(Received for publication, July 17, 1995; and in revised form, September 18, 1995)
From the
The urea-induced unfolding of the Escherichia coli tryptophan synthase -subunit is examined via fluorescence
measurements with tryptophan-containing
-subunit mutants,
constructed by in vitro mutagenesis. Early unfolding studies
with urea and guanidine suggested that the wild type protein unfolded
in a two-step process with a stable intermediate composed of a native
-1 folding unit (residues 1-188) and a completely unfolded
-2 folding unit (residues 189-268). Recently, more detailed
spectroscopic and calorimetric data from the Matthews and Yutani groups
indicate that such a structure for the intermediates seems unlikely.
Previously, we described the introduction of Trp residues as unfolding
reporter groups separately into each of the folding domains and showed
that these proteins are wild type enzymatically and in their stability
to urea. The unfolding behavior of these
-subunits, monitored by
fluorescence intensity changes at the discrete emission
for each, in both equilibrium and kinetic experiments, suggest
that: (a) both folding units commence unfolding simultaneously
(near 2 M urea); (b) the larger
-1 unit unfolds
in a multistep process, initially yielding a partially unfolded
intermediate form which subsequently appears to unfold progressively to
completion; and (c) the smaller
-2 unit unfolds in a
single step event. These results are also clearly incompatible with the
early proposals on the structure of the intermediate. It is suggested
here that the intermediate is heterogeneous, consisting of a stable,
partially unfolded form of
-1 attached to either a completely
folded or completely unfolded form of
-2. These results are
consistent with and provide an added dimension to the recent
description of the proposed structure of the intermediate.
The unfolding behavior of the -subunit of the bacterial
tryptophan synthase is characterized by two important features: there
appears to be two independent folding units within the protein (1) and there is a stable, populated intermediate in the
unfolding pathway(2, 3) . The structures of the
folding units are defined in terms of the intact native structure. The
-subunit structure is a variant (4) of the classic
/
barrel structure in that it has several extra
-helices(5) . The amino-terminal (188-residue)
-1
folding unit consists of an
form;
the carboxyl-terminal (80-residue)
-2 folding unit is an
structure. The unfolding pathway,
experimentally inducible by a variety of denaturants (heat, urea,
guanidine), has long been known to be a multistep process. However, the
structure of the major intermediate in this pathway, detected by
calorimetry, circular dichroism, UV absorbance, hydrogen exchange,
proton NMR, and tyrosine fluorescence measurements, has not yet been
clearly defined.
Interest in this laboratory stems from a
calorimetric analysis of nearly 75 mutant -subunits of the Escherichia coli enzyme obtained by in vitro mutagenesis and containing single amino acid
substitutions(6) . The broad spectrum of stability (i.e. unfolding) properties exhibited by these mutant proteins presented
the opportunity to explore the unfolding pathway in more detail. The
approach initiated here was to introduce a single Trp residue
separately into each of the folding units to use as reporter groups for
the fluorescence-monitored unfolding of each unit. The wild-type
-subunit does not contain tryptophan. Three
-subunits (F139W,
F258W, and F139W/F258W) were constructed in which Phe residues were
substituted singly at positions 139 (in
-1) and 258 (in
-2)
and at both sites(7) . The positioning of the Trp residues was
such that all of the existing mutational alterations can be engineered
into either of these tryptophan-containing host
-subunits. Another
critical property of these host proteins is that the Trp substitutions
do not affect their enzymatic or stability behavior(7) . This
report describes the urea-induced unfolding pathway for these wild
type-like proteins as monitored by tryptophan-specific fluorescence
measurements and provides additional information on the structure of
the unfolding intermediate.
On-line formulae not verified for accuracy
where A(t) is the fluorescence intensity at a
given time t, A() is the intensity when no
further change is observed, k
is the apparent
first order rate constant of the ith kinetic phase, and A
is the amplitude of the ith phase. The
kinetic data were fit to one or more exponentials as appropriate, and
the relaxation times and associate amplitudes were extracted with a
nonlinear least-squares fitting program of Sigma Plot v.4.1 (Jandel
Scientific).
Figure 1:
Fluorescence spectral
changes for the F139W (A) and F258W (B)
-subunits in urea. The protein concentrations were 5
µM. The dashed line is for 5 µML-tryptophan in 6 M urea.
Given the fact that no further
intensity changes are observed between 6 M and 8.5 M urea, it is assumed that the fluorescence intensity changes
achieved at 6 M urea represent those for a total unfolding of
the respective folding units. Utilizing the intensity decreases at the
for the native form of each
-subunit, the
fractional unfolding, F
, can be determined as a
function of urea concentration. The results for the F139W
-subunit
are shown in Fig. 2A. Unfolding begins at
2 M urea, and apparent complete unfolding in the region around Trp-139
is seen at 5-5.5 M, a concentration similar to that
required for complete unfolding when UV differences are
monitored(7) . Reversibility data are shown by the open
circles.
Figure 2:
Urea-induced equilibrium unfolding of
the F139W -subunit monitored by fluorescence at 328 nm. A, fractional unfolding, F
(F
= (I - I
)/(I
- I
) where I is the observed intensity at
some urea concentration, and I
and I
are the intensities of native and unfolded
species (closed circles); open circles represent the F
observed upon dilution from 6 M urea
to the indicated urea concentration. B, fraction of native (f
), intermediate (f
), and
unfolded (f
) forms (the dashed lines are
the values obtained from UV absorbance measurements, (7) ). C, the dashed and solid lines are the
calculated F
values for a one- or two-step
transition, respectively.
Previous UV absorbance data obtained with the F139W
-subunit (7) suggested a three-state, two-step unfolding
model, namely
On-line formulae not verified for accuracy
The F139W -subunit fluorescence-based denaturation curve (Fig. 2A) is relatively broad over the transition
range, and deviations from linearity for the
G versus urea concentration plot (8) are also observed. In
addition, an examination of the intersecting fluorescence spectra found
during the N
U transition (Fig. 1A) reveals no
clear isosbestic point. These observations also suggest a multistep
unfolding process for this protein when monitored by fluorescence
measurements. A two-step model can be described as before (7) by
On-line formulae not verified for accuracy
where K and K
are the
equilibrium constants for the N
I and I
U transition,
respectively, and Z (the fractional change in intensity for
the intermediate form) = (I
- I
)/(I
- I
) where I
, I
, and I
are, respectively,
the intensities of the intermediate, unfolded and native forms. The
fluorescence equilibrium data were fit statistically to a one-step (Fig. 2C, dashed line) or a two-step (Fig. 2C, solid line) model over the
transition range. From these results, a two-step transition seems more
likely. Assuming a linear dependence of free energy on urea
concentration, the free energies of unfolding in the absence of urea
for each step,
G
and
G
, were
estimated according to Pace(8) :
G
=
G
+ m
C and
G
=
G
+ m
C (where m
and m
are the cooperativity factors and C is
the urea concentration). The parameters, Z,
G
,
G
, m
, and m
, are, respectively,
0.33, 5.6 ± 0.8 kcal/mol, 4.9 ± 0.9 kcal/mol, -2.3
± 0.3 kcal/mol/M and -1.3 ± 0.2
kcal/mol/M. The fractional distribution of native,
intermediate, and unfolded forms for the F139W
-subunit are given
in Fig. 2B. The concentration of intermediate form
reaches a maximum at
3 M urea and is subsequently
converted to more unfolded forms. This pattern is quite similar to that
obtained for this protein when more global UV absorbance differences
were employed (7) to monitor unfolding (Fig. 2B, dashed lines).
A similar
treatment of the equilibrium unfolding data for the F258W -subunit
is given in Fig. 3. The unfolding transition initiates at
2 M urea, and apparent complete unfolding in the region around
Trp-258 is observed at 4-4.5 M (Fig. 3A). The transition range for denaturation
is more narrow (Fig. 3A) than that for the F139W
-subunit, and unfolding is fully reversible. Although a two-step
unfolding transition was observed for the F258W
-subunit when UV
absorbance measurements were employed(7) , attempts to fit the
fluorescence data to a one-step (Fig. 3B, dashed
line) or two-step (Fig. 3B, solid line)
process do not provide a mutually exclusive case for either. A plot of
G versus urea concentration can best be judged as linear
and indicative of a simple one-step unfolding process. This conclusion
also appears consistent with subsequent kinetic data. Based on this
interpretation, estimated
G
and m values are 7.0 ± 0.7 kcal/mol and
-2.5 ± 0.4 kcal/mol/M, respectively.
Figure 3:
Urea-induced equilibrium unfolding of the
F258W -subunit monitored by fluorescence at 341 nm. A and B correspond to A and C of Fig. 2.
Figure 4:
Kinetics of the urea-induced unfolding of
the F139W -subunit. A-E, results of typical urea
concentration jumps from 0 M
xM urea; F-J, the results of typical urea
concentration jumps from xM
6 M urea. I
and I
are the observed intensities at time 0 and t,
respectively, and I
is the intensity where
no further change is observed.
Fig. 4, A and B, shows that the kinetics of typical urea
concentration jumps from 0 M to 4 M urea. A
simple linear relationship suggests a single kinetic event. In
contrast, typical results for urea concentration jump from 0 M to
4 M (Fig. 4, C-E) indicate
two kinetic events, a fast and slow step. The amplitudes of the changes
for the fast phases are
90%, and those of the slow phase are
10%. The rate constants for these changes are given in Fig. 5A (solid and open circles are for the
fast and slow phases, respectively). Similar results are seen in urea
concentration jumps from xM to 6 M urea (Fig. 4, F-J). In these experiments, the protein
was allowed to reach equilibrium at a specified urea concentration
(usually for 12 h) and subsequently rapidly adjusted to a final
concentration of 6 M urea. Fig. 4, F-H,
gives typical results of urea concentration jumps from 0-2.75 M to 6 M. Two phases are again observed. Fig. 5B gives the apparent rate constants for the fast (closed circles) and slow (open circles) phases. As
shown in Fig. 5C, the amplitude of the fast phase is
90% (closed circles) and
10% for the slow phase (open circles). In contrast to these data, the results of urea
concentration jumps from
3 M to 6 M indicate an
abrupt loss of the fast phase (Fig. 4, I-J). The
amplitude of the fast phase decreases to a point where the slow phase
only accounts for essentially all of the observed intensity change (Fig. 5C). The apparent rate constants shown for the
fast phase also appear to exhibit a decrease (Fig. 5B)
in these experiments, but the estimation of these rate constants is
problematic because of the decreased amplitudes. The apparent rate
constants for the slow phase do not appear to change.
Figure 5:
Summary of the unfolding kinetics for the
F139W -subunit. A, k values for urea
concentration jumps from 0 M
xM urea. B, k values for urea concentration jumps
from xM
6 M urea. C, the
relative amplitudes of the fast and slow steps for the xM
6 M urea concentration jumps. The closed and open circles are for the fast and slow
steps, respectively.
Both sets of
experiments agree well and are consistent with the equilibrium data
which indicate that the N I transition occurs over the urea
concentration range of 0-3 M and the I
U
transition proceeds over the 3-6 M urea concentration
range. The kinetics indicate that urea concentration jumps within the 0 M to 3-4 M range (Fig. 4, A and B) and within 3-4 M to 6 M range (Fig. 4J) each exhibit essentially single kinetic
events. Urea concentration jumps that span the 3-4 M range (Fig. 4, C-E, F-I)
exhibit two events, a fast phase with an amplitude of
90% followed
by a slower phase with an approximate amplitude of 10%.
Comparable
kinetic experiments with the F258W -subunit employing urea
concentration jumps either from 0 M to xM urea (Fig. 6, A-D) or from xM to 6 M urea (Fig. 6, E-H) indicate
that a single kinetic event is sufficient to achieve
98%
unfolding. The rate constants obtained for both sets of experiments are
given in Fig. 7, A and B.
Figure 6:
Kinetics of the urea-induced unfolding of
the F258W -subunit. A-D, results of typical urea
concentration jumps from 0 M
xM urea; E-H, results of typical concentration jumps
from xM
6 M urea.
Figure 7:
Summary of the unfolding kinetics for the
F258W -subunit. A, k values for urea
concentration jumps from 0 M
xM urea. B, k values for urea concentration jumps
from xM
6 M urea.
The unfolding properties reported here involving the regions
surrounding Trp-139 and Trp-258 are clearly different and are
consistent with the notion of two independent folding units in the
-subunit. Neither here nor in many other folding studies with this
protein is there any unequivocal indication that such folding units
cannot function as such although the initial experiments defining them
remain the strongest evidence that they exist. We have interpreted our
results in these terms, namely, that the fluorescence-monitored
unfolding properties of the F139W and F258W
-subunits reflect some
of the unfolding properties of the
-1 and
-2 folding units,
respectively.
Regardless of whether or not there are independent
folding units, there is little doubt concerning the presence of the
relatively stable unfolding intermediate for this protein. Initial
conclusions that it consisted of an intact -1 unit and an unfolded
-2 unit have been largely discarded by recent evidence from both
the Matthews and Yutani
groups(9, 10, 11, 12, 13) .
Evidence here also support this conclusion. Unfolding of the region
surrounding both Trp-139 (in
-1) and Trp-258 (in
-2) commence
unfolding nearly simultaneously at urea concentrations near 2 M. Thus, no completely intact structure in either folding unit
can exist during the unfolding transitions.
The structural
description of this intermediate here is based on the combined
fluorescence equilibrium and kinetic data from both F139W and F258W
-subunits. Maximum urea denaturation (5-6 M urea
for F139W and 4-5 M for F258W) leads to similar spectra
for both of these proteins and free L-tryptophan. Small
intensity differences remain, however, and there is a question of
whether or not there is complete unfolding for the respective folding
units. NMR data (10) indicate that pockets of local native
structure may remain at 6 M urea. His-92, for example, becomes
totally exposed only at 6.5-7 M urea. Although our data
alone cannot prove complete unfolding of each unit, it is clear that no
further fluorescence spectral changes are seen at urea concentrations
as high as 8.5 M. Thus, the final fluorescence intensity
levels for each tryptophan-containing
-subunit are considered to
represent those for complete unfolding. With these caveats in
mind, together with the basic assumption that the regions surrounding
Trp-139 and Trp-258 represent, respectively, the
-1 and
-2
folding units, a relatively simple unfolding scheme for the
-subunit is suggested (Fig. 8). Although previous data (7) have indicated a strong similarity between the wild type
and the Trp-containing
-subunits, it must be noted that the
relaxation times shown here apply specifically to the F139W and F258W
-subunits. These time constants, however, might be expected to be
at least qualitatively similar to those for the wild type protein. It
can be seen that, although incompatible with the initial ideas of the
structure for the major unfolding intermediate as noted above, this
pathway is consistent with other more recent data and illustrates the
potential value in introducing specifically positioned reporter
residues.
Figure 8:
Schematic diagram of a suggested unfolding
pathway for the -subunit. Circles indicated the native
structure, hexagonals and
-1* indicate the partially
unfolded intermediate for the
-1 folding unit, and carets indicate unfolded forms. The relaxation times are those obtained
with the F139W and F258W mutant
-subunits.
The -1 folding unit (as exemplified with the F139W
-subunit) appears to unfold in multiple steps starting at
2 M urea. The equilibrium unfolding data are most consistent
with a simple two-step (N &rlhar2; I &rlhar2; U) unfolding process in
which the intermediate form appears maximally at
3 M urea. The kinetic data also indicate at least two kinetic events
(fast and slow) for the N
U transition. The assignment of the
fast and slow steps to the N
I and I
U transitions,
respectively, is based on the two types of urea concentration jump
experiments. Urea concentration jumps from 0
4 M and from
4 M
6 M exhibit a single
fast and a single slow rate, respectively. Maximum values for the fast
and slow rates correspond to relaxation times of 12-16 s and
125-130 s, respectively, with the fast step accounting for
90% of the total observed change. From a statistical analysis of
the equilibrium experiments, the predominant unfolding reaction below 3 M is the N
I transition and above 3 M is the I
U transition.
The -2 folding unit (i.e. as
exemplified with the F258W
-subunit) also initiates unfolding at
2 M urea. The equilibrium data can be fit to a one-step
(N &rlhar2; U) process, and, consistent with this, the kinetics of urea
concentration jumps over any concentration range can be described by a
single, uniform rate with maximum value corresponding to a relaxation
time of 54-58 s. By these criteria, it appears that at any
denaturing urea concentration, the
-2 folding unit is either
completely native or completely unfolded.
Thus, these fluorescence
studies also suggest that there is a major -subunit unfolding
intermediate at about 3 M urea as others have proposed. At 3 M urea, the
-1 folding unit appears to exists primarily
in a stable, partially unfolded state. The
-2 unit is either
native or completely unfolded at all denaturant concentrations, and, at
3 M urea, it is
50% native and
50% unfolded. This
leads to our suggestion that the intermediate is heterogeneous,
consisting of a stable partially unfolded
-1 folding unit joined
to either a totally unfolded or totally folded
-2 folding unit.
These conclusions are not inconsistent with Yutani's (12, 13) structural description of the intermediate as
a molten globule form nor with the more detailed picture by Matthews
group(9, 10, 11) . In the latter description,
for example, the
-2 folding unit is thought to remain partially
organized. We have concluded that the
-2 unit exists as both
completely folded and completely unfolded in the intermediate
structure. Secondly, the intermediate is proposed to be an
``opened'' form of the barrel structure in which
40% of
the secondary and tertiary structure is retained but the packing is
loosened. We suggest that the region surrounding Trp-139 has reached a
stable, partially exposed state in which
-1 is attached to either
a native or completely unfolded
-2. Thirdly, it is concluded that
there is partial contact between
-strand 6 (in
-1) and
-strand 7 (in
-2). Our conclusion is that a fraction of a
completely intact
-2 remains associated with a partially unfolded
-1 unit.
The structure of an additional, stable refolding
intermediate proposed from UV and CD spectroscopy by the Matthews group (10) is thought to contain little secondary structure with most
of its tyrosyl residues exposed but yet retaining some local (near
His-92) tertiary structure. Our fluorescence data do not clearly
indicate any additional intermediates although the slow step for
-1 unfolding may be related to the unfolding of the second
intermediate proposed by Matthews' group. The structural identity
of an additional potential additional folding unit within
-1 may
come from a closer examination of an hypersensitive trypsin site at
Arg-70(15) , similar to, although apparently less
hypersensitive than, the Arg-188 site. Whether or not this represents a
site between two smaller folding units within
-1 is, at best,
conjecture. This possibility is being explored by the approach used
here.
Given the sensitivity of fluorescence to environmental factors, a further exploration of the unfolding/refolding characteristics of these wild type-like proteins together with an examination of their behavior when our destabilizing/stabilizing amino acid substitutions are introduced should prove useful in resolving these events.
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