The folding of cardiotoxin analogue III (CTX
III), a small (60 amino acids), all
-sheet protein from the venom of
the Taiwan Cobra (Naja naja atra) is here investigated. The
folding kinetics is monitored by using a variety of techniques such as
NMR, fluorescence, and circular dichroism spectroscopy. The folding of
the protein is complete within a time scale of 200 ms. The earliest
detectable event in the folding pathway of CTX III is the formation of
a hydrophobic cluster, which possess strong affinity to bind to nonpolar dye such as 1-anilino-8-napthalene-sulfonic acid.
Quenched-flow deuterium-hydrogen exchange experiments indicate that the
segment spanning residues 51-55 along with Lys23,
Ile39, Val49, Tyr51 and
Val52 could constitute the "hydrophobic cluster."
Folding kinetics of CTX III based on the amide-protection data reveals
that the triple-stranded, antiparallel
-sheet segment, which is
located in the central core of the molecule, appears to fold faster
than the double-stranded
-sheet segment.
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INTRODUCTION |
The protein folding pathway is the kinetic process in which
disordered polypeptide chain proceeds to fold into its highly ordered
native structure (1). Folding of a polypeptide chain into its unique
three-dimensional structure is believed to be encoded in its amino acid
sequence (2). The transformation of the unfolded chain to its native
state obviously does not involve a random search of the conformational
space, because such a process would require too much of time (3). Most
proteins are known to fold within a time span of a few minutes if not
seconds (4). Therefore, it is obligatory that the initial events in
protein folding should limit the conformational space and thus specify the pathway for unfolded polypeptide chain to attain its native state.
Hence, it is important to understand the structural interactions that
come into play during the early events of folding (5, 6). These
interactions, consequently, predetermine the three-dimensional structure of the protein.
Folding to the native conformation is a highly cooperative process for
small, single domain proteins (7). Although, transient intermediates
have been observed during the folding process, it is still not clear at
what stage the cooperativity in the folding process arises.
Characterization of early folding intermediates provides vital clues(s)
to understand the cooperativity phenomenon observed in the folding of
small proteins. Recently, with the advent of powerful techniques such
as the quenched-flow deuterium-hydrogen exchange in conjunction with
two-dimensional NMR experiments, it has become feasible to characterize
the transient intermediates that occur in the early stages of folding
(8-10). The quenched-flow hydrogen-deuterium exchange technique is
particularly powerful, because it permits many specific sites within a
protein to be probed on the millisecond time scale (11). This technique
has been successfully used to study the folding pathway of several proteins (8). Interestingly, most of these proteins are composed of
helical conformation or possess both
-helical and
-sheet segments. Roder et al. (11), studying the early folding
events in cytochrome C, a predominantly helical protein, found that
helix formation occurs very quickly within the low millisecond time scale. The fast time scales of helix formation (in cytochrome C) are
not surprising, because the hydrogen bonding interactions involved in
helix formation are local. Since hydrogen bonding in
-sheet
formation involves interactions between distant parts of the
polypeptide chain, it is expected that the folding rates of
-sheet
segments would be slower than
-helices. In this context, we
investigate the early folding events in the folding pathway of a small,
all
-sheet protein such as cardiotoxin analogue III (CTX
III)1 from the Taiwan cobra
(Naja naja atra).
CTX III isolated from the venom of the Taiwan cobra (N. naja
atra) is a 60-amino acid, highly basic, all
-sheet protein
(12-14). The three-dimensional structure of CTX III shows that the
protein is "three-finger" shaped with three loops projecting from a
globular head (14, 15) as shown in Fig. 1. The protein is cross-linked by four disulfide bonds. The secondary structure of CTX III (14) includes five
-strands arranged to form double and triple-stranded antiparallel
-sheets (Fig. 1). In the
native state, CTX III does not show any helical segments, and
interestingly, secondary structure prediction analysis on the amino
acid sequence of CTX III shows that no portion(s) of the protein has
any propensity to adopt a helical conformation. Thus, CTX III is an
ideal choice to understand the early folding events in an all
-sheet
protein.

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Fig. 1.
MOLSCRIPT representation of the structure of
CTX III showing the overall solution backbone folding. The
ribbon arrows represent regions of the peptide backbone in
the -sheet conformation. S1-S5 represent the various
-strands (strand I to strand V) in the structure of CTX III. The
disulfide bonds in the protein are located between residues 3 and 21, 14 and 38, 42 and 53, and 54 and 59.
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In the present study, the kinetic folding pathway of CTX III is
examined using a variety of techniques such as stopped-flow circular
dichroism, stopped-flow fluorescence, and quenched-flow deuterium-hydrogen exchange. The results obtained herein are consistent with the hydrophobic collapse model of protein folding.
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EXPERIMENTAL PROCEDURES |
Materials--
CTX III was purified as per the method reported
earlier (14). 1-Anilino-8-napthalene sulfonic acid (ANS) was purchased
from Sigma. Guanidine hydrochloride (GdnHCl, ultrapure) was purchased from Merck, Germany. All other chemicals used were of high quality. Carboxymethylated CTX III was prepared as per the procedure reported by
Goldberg et al. (16).
GdnHCl-induced Denaturation--
Equilibrium unfolding was
monitored by far-UV and near-UV CD as a function of GdnHCl
concentration. CD measurements were made on a Jasco J720
spectropolarimeter. CD spectra were collected with the slit width set
to 430 µM, a response time of 1 s, and a scan speed
of 20 nm/min. Each spectrum was an average of at least five scans.
Secondary structure measurements were made at 213 nm with protein
concentrations of 160 µM in a 0.02-cm path length
cuvette. Near-UV CD measurements at 270 nm were carried out using a
protein concentration of 160 µM with a cuvette with a
path length of 0.1 cm. The sample temperature was maintained using a
Neslab RTE-110 circulating water bath at 25 ± 0.2 °C.
Data Analysis--
Equilibrium unfolding data obtained using
GdnHCl as denaturant were converted to plots of
FU, the fraction of protein in the unfolded
state, versus denaturant concentration using the
equation,
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(Eq. 1)
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where XO is the value of the spectroscopic
property measured at denaturant concentration [D].
XF and XU represent the
intercepts, and mF and mU
the slopes of the folded and unfolded base lines of the data,
respectively, and were obtained from linear least squares fits of the
base lines.
For a two-state F
U unfolding pathway, the free energy of
unfolding by denaturant (
Gu) at concentration
[D] is the related to FU by a transformation
of the Gibbs-Helmholtz equation in which the equilibrium constant of
unfolding in the transition zone, kapp = FU/(1
FU),
FU = e
GU/RT/(1 + e
GU/RT). It is
assumed that the free energy of unfolding,
GU, has a linear dependence on the
concentration of the denaturant [D],
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(Eq. 2)
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wherein
G(H2O) and
mG are the intercept and slope, respectively, of
the plot of
GU versus the
concentration of the denaturant. mG is the
measure of the cooperativity of the unfolding reaction and
GU(H2O) is the free energy
difference between the folded and unfolded states in the absence of any
denaturant.
Stopped-flow Fluorescence Measurements--
All stopped-flow
fluorescence measurements were performed at 5 ± 0.2 °C using a
SF-61 stopped-flow spectrofluorimeter (Hi-Tech Scientific Co.). The
concentration of ANS was varied between 50 and 250 µM by
appropriately mixing the stock solution of ANS (300 µM)
in the native buffer (10 mM sodium acetate buffer, pH 3.0). 1.6 mM of equilibrium-unfolded CTX III (in 6 M
GdnHCl) was diluted 10-fold into 270 µl of the ANS-containing native
buffer. This procedure ensured that the concentrations of the protein
(160 µM) and GdnHCl (0.6 M) were unchanged
during the refolding reaction in the presence of different
concentrations of ANS. The binding of ANS was monitored by setting the
excitation and emission wavelengths at 418 and 497 nm, respectively.
The bandwidth used in all the measurements was 2 mm. The dead time of
stopped-flow mixing device was 4.0 ms.
Stopped-flow CD Measurements--
Stopped-flow CD measurements
were carried out using a stopped-flow apparatus from Biologie attached
to a Jasco J-720 spectropolarimeter. The refolding reaction was
initiated by a 10-fold dilution of the GdnHCl-denatured CTX III sample.
The path length of the optical cell used for all CD measurements was 1 mm. All data were acquired at 5 °C, and the change in ellipticity at
appropriate wavelengths (213 and 270 nm) was monitored as a function of
time. The kinetic data were expressed as a sum of exponential terms.
Nonlinear least squares curve fitting was used to obtain the number of
exponential phases with their amplitudes and apparent rate
constants.
Quenched-flow Experiments--
All experiments were carried out at
5 ± 0.1 °C using a RQF-63 rapid mixing quenched-flow apparatus
(Hi-Tech Scientific Co.). Complete denaturation and exchange of the
backbone amide protons with deuterium was achieved by dissolving CTX
III (30 mg/ml) in 6 M deuterated guanidine hydrochloride in
D2O at pH 6.6 ± 0.2. Deuterated guanidine
hydrochloride was obtained through repeated cycles of dissolving
ultrapure GdnHCl in D2O followed by repeated lyophilization. Refolding of the denatured protein was initiated by
10-fold dilution with 50 mM
glycine-d5 (pH 3.0) in H2O. At this
pH, negligible labeling occurred. After variable refolding times
ranging from 9.8 to 190.4 ms, the solution was diluted again to 10 times the initial protein volume with 0.2 M sodium borate (pH 9.6) to initiate labeling of the deuterated amides in CTX III with
protons. After a lapse of 10 ms, the labeling pulse was stopped by a
further 3.4-fold dilution of the initial protein volume with 1 M HCl in water. The final pH was about 3.2, at which the
hydrogen/deuterium exchange (for the amides in native CTX III) was
minimal. It should be mentioned that there were no signs of
intermolecular aggregation of the protein during any of the refolding
steps in the quenched-flow experiment. This was ensured from the steady
state CD (in the far UV region) and 1H one-dimensional NMR
spectra of the refolded protein sample. It should be mentioned that
absorbance of the refolded protein sample(s) at 300 nm also did not
indicate aggregation of the protein during refolding. After the final
step of refolding, the protein samples were concentrated by
ultrafiltration at 4 °C.
A magnitude COSY spectrum of each sample was recorded on a 600-MHz NMR
spectrometer at 5 ± 0.1 °C. 256 increments over 1024 data
points were collected. All of the NMR data were processed using UXNMR
software on a Silicon Graphics workstation. The intensities of the
intraresidue (C
H, NH) cross-peaks were normalized based
on the aromatic cross-peak (C
H, C
H)
intensities of Phe25 and Tyr22. The proton
occupancy of individual amides at the zero time point of refolding were
obtained by a 10-fold dilution of the 6 M GdnDCl-denatured CTX III (in D2O) with the pulse labeling borate buffer in
H2O at pH 9.6. It is important to mention that the first
step of initiation of folding (without the exchange) at pH 3.6 using
HCl-glycine buffer used at other refolding time points is eliminated
for the zero time point proton occupancy estimation. The pulse labeling step using the borate buffer (pH 9.6) process ensures exchange of all
deuterium atoms by protons and also the protein folds back to its
native state. The intensities of the cross-peaks of the residues in the
fingerprint region of the COSY spectrum at this time point (the zero
time point) represent the initial proton occupancy. It is pertinent to
mention that the relative percentage proton occupancy of individual
residues at the zero time point (and in subsequent refolding times) was
estimated by comparing with the amide proton intensities of the
corresponding residues in the fingerprint region of the COSY spectrum
of the protein dissolved in D2O at pH 3.6. The intensities
of the cross-peaks of the individual amide protons of the native
protein dissolved in D2O at pH 3.6 are assumed as 100%.
The time courses of change in proton occupancies were fitted to a
single exponential decay (y = A
exp
kt + C, where A is the amplitude of
the phase, k is the apparent rate constant, and C
is the final amplitude) by the Levenberg-Marquardt nonlinear least
squares method, yielding rate constants and phase amplitudes in the
kinetic folding experiment. All data analysis were performed using
Kaleidagraph software (Synergy Software).
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RESULTS |
Equilibrium Unfolding--
Equilibrium unfolding as a function of
GdnHCl concentration was monitored by far and near UV CD spectroscopy.
CTX III is an all
-sheet protein lacking in tryptophan residues.
Hence, the ellipticity changes at various concentrations of GdnHCl were
examined at 213 nm (far UV region) and 270 nm (near UV region). It was ensured that the protein is completely unfolded at 6 M
GdnHCl. The ellipticity changes can be fitted to a two-state model
(native
unfolded) for unfolding (Fig.
2). Fig. 2 shows the equilibrium denaturation curve obtained for CTX III at pH 6.8, 25 °C, using GdnHCl as the denaturant. In both far UV (213 nm) and near UV (270 nm),
CD gave nearly superimposable denaturation curves, and therefore the
unfolding process of CTX III has been assumed to fit to a two-state
model (data not shown). The free energy of unfolding in the absence of
denaturant was obtained by linear extrapolation of
G(D)
to zero denaturant concentration. The values obtained for
GU(H2O) from the GdnHCl
denaturation profile monitored at 213 and 270 nm were similar within
experimental error. The mG value, which is a
measure of the cooperativity of the unfolding reaction, was found to be
0.67 kcal·mol
1·M
1. The
concentration of denaturant (Cm) at which the
protein (CTX III) is half-unfolded (when
GU = 0) is estimated to be 4.75 M, by monitoring the changes in
ellipticity at 213 nm. The renaturation of CTX III from the 6.0 M GdnHCl obtained by monitoring the ellipticity changes at
213 nm is depicted in the inset of Fig. 2. It can be deduced
from the refolding curve that the unfolding CTX III by GdnHCl is
perfectly reversible (Fig. 2, inset), and this aspect helps
us to understand the molecular events in the folding pathway of the
protein (CTX III).

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Fig. 2.
GdnHCl-induced denaturation of CTX III at pH
6.8. Denaturation was monitored by measurement of mean residue
ellipticity at 213 nm. The inset depicts the renaturation
profile of CTX III denatured by 6 M GdnHCl using the 213-nm
mean residue ellipticity.
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Refolding Kinetics Monitored by Optical Stopped-flow
Measurements--
The refolding kinetics of CTX III was monitored by
using stopped-flow fluorescence and stopped-flow CD techniques. The
lack of tryptophan in the protein necessitated the use of ANS as an extrinsic probe to monitor the folding kinetics by stopped-flow fluorescence. When ANS is present during refolding, it is found to bind
strongly to the protein within 8 ms (Fig.
3), which is close to the dead time of
mixing in the stopped-flow instrument. As a consequence, the rise in
ANS emission cannot be monitored accurately. As the folding of protein
proceeded, the emission intensity of ANS increased in the burst phase
and then decreased steadily in the slow phase (Fig. 3,
inset). The decrease in the ANS fluorescence signal is
triphasic with time constants of
1 = 50.1 ± 7.0 ms,
2 = 291.5 ± 4.0 ms, and
3 = 5.5 ± 1.8 s. The kinetic curve reveals that most of the
changes in the ANS fluorescence are complete within 200 ms (Fig.
3).

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Fig. 3.
Stopped-flow kinetics of refolding of CTX III
monitored by ANS fluorescence. The final protein concentration was
160 µM. The fluorescence ( ex = 418 nm,
em = 497 nm) was monitored for 1.5 s. Nonlinear
least squares fits of the data give values of 1 = 50.1 ± 7.0 ms, A1 = 28.7 ± 3.0;
2 = 291.5 ± 40.0 ms, A2 = 37.4 ± 2.0; 3 = 5.5 ± 1.8 s, and
A3 = 29.7 ± 7.5. The inset
clearly depicts the hydrophobic collapse that occurs in the burst phase
of folding.
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Stopped-flow CD experiments were carried out to complement the
fluorescence data by investigating the formation of secondary structure
(far UV CD) and tertiary structure (near UV CD) during the folding
process of CTX III. The kinetics of refolding measured from ellipticity
change(s) at 270 nm arising from the tyrosine residues are shown in
Fig. 4A. This figure shows
that most of the changes are over in the first phase of refolding,
which occurs within 100 ms. The 270-nm ellipticity changes have been
fitted to a three-exponential decay function. An initial phase starting at 12.3 ms rapidly decays within 20 ms. To overcome the technical problems of curve fitting, we have ignored fitting the initial phase.
The ellipticity changes occurring at higher refolding times have been
fitted by shifting the zero time point to 20 ms. The remainder of the
CD signal evolves within about 200 ms. The refolding kinetics of CTX
III monitored in the far UV region at 213 nm (Fig. 4B)
presents several interesting features. In the time range (20 ms) just
above the dead time of the stopped-flow CD instrument, the ellipticity
value at 213 nm decreased significantly (Fig. 4B). During
this phase, the changes in the ellipticity at 270 nm (Fig.
4A) are minimal, are similar to the ellipticity values during this time period, and are similar to those obtained for completely unfolded CTX III at this wavelength (270 nm). After the
burst phase decrease, the ellipticity value at 213 nm increased slowly
to attain the value of the native protein. Such an "overshoot" behavior as shown in Fig. 4B has been reported in several
other proteins (17) and has been attributed to the formation of nascent nonnative helix formation during the burst phase of folding of proteins
(17, 18).

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Fig. 4.
Stopped-flow kinetics of refolding of CTX III
monitored by circular dichroism at 270 nm (A) and 213 nm
(B). Nonlinear least squares fits of the data obtained
at 270 nm yield values of 1 = 48.7 ± 0.8 ms,
A1 = 29.0 ± 1; 2 = 592 ± 234 ms, A2 = 33 ± 7.5;
3 = 5.0 ± 0.12 s, and
A3 = 37.0 ± 7.5.
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The Anomalous CD Overshoot Phenomenon--
To examine if the
overshoot in CTX III is due to the formation of nascent, nonnative
helix formation, we carried out refolding experiments in a helicogenic
solvent such as 2,2,2-trifluoroethanol (TFE). CTX III is an all
-sheet protein, and its three-dimensional solution structure reveals
that no portion of the polypeptide chain of CTX III exists in helical
conformation. Recently, it has been demonstrated that TFE
nonspecifically induces helix conformation in CTX III (13) at higher
concentrations (
90%, v/v). It can be argued that if the CD overshoot
phenomenon (as observed in CTX III) were due to the formation of
nonnative helix, then refolding CTX III in TFE-water mixtures at
concentrations less than 90% (v/v) would be expected to cause the
intensity of the CD overshoot to increase due to the stabilization of
the nascent, "non-native" helix. From the curves depicting the
changes in the 222-nm ellipticity upon refolding CTX III in the buffer
alone and in the buffer containing various TFE concentrations (Fig.
5), It is found that the CD overshoot signal in the burst phase of refolding does not show the expected increase in the ellipticity at 222 nm when the refolding of the protein
is carried out separately in the refolding buffer containing 10 and
50% (v/v) concentrations of TFE (Fig. 5). Based on these results, we
believe that the CD overshoot signal observed in CTX III may not be
linked to the formation of transient, nonnative helical conformation.
Chaffotte et al. (20), investigating the overshoot
phenomenon in the early stages of refolding of hen egg white lysozyme
reported that the "burst" of ellipticity (in the far UV CD region)
that occurs in the dead time of the experiments does not reflect
significant changes in the secondary structure of the protein. They
believe that the CD overshoot essentially reflects changes in the state
of side chains, with an important contribution arising from disulfide
bonds (21). Interestingly, Varley et al. (22), studying the
kinetics of folding of interleukin-1
, which is an all
-sheet
protein but lacks disulfide bridges, did not observe the overshoot
phenomenon. Similarly, in proteins such as cytochrome C, ubiquitin, and
staphylococcal nuclease, which are also characterized by the lack of
disulfide bridges, the anomalous far UV CD ellipticity overshoot
phenomenon is not observed (20). It appears that there exists a strong
correlation between the overshoot of CD ellipticity in the burst phase
of refolding and the presence of disulfide bonds in the native state of
the protein. In conclusion, the results presented thus far clearly
indicate that the CD overshoot phenomenon is in some way related to the attainment of asymmetry of the disulfide bonds during the process of
protein folding. To understand the contribution of the disulfide bonds
to far UV CD overshoot phenomenon observed during the refolding of CTX
III, we studied the "refolding" of S-carboxymethylated CTX III. There were no significant change(s) in the ellipticity (at 213 nm) values upon refolding. Importantly, the overshoot in the 213-nm
ellipticity is not observed when the S-carboxymethylated CTX
III sample dissolved in 6 M GdnHCl is diluted (refolded) 10 times with the refolding buffer. Thus, the results of these experiments unambiguously demonstrate that the far UV CD overshoot phenomenon observed during the refolding of CTX III is significantly linked with
the asymmetrization of the disulfide bridge(s) in the protein. It is of
interest to note that the CD overshoot phenomenon is not observed when
CTX III treated with 2 M GdnHCl (at this concentration of
GdnHCl, the structure of CTX III is unperturbed) is refolded with the
refolding buffer, indicating that the overshoot in the CD signal is
connected with the reorganization of tertiary structural interactions
during the refolding of CTX III (data not shown). The 270-nm tyrosine
ellipticity evolves completely only after 50 ms (Fig. 4A),
and hence the possibility of the far UV CD overshoot being associated
with the attainment of the asymmetric environment of the optically
active aromatic residues is remote. However, at the present juncture,
we cannot totally exclude this possibility. In addition, our results
show that, at least in the refolding of CTX III, no transient,
nonnative helix conformation(s) is formed during the early stages of
protein folding.

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Fig. 5.
Refolding of CTX III monitored by
stopped-flow circular dichroism at 222 nm in various concentrations of
TFE. Refolding of CTX III in the presence of even 50% TFE (v/v)
did not result in any significant increase in the intensity of the CD
signal (at 222 nm) during the burst phase of refolding.
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Kinetics of Amide Proton Protection--
Since the amide protons
in CTX III have been assigned in the two-dimensional COSY NMR spectrum
(14, 15), it is possible to unambiguously follow the kinetic protection
for 43 separate amide residues involved in secondary and tertiary
structural interactions (Fig. 6). These
amide protons are distributed throughout the molecule. All the residues
were adequately described by a single-exponential decay (Fig.
7). The time constants
(the inverse
of rate constant, k) of refolding of the various residues
are listed in Table I. If we arbitrarily
subdivide the time constants in Table I into two classes, fast (
< 20 ms) and slow (
> 40 ms), then the residues in the fast class
appear to be primarily associated with the hydrophobic core of the
molecule, whereas those in the slow class are mostly located on the
periphery of the folded protein structure. Comparison of the time
constants of the various residues listed in Table I suggests that there
could be initial rapid hydrophobic collapse occurring during the
refolding of the protein. Interestingly, the time constants of
Lys23, Ile39, Val49,
Tyr51, Val52, Cys53,
Asp57, and Arg58 are less than 20 ms (Table I).
In the native state of the protein, these residues are distributed in
both the structured and unstructured regions of the CTX III molecule.
The small refolding time constant of Lys2 (
= 21.0 ms)
is worth mentioning. The solution structure of CTX III (12, 15) reveals
that the amide proton of Lys2 is hydrogen bonded to the
carbonyl group contributed by Arg58. This interaction
tethers the N- and C-terminal ends of the CTX III molecule together.
Comparing the time constants of folding of Lys2 with those
of the residues involved in the initial "hydrophobic collapse," it
appears that the clustering of hydrophobic residues in the early stages
of folding (<20 ms) helps the N- and C-terminal ends of the molecule
come to close proximity. This aspect favors a hydrogen bond formation
between Lys2 NH and Arg58 CO, which could
promote the formation of the native tertiary structural interactions in
the subsequent stages of folding. In the native state of the molecule,
the amide proton of Val49 is involved in hydrogen bonding
with the carbonyl group of Thr46, forming a type 1
-turn
(12, 15). The significantly small folding time constant for
Val49 (
= 5.5 ms) could be attributed to the very early
formation of the type 1
-turn in the backbone of the polypeptide
chain between Thr46 and Val49 (Table I).

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Fig. 6.
Magnitude COSY spectra of CTX III samples
prepared by quenched-flow hydrogen exchange methods at various pulse
labeling time periods. The labeled NH-C H cross
peaks represent residues that have undergone the amide deuterium-proton
exchange at various refolding time periods. The cross-peak labeled
as × originates from the protonated contaminant(s) in
glycine-d5 used in the refolding buffer.
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Fig. 7.
Comparison of the observed time courses of
amide proton occupancy of a selected residues as a function of the
length of the folding time. All residues were fit to a first order
exponential decay (for further details, see "Experimental
Procedures"). Panel A depicts the percentage of proton
occupancy of residues located in the secondary structure region.
Lys2 (K2) belongs to the double-stranded
-sheet segment. Cys21 (C21),
Ile39 (I39), and Val52
(V52) belong to the triple-stranded -sheet segment. The
percentage of proton occupancy for residues located in the unstructured
regions (Leu6 (L6), Lys18
(K18), Thr29 (T29), and
Val41 (V41)) are represented in panel
B. Since the changes in the proton occupancy are not completed
within the refolding time of 190.4 ms for the residues given in
panel B, a clear time-dependent asymptote
(C) could not obtained for the curves representing these
residues. Hence, we used the exponential function, y = Ae kt (by eliminating the asymptote
(C = 0)) to fit time course changes in the amide proton
occupancy of these residues (given in panel B)).
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It can be seen in Fig. 7 that the residues that occupy the relatively
unstructured portions of the molecule (such as Leu6,
Lys18, Thr29, and Val41) show high
refolding time. In addition, the rate of the deuterium-hydrogen exchange is slower in these residues. This indicates that these residues belong to the slow folding category. The local structures (wherein some of these slow folding residues are thought to be located)
in the native protein generally tend to form at longer refolding times
(8). In contrast to the slow folding residues, the residues involved in
the secondary structure formation in the native state of the protein
show proton occupancy in the range of 10-20% after 100-ms
refolding.
Comparison of the kinetics of the amides in the various
-strands
comprising the double and triple-stranded
-sheets in CTX III (Table
II) shows that there is a clear pattern
in the rate(s) of formation of the five
-strands. The average
folding time constants of the
-strands (Table II) in the protein are
as follows: strand I,
= 34.8 ms; strand II,
= 35.1 ms; strand
III,
= 23.6 ms; strand IV,
= 18.3 ms; and strand V,
= 17.2 ms. Formation of strand II and strand I comprising the double-stranded
-sheet segment appears to occur almost simultaneously. The average
time constant of residues in the double-stranded
-sheet is 35.0 ms (Table II). The triple-stranded
-sheet segment appears to fold faster than the double-stranded
-sheet segment. The average time constant of residues comprising the triple-stranded
-sheet segment is 19.7 ms. Among the
-strands (strands III, IV, and V) constituting the triple strand, residues in strand V fold rapidly with an average time constant of 17.2 ms (Table II). Strand IV (
18.3 ms)
appears to fold faster than strand III (23.6 ms). In summary, it
appears that the formation of the triple-stranded
-sheet domain
occurs before the double-stranded
-sheet segments, as shown in Fig. 8. It should be mentioned that the
difference(s) in the values of the time constants of folding of
residues in the structured and unstructured regions of the protein
molecule is not very distinct (Table I). However, considering the small
size of the protein and the extensive cross-linking of the molecule by
the occurrence of four disulfide bridges, the observed narrow
difference(s) in the time constants of refolding between the residues
involved in the secondary structure of the molecule and those of
residues in the unstructured portion(s) of the CTX molecule is quite
significant.

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Fig. 8.
Time courses for the protection of amides
from exchange of the residues involved double- and triple-stranded
-sheets during the refolding of CTX III. The curves
drawn represent the average of single exponential fits to the time
course for individual amides in the double and triple-stranded
-sheet segments. Nonlinear least square fits of the data give values
of 19.7 ms (filled circles) and 35.0 ms (open
circles) for the average time constants of folding of the triple-
and double-stranded -sheet domains, respectively. Time courses of
five and nine residues were averaged to trace the kinetic curves for
the double- and triple-stranded -sheets, respectively.
|
|
 |
DISCUSSION |
CTX III belongs to the class of three-finger proteins. The
solution structure of the protein shows that three loops emerge from
the globular head (14, 15). The secondary structure of the protein is
exclusively
-sheet (hence all
-sheet protein) with no portion of
the polypeptide chain in helical conformation (Fig. 1). The
antiparallel double- and triple-stranded
-sheet segments in CTX III
are stabilized by the four disulfide bridges. It is a highly stable
protein, and thermal denaturation studies have demonstrated that CTX
III melts only at temperatures greater than 90 °C (23). In addition
to the disulfide bridges, the protein is strongly stabilized by a core
of hydrophobic residues. It is widely believed that the hydrophobic
core bestows extraordinary structural stability to the protein (12).
Recently, interesting studies on the protein folding aspects of snake
venom cardiotoxins have been reported. Partially structured
intermediate states have been identified along the alcohol- (13) and
acid-induced (24) unfolding pathways of CTX III. In pursuit of our
ultimate goal of understanding the folding/unfolding pathway(s) of CTX
III, we have embarked on examining the kinetic events in the folding pathway(s) of this protein.
The Hydrophobic Collapse Detected by ANS Fluorescence--
ANS has
been proven to be a sensitive probe for monitoring the folding of
globular proteins (25). The intense ANS fluorescence signals observed
in the burst phase of folding are believed to be primarily due to the
binding of ANS to nonpolar surfaces, presumably in the hydrophobic
core, which in folding intermediates may be solvent-accessible. Such
binding is therefore commonly used to measure the compactness of the
bound intermediates (26). As described earlier, the burst phase of
refolding of CTX III is characterized by the presence of highly intense
ANS fluorescence (Fig. 3). We believe that the strong ANS fluorescence
signal observed during the burst phase is due to the binding of the
nonpolar dye to the hydrophobic clusters in the very early stages of
folding of CTX III. Interestingly, monitoring the 270-nm ellipticity, which signifies the asymmetric environment of the tyrosine residues in
the protein, shows that CTX III regains less than 10% of the final
ellipticity in native protein (at 270 nm) during the burst phase of
folding (Fig. 4A). These results reveal that the specific tertiary structural interactions pertaining to the aromatic
chromophores (tyrosines in this case) are not formed during the burst
phase of folding. It is important to mention that the near UV CD signal at 270 nm for CTX III primarily stems from the tyrosine residue located
at position 22. This residue contributes significantly to the
stabilization of the native state of the protein. In this context, it
would be of interest to note that Agashe et al. (26), examining the folding pathway of barstar, demonstrated that the protein
collapses (during the burst phase of refolding) to a compact globule
with a solvent-accessible hydrophobic core with no optically active
secondary or tertiary structure.
Folding Events Detected by Amide Proton Pulse Exchange--
It
would be of interest to trace the chronological hierarchy of the
formation of the various secondary structural segments in the refolding
pathway of CTX III. The fifth
-strand (strand V) spanning residues
50-55 is largely hydrophobic. This
-strand is a component of the
triple-stranded
-sheet segment in CTX III. It appears that this
portion of protein along with other hydrophobic residues located at the
C terminus forms the hydrophobic cluster that occurs in the burst phase
of the folding pathway. Based on the small folding time constant (Table
I), the residues such as Lys23, Ile39,
Val49, and Asp57 along with the residues
comprising strand V are believed to collapse into a hydrophobic
cluster. The folding time constants of these residues is significantly
small (
< 20 ms). The small folding time constant of
Lys2 (
= 21.0 ms) is interesting (Table I and Fig. 7).
As stated earlier under "Results," a hydrogen bond between the
amide proton of Lys2 and the carbonyl group of
Arg58 brings the N- and C-terminal ends of the backbone of
the CTX III molecule spatially close. It appears that the formation of the "hydrophobic cluster" in the burst phase (<20 ms) facilitates the formation of hydrogen bonding between Lys2 NH and
Arg58 CO. The formation of this crucial hydrogen bond
appears to initiate the subsequent events of folding in CTX III.
Interestingly, the hydrogen bonding partners (carbonyl groups) for the
amide groups of the residues in strand V are located in strand III of
triple-stranded
-sheet. However, the average folding time constant
for residues in strand III is 23.6 ms and is greater than that of the
residues in strand V (17.2 ms, Table II). This implies that strand V
could be formed before strand III. In the absence of the interstrand hydrogen bonding with strand III, the amide deuterium in strand V is
protected due to the formation of the hydrophobic cluster. Formation of
such a nonpolar cluster probably secludes these residues in strand V
from the solvent water, resulting in the observed slow exchange of
amide deuterium with the solvent protons (27). On the whole, it is
clear that the triple-stranded
-sheet segment forms early during the
refolding of CTX III. Based on the average time constants of residues
in the double- and triple-stranded
-sheet segments, it appears that
the triple-stranded
-sheet (
= 19.7 ms) is formed before the
double-stranded (
= 35.0 ms)
-sheet segment (Table II and Fig.
8). Partially structured intermediates have been identified and
structurally characterized along the alcohol- (23) and acid-induced
(24) unfolding pathways of CTX III. Interestingly, it is found that the
triple-stranded
-sheet segment is persistently found in the
intermediate states. To this extent, the results obtained in the
kinetics folding pathway of CTX III are consistent with those obtained
from equilibrium unfolding studies.
A Global View of the Folding Events in CTX III--
The kinetic
events in the refolding pathway of CTX III as monitored by stopped-flow
fluorescence clearly indicate the presence of a transient intermediate,
which is characterized by the intense ANS fluorescence signal in the
burst phase of folding. Intermediates with such spectral (intense ANS
signal) characteristics have been detected in the burst phase of
refolding of several proteins. The high affinity for ANS for these
intermediates is thought to stem from a hydrophobic clustering of
residues. Interestingly, the refolding kinetics followed by
quenched-flow deuterium-hydrogen exchange of the amide protons reveals
that the residues that possess refolding time constants less than 20 ms
(fast folding residues) are apolar residues concentrated in the
triple-stranded
-sheet segment and C-terminal loop. It appears that
clustering of the hydrophobic residues in this portion of the CTX III
molecules creates local structural geometry that is conducive for
strong ANS binding observed in the burst phase of folding. The far UV CD overshoot phenomenon observed in the burst phase (8.0 ms) of refolding of CTX III is found to be associated with the attainment of
asymmetry of the disulfide bond(s) in the protein. The head region of
the CTX III molecule, wherein most of the apolar residues involved in
the burst phase hydrophobic collapse are located, is extensively
cross-linked by disulfide bridges. It is possible that the regaining of
asymmetry of the disulfide bridge(s) in the protein could aid in the
clustering of the hydrophobic residues (observed in the burst phase of
folding) in the head region of the molecule. It appears that the
"collapsed" intermediate observed in the burst phase of folding
lacks most of the native tertiary structural interactions. This is
exemplified by the fact that only about 10% of the total expected
ellipticity at 270 nm is evolved in the burst phase of refolding (Fig.
4A). It is well known that the near UV CD signal (at 270 nm)
in CTX III is mostly due to Tyr22, and it appears that this
residue does not attain its asymmetric environment during the burst
phase of refolding of the protein. This inference is supported by the
high folding time constant (31 ms) obtained for Tyr22 using
the quenched-flow deuterium-hydrogen experiments.
A common 50-ms phase is observed during the refolding of CTX III using
the stopped-flow CD and fluorescence experiments. This phase is
predicted to represent the formation of most of the native secondary
and tertiary structural interactions in the protein. This formation is
signified by the fact that most of the residues (monitored by the
quenched-flow deuterium-hydrogen exchange experiments) involved in the
secondary structural interactions show folding time constants in the
range of 30-50 ms. The slower phases observed probably represent the
formation of local tertiary structural interactions and
cis-trans proline isomerization.
Comparison with Folding Kinetics of Interleukin-1
--
The
folding pathway of interleukin-1
, another all
-sheet protein, has
recently been studied using a variety of spectroscopic techniques
including quenched-flow deuterium-hydrogen exchange experiments (22).
It is quite informative to compare these results with those obtained in
the present study on CTX III. In interleukin-1
, the complete folding
of the protein occurs on a time scale greater than 25 s (22). The
slow folding rate of interleukin-1
is in complete contrast to that
observed for CTX III, wherein the folding is complete within 200 ms.
The difference in the folding rates could be due to the difference in
the molecular mass of two proteins. CTX III is 6.5 kDa, whereas
interleukin-1
is about 85 kDa. CTX III is cross-linked by four
disulfide bridges located between residues 3 and 21, 14 and 38, 42 and
53, and 54 and 59. These disulfide bonds have been shown to be crucial
for the maintaining the structural integrity of CTX III. The reduction
of the disulfide bridges invariably results in the complete unfolding
of the protein (CTX III). The solution structure of CTX III shows that
the disulfide bonds in the protein stabilize the
-sheet element in
the protein. The residues that are contemplated to be involved in the
initial hydrophobic collapse are mostly located in the head portion of the molecule. This region is extensively cross-linked by disulfide bridges, and this cross-linking, we believe, could facilitate the
clustering of hydrophobic residues observed in the early stages of
folding of CTX III. The absence of disulfide bridges in
interleukin-1
, on the other hand, could be responsible for its
slower refolding rate. Interestingly, just as in CTX III, the folding
pathway of interleukin-1
is characterized by the formation of
intermediate state(s) (in the burst phase of folding) that exhibit high
affinity for binding to ANS (22). One important difference between the folding kinetics of CTX III and interleukin-1
is the rate at which
the
-sheet conformational elements are formed. The formation of
stable native secondary structure in interleukin-1
, as measured by
quenched-flow deuterium-hydrogen exchange experiments begins only after
1 s (22). Thus, the comparison of these two all
-sheet proteins
shows that rates of protein folding do not only depend on the type of
secondary structural conformation in the protein(s) but also strongly
depend on other local or tertiary structural interactions of the
protein in context.
Significance of the Hydrophobic Collapse--
It is important to
understand the significance of the hydrophobic collapse that occurs in
the burst phase of refolding of several proteins including CTX III.
Gutin et al. (28), examining the folding of proteins using a
simple lattice model, showed that, depending on the interaction among
residues, two different regions of folding are possible. A two-stage
kinetics is observed when strong attraction among residues dominates in
the folding process. The first stage is fast hydrophobic collapse into
a compact nonnative globule, and the second stage is a slow search for
the native conformation among these compact globules. They find that
folding into the native state from the compact intermediate is an
all-or-none transition. Gutin et al. (28) observed that
native structure formation and compaction proceeded simultaneously from
the random coil as an all-or-none transition when there was repulsion
among residues. In real proteins, the interaction
(attraction/repulsion) strongly depends on the physical conditions
(concentration of denaturants, pH, and temperature) used during
folding. In addition, the disulfide bonds and hydrophobic residues in
the protein tend to increase the average attraction among the residues
and consequently favor the compaction of the molecule during refolding.
CTX III is a highly hydrophobic molecule extensively cross-linked by
four disulfide bridges. These features of the protein probably enable the polypeptide chain to undergo compaction during refolding prior to
the assumption of the native conformation.
Camacho and Thirumalai (29) recently proposed that folding proceeds by
a stagewise decrease of the number of available conformations. They
suggest that at the first stage of folding, the polypeptide chain
undergoes overall compaction and subsequently falls into one of the
compact energy-minimized conformation. The native conformation is
realized later by a search over a small number of energy-minimized compact conformation(s). However, several kinetics studies of random
systems (30) have indicated that these minimum energy misfolded
("glassy") states represent deep traps and that escaping is
extremely slow. Thus, it is believed that compaction itself may not
increase the folding rate because it does not decrease the energy
barriers en route to the native conformation (29, 30). Compaction is
contemplated to slow down protein folding by decreasing the number of
conformations available and, therefore, prohibits some conformations
that form pathways with lower barriers (28).
Does Hydrophobic Collapse Precede the Formation of Secondary
Structure?--
It is worthwhile to address the question of whether
hydrophobic collapse precedes secondary structure formation or
vice versa. Contrasting reports exist in the literature on
this question. Studies with isolated peptide fragments reveal that the
secondary structure is only marginally stable in the absence of
stabilizing tertiary structural interactions (31), which indicates that the formation of collapsed state(s) requires prior formation of secondary structure. On the contrary, Agashe et al. (26)
demonstrated that the hydrophobic collapsed state exists in barstar,
which lacks secondary structural interactions, suggesting that
collapsed states in kinetic or equilibrium intermediates can exist even in the absence of secondary structure.
The results of the present study on CTX III indicate that the
hydrophobic cluster is formed in the very early stages of the folding
process. The formation of this hydrophobic cluster is primarily due to
the coalescence of nonpolar residues located in strand V of the
triple-stranded
-sheet segment. However, based on the results
obtained in the present study, it is difficult to predict the
hydrophobic clustering of residues. Since the residues comprising the
hydrophobic cluster also form a part of the secondary structural
element (strand V), the clustering hints at the possibility of the
event of the hydrophobic collapse and the formation of secondary
structure occurring simultaneously. However, there are obvious
limitations to this conclusion, because there are residues that are
involved in the secondary structure formation but fold on a slower time
scale than residues involved in the hydrophobic collapse. We believe a
detailed submillisecond kinetic study is required to obtain a
definitive answer to this question (32). Recently, CTX III has been
cloned and expressed in high yields (19), and detailed folding studies
with appropriate mutants could provide useful clues to understand the
structural interactions that come into play during the early events of
folding of CTX III.
We sincerely thank the anonymous referees for
useful comments on an earlier version of the manuscript. We thank the
Regional Instrumentation Center at Hsinchu (Taiwan) for use of the
600-MHz NMR facility.