to require the
formation of a compact tertiary non-native structure
most likely devoid of interstrand b-sheets, that must
rearrange to form such a complex and intertwined
b-strand based interface. A similar picture emerged
Figure 9. Folding mechanism of HPV-16 E2C (see
Discussion).
Folding of a Dimeric b-Barrel
from the comparison of folding from fragments
in two paradigmatic small proteins, barnase and
CI2.28
Figure 9 shows the simplest model compatible
with our results at this stage. The unfolded state
ensemble in urea has local residual structure15 but
we cannot rule out discrete sub-populations of
isomers that may direct the formation of a
monomeric or dimeric intermediate. Focusing on
the main pathway at low protein concentration, we
determined the kinetic stability from ku1/k1k2, to be
12 kcal molK1, compared to 11 kcal molK1 from
equilibrium.13 If the minor unfolding phase was
sequential, i.e. an intermediate, then the overall
process should be ku2ku1/k1k3, and this yields an
absurd value.
Our results so far suggest that the early dimerization takes place from the monomeric compact
intermediate. Thus, depending on the protein
concentration, the folding pathway will take place
predominantly through a first-order or secondorder reaction. These parallel channels yield
possibly two folded conformers, where the actual
structural change could be very small, i.e. the
position of a side-chain. We propose that there is a
slow pH-dependent equilibrium around a pKa
corresponding to histidine groups.13 There is a
major phase of unfolding, indicative of no intermediate in the reverse reaction. It remains to be
established if the minor fast unfolding phase takes
place in parallel or if it corresponds to an unfolding
intermediate; so far, everything suggests that it is a
parallel unfolding pathway. Ongoing mutagenesis,
DNA binding, protein truncation and NMR studies
of the folded and unfolded states will complete the
folding pathway of this model protein with a
unique folding topology.
Materials and Methods
The expression in Escherichia coli as well as the
purification of the E2 DNA binding domain from HPV16 were as described.13 An extinction coefficient of
41,900 MK1 was used for determining the concentration
of the dimer.29,30 Refolding and unfolding experiments
were performed using a DX-18MV stopped-flow
equipment from Applied Photophysics, and an Aminco
Bowman Series 2 spectrofluorimeter.
Fluorescence experiments were carried out at fixed
excitation (280 nm) and measuring the emission at
335 nm, except where the wavelength is the variable.
The unfolded protein was obtained either by lowering the
pH to 1.7 with HCl or at 4.0 M urea, depending on the
type of experiment. Refolding buffer was 100 mM Mes–
NaOH buffer (pH 6.1), and 1 mM DTT, unless otherwise
stated. All experiments were carried out at a fixed
temperature of 25(G0.1) 8C, unless indicated otherwise.
Double jump refolding/unfolding experiments were
started from 8 mM acid unfolded E2C, mixed with
refolding buffer for 100 ms or the indicated delay time
as the variable and then unfolded with different
concentrations of urea in 100 mM Mes buffer (pH 6.1),
and the fluorescence change monitored. For unfoldingrefolding double jump experiments, 8 mM E2 was mixed
681
Folding of a Dimeric b-Barrel
1:1 with 8 M urea, both in Mes buffer, in the first mixture
to give 4 mM and 4.0 M concentrations of protein and
denaturant, respectively. After variable times of unfolding, E2 was diluted to 0.4 M urea, and the fluorescence
monitored. We previously showed that the refolding of
acid unfolded E2 domain is decomposed into a fast
concentration-independent phase (k1), and a slow association/folding phase with more than one component.14
For the data analysis, we used a first-order approximation
(two exponentials), which showed a strong concentration
dependence in one phase and a less pronounced
dependence on the other (k2 and k3, respectively). Since
the rates are only fourfold different, it is extremely
difficult to assess whether the slower rate (k3) changes
with concentration or it is affected by the faster secondorder rate (k2). Since in the present work, we analyze the
effect of several factors on the folding, we simplify the
analysis by treating the rates as single-exponential
decays, as opposed to using a sum of second-order
equations, not amenable for an accurate mathematical
analysis. k2 and k3 are thus apparent rates, but are
nevertheless useful to describe the effects studied here.
The association phase at a single concentration was
analyzed using a second-order equation with an exponential component as previously described,15 and gives a
value identical to that from the slope of the observed
rates, validating the approach.
Circular dichroism spectra were recorded on a Jasco
J810 instrument in 0.1 cm path cuvettes, at 25(G0.1)8 C at
10 mM protein concentration, and averaging ten scans. For
urea unfolding denaturation, folded E2C was diluted into
a 4.0 M urea solution in 25 mM BisTris–HCl (pH 7.0) and
the ellipticity recorded immediately after.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgements
We thank Lisa Gloss for helpful criticism of the
manuscript. A.D.N., L.G.A., and D.U.F. hold PhD
fellowships from CONICET. This work was supported by the Wellcome Trust, CRIG OIA U41
RG27994. The authors thank the support of Agencia
Nacional de Promocion Cientı́fica y Tecnológica
(ANPCyT, Argentina).
15.
16.
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Edited by F. Schmid
(Received 7 April 2005; received in revised form 10 May 2005; accepted 31 May 2005)
Available online 1 July 2005