From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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The productive folding pathway of
cytochrome c passes through an obligatory HW intermediate
in which the heme is coordinated by a solvent water molecule and a
native ligand, His-18, prior to the formation of the folded HM state
with both the native His-18 and Met-80 heme coordination. Two off
pathway intermediates, a five-coordinated state (5C) and a
bis-histidine state (HH), were also identified during the folding
reaction. In the present work, the thermodynamics and the kinetics of
the unfolding reaction of cytochrome c were
investigated with resonance Raman scattering, tryptophan fluorescence
spectroscopy, and circular dichroism. The objective of these
experiments was to determine if the protein opens up and diverges into
the differing heme ligation states through a many pathway mechanism or
if it passes through intermediate states analogous to those observed
during the folding reaction. Equilibrium unfolding results indicate
that, in contrast to 5C, the stability of HH with respect to HW
decreases as the concentration of GdnHCl increases. The difference in
their response to the denaturant indicates that the polypeptide
structure of 5C is relatively loose as compared with HH in which the
polypeptide is misfolded. Time-resolved resonance Raman measurements
show that strikingly similar ligand exchange reactions occur during
unfolding as were observed during folding. Combined with fluorescence
data, a kinetic model is proposed in which local structural
rearrangements controlled by heme ligand exchange reactions appear
prior to the global relaxation of the polypeptide chain.
To perform its biological function, a nascent polypeptide produced
at the ribosome needs to fold to its native structure accurately and
efficiently. The "folding codon" encoded in its amino acid sequence
and the mechanism by which the polypeptide folds to its native
structure remain central problems in biology (1-7). Recent advances
have shed light on the nucleation condensation model in which the
growth of the polypeptide architecture of a small single domain protein
starts in a particular region of the molecule, the folding nucleus, and
subsequently propagates throughout the rest of the molecule until the
native conformation is reached (3, 8, 9). The reversible folding often
appears as two-state cooperative processes in which only the totally
unstructured unfolded state
(U)1 and the well ordered
folded state (F) are populated. The folding transition state (
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
)
relies on the formation of the folding nuclei (Equation 1) and may be
determined by site-directed mutagenesis along with kinetic analysis
(8).
The folding of larger proteins, on the other hand, is much more
complex. The polypeptide commonly passes through various intermediates
(Ii) prior to reaching the unique native structure (Equation 2).
(Eq. 1)
In addition, the productive folding pathway sometimes needs to
compete with nonproductive off pathway reactions.
(Eq. 2)
The off pathway intermediates (Di) are
dead-end products that may form deep kinetic traps during folding and
have been reported to be linked to human pathogenesis (10, 11). To
unify the kinetic studies from a large variety of proteins, a "new
view" of protein folding has been postulated in which unique folding
channels are replaced by a statistical distribution of many pathways
(4, 12-14). Although there is little direct experimental evidence for
the statistical nature of the folding reactions, a diversity of
unfolding pathways has been postulated for chymotrypsin inhibitor 2 by
molecular dynamics simulations (13).
(Eq. 3)
In contrast to the folding reactions, the unfolding transition in single domain proteins usually appears to be a cooperative two-state process regardless of the size of the protein. It is generally considered as a "melting" of the protein crystal, because the closed packed interior of a protein resembles the ordered structure of molecular crystals. In this report, we use cytochrome c (cyt c) as a model to unravel the statistical nature of the unfolding reaction. Cyt c is a convenient model for folding and unfolding studies, because the native structure is well known and the folding kinetics can be followed by various spectroscopic techniques (15-20). In addition, the protein contains a heme prosthetic group that can be used as a probe for the local structure of the protein during the folding reaction (21). We have demonstrated in previous reports that the folding reaction of cyt c follows a two-step mechanism (15, 21-23). In the nascent phase of folding, cyt c collapses from a random coil-like structure to a semi-compact form that involves little or none of the specific tertiary interactions present in the native structure. This collapsing process is kinetically controlled and thus happens very fast, in less than 100 µs (21, 24, 25). The size of the conformational space is dramatically reduced during this phase. It facilitates the searching of the native structure in the subsequent phase, which is a thermodynamically controlled heme ligand exchange phase that is regulated by random structural fluctuations biased to the native polypeptide fold. It is at this stage when the specific native contacts are established (15).
The productive folding pathway of cyt c, as described in
Scheme 1, starts from the totally
unstructured polypeptide state, U, and passes through an intermediate,
HW, in which His-18 and a water molecule are coordinated to the heme,
and then culminates in the native His-18-Met-80 heme-coordinated form
(HM).
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(Eq. 4) |
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MATERIALS AND METHODS |
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Horse heart ferricytochrome c (type IV from Sigma) was treated with ferricyanide to oxidize the residual ferrous form and purified on a Sephadex G-25M column. For the equilibrium studies, the protein was diluted into pH 3.0 or 5.9 acetate buffer (0.1 M) at the desired concentration of guanidine hydrochloride (GdnHCl, ultra pure grade from Pierce) a few hours prior to the experiments. The final protein concentration was 20 µM. The fluorescence measurements were recorded with a SLM Aminco fluorometer (Urbana, IL). The excitation wavelength was 295 nm, and both slits were set to 4 nm. The CD measurements were performed on a JASCO model J720 spectrometer (Easton, MD).
For the kinetic studies, the unfolding of cyt c was initiated by a 6-fold dilution of the protein with 4.8 M GdnHCl in 0.1 M acetate buffer solution in a rapid solution mixer with a mixing dead time of ~100 µs (21). The final protein concentration was 10 µM. The two parent solutions were pressurized by a syringe pump and introduced into the T-shaped mixing nozzle, which is in direct contact with a quartz flow cell (250 × 250 µm). The details of the characterization of this mixer were described previously (21). The time-resolved resonance Raman and fluorescence spectra were obtained by moving the quartz cell relative to the laser focusing point along the flow direction. The position of the flow cell was controlled by a micrometer on a translational stage, such that an accurate time domain could be calculated from the solution flow rate.
The Raman measurements were made with previously described instrumentation (21). The output at 407 nm from a krypton ion laser (Spectra Physics) was focused to an ~30 µm spot (power ~100 milliwatts) on the continuously flowing sample in the flow cell. The scattered light was collected and focused on the entrance slit of a 1.25-m polychromator (Spex) where it was dispersed and then detected by a charge-coupled device camera (Princeton Instruments). The Raman spectrum at each time point was deconvoluted into contributions from HH, HW, 5C, and HM based on the procedure published elsewhere (21).
For the tryptophan fluorescence measurements, the output at 264 nm from
a frequency-doubled argon ion laser (Coherent) was focused to an ~30
µm spot on the continuously flowing sample in the observation cell.
The fluorescence was collected and focused on the entrance slit of a
0.27-m polychromator (Spex) where it was dispersed and then detected by
a charge-coupled device camera (Photometrics).
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RESULTS AND DISCUSSION |
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Equilibrium Unfolding Studies--
GdnHCl-induced unfolding of cyt
c was monitored by far UV CD and tryptophan fluorescence
spectroscopy as a function of denaturant concentration. Far UV CD
monitors the -helix content of the protein (30). The tryptophan
fluorescence (31, 32), on the other hand, probes the average distance
between the fluorescent chromophore, Typ-59, and the intrinsic
quencher, the heme prosthetic group, which is covalently linked to
Cys-14 and Cys-17 through thioether linkages. At pH 5.9, the
equilibrium titration curve from CD measurements coincides well with
that from the fluorescence measurements with a midpoint of
approximately 2.5 M as shown in Fig.
1A.
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To gain more structural information, the equilibrium unfolding curves were investigated with resonance Raman scattering. The native HM form converts to HH and HW as the concentration of GdnHCl increases with a preference of HH over HW (Fig. 1B). Based on the populations of these three species, a representation of their free energy changes relative to HW as a function of the GdnHCl concentration was constructed and is shown in Fig. 1C. In the absence of GdnHCl, the relative stabilities of the intermediates are HM > HH > HW (22). In the presence of 2.4 M GdnHCl, the population of HM is equal to that of HH (Fig. 1B), suggesting that the free energies of HM and HH are identical. Furthermore, the energy level of HW lies above that of HM and HH, because the population of HW is less than that of HM and HH (Fig. 1B). Likewise, the free energies of HM and HW are identical at 2.7 M GdnHCl and that of HH lies below those of HM and HW. It is concluded from Fig. 1C that both the native state, HM, and the off pathway intermediate, HH, are destabilized by GdnHCl with respect to HW. This result is consistent with the observation that the presence of GdnHCl in the refolding solution destabilizes HH and assists the conversion of HH back to the obligatory intermediate HW during the folding reaction (23).
It is intriguing that the transition curve from the resonance Raman
scattering measurements occurs slightly earlier (lower GdnHCl
concentration) as compared with those from the CD and fluorescence measurements as shown in Fig. 1A. This is most evident in
the pre-transition region, where at 2 M GdnHCl the
population of HM determined from the resonance Raman measurements has
dropped by nearly 20% in contrast to no reduction of the native
-helical content and 8% reduction of the molecular size based on
the CD and fluorescence measurements, respectively. The difference
between these physical measurements suggests that under low GdnHCl
concentrations, the polypeptide backbone structure changes, associated
with the changes in heme ligation states, are localized and the
thermodynamic intermediates are compact with most of the native
secondary structure preserved. Similar conclusions were drawn from
equilibrium hydrogen exchange experiments (29). The merger of the HM
curve with those from CD and fluorescence measurements in the
post-transition region implies that the residual secondary and tertiary
structure in HH and HW is eventually lost in the presence of high
concentrations of denaturant.
At pH 3.0, the denaturation transition curves measured by CD and
tryptophan fluorescence appear much broader than that of pH 5.9 with
the transition midpoint shifted from 2.5 to 2.0 M (Fig.
2A). It is plausible that the
positive charges distributed over the polypeptide chain resulting from
the protonation of certain amino acid residues introduce electrostatic
repulsion between polypeptide segments. They destabilize the molecule
and loosen the structure. In contrast to the cooperative nature of the
sharp transition observed at pH 5.9, the perturbation induced by GdnHCl in one part of the molecule is not readily transmitted to other parts
of the molecule. This results in a broader transition with a midpoint
shifted to a lower GdnHCl concentration as shown in Fig.
2A.
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The change of the ligation states in response to the GdnHCl
concentration is much more complex under acidic conditions than that at
the higher pH as shown in Fig. 2B. The population of HM from
the resonance Raman measurements deviates substantially from the CD and
fluorescence titration curves in the pre-transition region. Only one
unfolding intermediate, HW, was detected at a GdnHCl concentration of
1.5 M or less. The conversion from HM to HW is extremely
efficient; approximately 20% of HM is converted to HW in the presence
of only 0.1 M GdnHCl. The sensitivity of the
iron-methionine bond to a small amount of GdnHCl cannot be attributed
to a direct weakening of this bond under acid conditions. We postulate
that the perturbation in the -loop structure, which holds Met-80 in
a favorable position for ligation, introduced by protonation
destabilizes the iron-methionine bond.
At concentrations above 1.5 M GdnHCl, where the population of HW reaches a maximum, another folding intermediate, 5C, becomes visible at the expense of HW. The population of 5C levels off at 2.5 M GdnHCl. As the GdnHCl concentration increases from 2.5 to 4.0 M, the ratio of HW/5C stays constant, whereas the amount of secondary structure and the compactness of the protein continue to decline (Fig. 2A). This suggests that the structures of both HW and 5C are loosened steadily by the addition of denaturant. The polypeptide structures associated with HW and 5C are not totally unfolded until the concentration of GdnHCl is greater than 4.0 M at which point more conversion from HW to 5C is observed.
Based on the populations of HM, HW and 5C shown in Fig. 2B, a pictorial illustration of their free energy changes relative to HW as a function of the GdnHCl concentration, was constructed (Fig. 2C). In the absence of GdnHCl, HM is more stable than HW, and HW is more stable than 5C (23). In the presence of 1.2 M GdnHCl, the population of HM is equal to that of HW (Fig. 2B), suggesting that the free energies of HM and HW are identical. In addition, the energy level of 5C lies above that of HM and HW, because the population of 5C is less than that of HM and HW. Analogously, the free energies of HM and 5C are identical in the presence of 2.0 M GdnHCl and that of HW lies below those of HM and 5C. The free energies of HW and 5C are identical in the presence of 2.3 M GdnHCl, and that of HM lies above those of HW and 5C. It can be concluded from Fig. 2C that in contrast to the native HM state, 5C is stabilized by GdnHCl with respect to HW.
Although both HH and 5C are off pathway intermediates, their response to denaturant is dramatically different as demonstrated in Figs. 1C and 2C. This difference can be attributed to the nature of the peptide backbone conformations associated with the two species. HH is a misligated state with the native His-18 and non-native His-33 as the two axial ligands. His-26 is also a potential heme ligand, but it was shown recently that His-33 is the primary ligand in the misligated state (33). The coordination of His-33 to the heme stitches the wrong parts of the peptide fragments together and traps the protein in a misfolded structure. It has been demonstrated that small amounts of GdnHCl accelerate the conversion of HH to the productive folding intermediate, HW (23). This is consistent with the destabilization effect of GdnHCl on HH reported here. On the other hand, the peptide backbone structure of 5C is rather floppy as compared with that of HH, because the intramolecular cross-linking between both His-18 and Met-80 through the heme is broken. The 5C intermediate is more stable in the presence of higher GdnHCl concentrations.
Unfolding Kinetics--
Fig. 3 shows
the unfolding kinetics of cyt c at pH 3.8 and 5.8 monitored
by resonance Raman scattering. The time-dependent populations of the various folding intermediates in these two reactions
are strikingly similar to those observed in the folding reactions. At
pH 3.8, HM steadily converts to HW with a half-life of just under a
millisecond (Fig. 3A). HW reaches a maximum (~80%) at 5 ms and decays to ~60% at 40 ms. 5C appears at about 1 ms and reaches
steady state at approximately 40 ms. The transient population of HW
suggests that HW may be the precursor of 5C. At pH 5.8, the unfolding
reaction is much slower than that at pH 3.8. HM gradually converts to
HH and HW (Fig. 3B). The reaction reaches steady state at
about 80 ms.
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To gain more insight into the unfolding mechanism, several kinetic
models were tested to account for the time-dependent data shown in Fig. 3. The model, which best fits the data, is illustrated in
Scheme 2, which surprisingly follows the
reverse sequence of the folding reaction (Scheme 1).
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The relaxation of the tertiary structure surrounding the heme during
the unfolding of cyt c at pH 5.8 (Fig. 3B) was
monitored by the decrease in the 397-cm1 line in the low
frequency Raman spectrum. It has been shown that the heme prosthetic
group in cyt c is highly distorted because of the local
environment of the native polypeptide fold (34, 35). This tension is
totally released as the polypeptide chain unfolds. The Raman line at
397 cm
1, assigned as a bending mode of the sulfur atom of
Cys-14/17, the
-carbon atom of the heme, and the atom bridging these
two (36), was established as a marker line for the formation of the
native tertiary structure of the heme pocket, because the heme is
covalently linked to the polypeptide backbone through these two
cysteine residues (22). Interestingly, the time-dependent changes of the 397-cm
1 line coincide well with the decay
of the native HM structure. This result suggests that the dissociation
of Met-80 from the heme is concerted with the disruption of the heme
pocket, the same type of behavior as has been observed during folding
(22).
The global structure changes associated with the ligand exchange reactions were investigated further with tryptophan fluorescence spectroscopy. In the native state, the fluorescence from Trp-59, the only tryptophan residue in cyt c, is totally quenched by the spatially nearby heme prosthetic group. The majority of the fluorescence intensity from Trp-59 can be recovered as cyt c unfolds because of the increase in the distance between Trp-59 and the heme that is anchored at residue positions 14 and 17. Tryptophan fluorescence therefore provides a qualitative estimate of the global size of the protein. The recovery of the fluorescence intensity during the unfolding reactions of cyt c at pH 3.8 and 5.8 agrees well with the disappearance of HM as shown in Fig. 3.
Several conclusions can be drawn from these experiments. First, the
fluorescence data shown in Fig. 3 suggest that the global molecular
size of cyt c is not completely relaxed until the ligand exchange reaction reaches steady state. In the presence of GdnHCl, at
concentrations of 4 M or more, the polypeptide chain of cyt c is totally unfolded and is free from conformational
constraints. The potential heme ligands (His-18 or His-33), covalently
linked to the polypeptide chain, can freely exchange with one another or a solvent water molecule with rates >10,000 s1
evident from the pH jump experiments (data not shown). These rates are
much faster than those observed for the unfolding reactions (Table I)
suggesting that the polypeptide backbone is in a semi-compact form
during the ligand exchange phase, such that the reactions are
constrained by residual tertiary structure just as that observed during
the folding of cyt c. Second, the Met-80 off rate
(k4) is much faster at low pH than that at high
pH (800 versus 70 s
1). This is because of
destabilization of the native polypeptide structure at low pH by the
electrostatic repulsion resulting from the protonation of certain amino
acid residues as discussed earlier. Third, the His-33 on rate
(k1) is 0 at pH 3.8 but 170 s
1 at
pH 5.8. This contrast is because of the lack of unprotonated His-33
available for heme coordination at low pH as demonstrated in the
folding reaction (22). On the other hand, the formation rate of 5C
(k5) is 0 at pH 5.8 because of the instability
of 5C at this pH. Lastly, analogous to the folding reaction, as may be
seen in Scheme 2, HW is an obligatory unfolding intermediate leading to
the formation of the HH and 5C forms.
Folding Kinetics--
For comparison, Fig.
4 shows the folding reactions of cyt
c under similar conditions as those applied in Fig. 3. The
solid lines are the fitted data with the model illustrated
in Scheme 1 with the rate constants listed in Table I. The His-18 on
rate (k6) and the His-33 on rate
(k1) are faster than those observed under
unfolding conditions because of the difference in the concentration of
denaturant in the solution. It is important to point out that although
the folding conditions are the same for both reactions shown in Fig. 4,
the overall folding rate slows down by more than an order of magnitude
when the pH of the unfolded protein is raised from 3.6 to 5.9. This is
a result of the kinetic trapping effect of the misfolded HH state,
which occupies more than 60% of the population during the nascent
phase of folding as shown in Fig. 4B. On the other hand, HH
is only transiently populated at approximately 10 ms following the
initiation of folding at pH 3.8. This comparison confirms the role of
HH as an off pathway intermediate and a deep kinetic trap.
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It is intriguing that in contrast to the reaction observed during unfolding, the folding kinetics measured by tryptophan fluorescence spectroscopy deviate significantly from those measured by the resonance Raman scattering as shown in Fig. 4. The loss of the fluorescence intensity appears much earlier than the formation of the native HM state. This is in good agreement with the model illustrated in Scheme 1, in which the protein collapsed to a semi-compact form during the nascent phase prior to the ligand exchange phase. The collapsing of the peptide backbone reduces the average size of the molecule before the ligand exchange equilibrium can take place; i.e. the native structure cannot be realized through ligand exchange reactions until the protein collapses to a semi-compact form. Conversely, the coincidence between the fluorescence and Raman data during unfolding (Fig. 3) suggests that the Met-80 heme linkage cannot be broken until the polypeptide architecture is loosened.
Concluding Remarks--
The folding of cyt c
follows a biphasic mechanism as illustrated in Scheme 1. In the nascent
phase of folding, the unfolded protein collapses to a semi-compact form
in a kinetically controlled process within 100 µs (15). It is
followed by a thermodynamically controlled heme ligand exchange phase
culminating in the native conformation (15). In this work, an analogous
biphasic mechanism with a reversed sequence was observed during
unfolding (Scheme 2). Unlike the fast ligand exchange rate
(>10,000 s1) for the totally unstructured polypeptide,
the ligand exchange rates observed during the unfolding reactions are
comparable to those observed during the folding reactions (Table I).
These results suggest that the ligand exchange phase occurring during unfolding takes place while the polypeptide chain is still in a
relatively compact state such that the conformations of the various
intermediates are partially constrained by the residual intramolecular
tertiary interactions. This proposal is confirmed by the results from
tryptophan fluorescence measurements, which indicate that only at the
termination of the ligand exchange phase does the protein expand fully
into a random coil-like unfolded state.
Based on this and prior studies (15, 21-23), the free energy changes
during the folding and unfolding reactions of cyt c can be
illustrated pictorially in Fig. 5. In
this cartoon, the free energy curves plotted against the global
molecular size under native (0.0 M GdnHCl) and denaturing
conditions (4.4 M GdnHCl) are shown as the dark blue
curve and the red curve, respectively. Under native
conditions, the energy of the unfolded protein (U') is higher than that
of the folded form (F) because of the exposure of the hydrophobic
residues to the solvent and the loss of the stabilization energy gained
from inter amino acid contacts (hydrogen bonding, electrostatic
interactions, hydrophobic-hydrophobic interactions, etc.). As the
unstructured polypeptide chain reduces its molecular size, the
associated free energy is reduced accordingly (dark blue
curve). It reaches an energy minimum when the polypeptide chain
assumes its native conformation. When the size of the polypeptide molecule is further reduced, the energy starts to climb because of the
repulsion imposed by steric interactions between the spatially neighboring amino acid residues. This free energy curve is analogous to
the attractive potential energy curve of a typical diatomic molecule,
in which the energy minimum of the potential curve is located at the
natural bond length of the given molecule. For simplicity, in this
energy diagram, the roughness of the potential energy surface is
ignored.
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On the other hand, under denaturing conditions, the unfolded polypeptide molecule (U) is more stable than its "folded" counterpart (F') because of the high entropy of the unstructured polypeptide backbone and the stabilization energy gained from denaturant-amino acid interactions. Unlike the unique folded state (F) observed under native conditions, there is no well defined folded state (F') under these conditions. As the size of the polypeptide chain is reduced, the energy increases because of the loss of entropy by the exclusion of the denaturant from the protein molecules. As the size of the molecule is further reduced, additional reduction of the stabilization energy is introduced by the repulsion between amino acid residues. The potential energy curve of cyt c under denaturing conditions therefore is analogous to the repulsive potential energy curve of a diatomic molecule.
When the unfolding reaction is initiated, shown as the green
arrow (1) in Fig. 5, the protein is rapidly promoted from the F to
the F' state. If the initiation of the reaction is fast enough with
respect to the conformational changes of the protein molecule, the
entire course of the unfolding reaction can be followed from the F' to
the U state (red curve). Likewise, the folding reaction from
the U' to the F state (dark blue curve) can be initiated in
a similar fashion indicated as the green arrow (2) in Fig. 5. The unfolding kinetics reported here demonstrate that the ligand exchange phase appears when the protein molecule is still in a semi-compact form, and it is followed by further relaxation of the
polypeptide chain into a random coil-like structure. In contrast, the
folding reaction follows a reversed biphasic process, in which molecular collapse occurs prior to the ligand exchange phase, again
located in the shaded area. One appealing feature in Fig. 5
is the presence of a crossing point between the dark blue
curve and the red curve, in which the energies of the
polypeptide molecules are the same in the absence and in the presence
of denaturant. In other words, the folding and refolding reactions must
share a common intermediate with a characteristic molecular size, which could exhibit a molten globule-like structure.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Research Grants GM-54806 and GM-54812.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 718-430-4234;
Fax: 718-430-4230; E-mail: SYEH{at}AECOM.YU.EDU.
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ABBREVIATIONS |
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The abbreviations used are: U, unfolded protein; F, folded protein; cyt c, cytochrome c; HW, cytochrome c with heme coordination by His-18 and a water molecule; HM, cytochrome c with heme coordination by His-18 and Met-80; 5C, five-coordinated cytochrome c with heme coordination by only a water molecule; HH, cytochrome c with heme coordination by bis-histidine; GdnHCl, guanidine hydrochloride; CD, circular dichroism.
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REFERENCES |
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