PROTEINS: Structure, Function, and Genetics 53:731–739 (2003)
Many Residues in Cytochrome c Populate Alternative States
Under Equilibrium Conditions
Michael P. Williamson*
Krebs Institute, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom
ABSTRACT
A curved temperature dependence
of an amide proton NMR chemical shift indicates
that it explores discrete alternative conformations
at least 1% of the time; that is, it accesses conformations that lie within 5 kcal/molⴚ1 of the ground state.
The simulations presented show how curvature varies with the nature of the alternative state, and are
compared to experimental results. From studies in
different denaturant concentrations, it is concluded
that at least 25% of residues in reduced horse cytochrome c, covering most of the protein, with the
exception of the center of the N- and C-terminal
helices, visit alternative states under equilibrium
conditions. The conformational ensemble of the protein therefore has high structural entropy. The
density of alternative states is particularly high
near the heme ligand Met80, which is of interest
because both redox change and the first identified
stage in unfolding are associated with change in
Met80 ligation. By combining theoretical and experimental approaches, it is concluded that the alternative states each comprise approximately five residues, have in general less structure than the native
state, and are accessed independently. They are
therefore locally unfolded structures. The locations
of the alternative states match what is known of the
global unfolding pathway of cytochrome c, suggesting that they may determine the pathway. Proteins
2003;53:731–739. © 2003 Wiley-Liss, Inc.
Key words: protein conformation; chemical shift;
NMR; unfolding; temperature dependence; guanidinium hydrochloride
INTRODUCTION
The typical view of a protein is as a static structure. Of
course, we know that, in reality, proteins are dynamic.
They have rapid, small-scale oscillations about the equilibrium structure, and they occasionally undergo larger scale
and less frequent deformations. From the energy barriers
to unfolding, one can calculate that proteins must also
undergo reasonably frequent global unfolding and refolding. There are a very small number of proteins (such as
hemagglutinin1) whose function requires them to have a
metastable conformation, different from the ground state.
However, it is far from clear whether most proteins can
access any other states that can be considered as defined
alternative states. To a large extent, this is because few
techniques allow us to probe such structures: Because of
©
2003 WILEY-LISS, INC.
the highly cooperative nature of protein folding, alternative partially folded states will almost always be populated
to very low levels.2 Even if proteins can populate higher
energy alternative structures, one would not expect to be
able to crystallize them in such states. NMR potentially
can and occasionally has observed alternative states.3– 6 In
recent years, there has been very extensive use of amide
exchange experiments, largely by NMR,7–13 which can
provide information on non-native states, but these tend to
be very high energy (and probably require extensive
structural rearrangement, to allow access of water to the
interior) and should be considered more as unfolding
intermediates rather than alternative structures.
A better understanding of alternative states would be
very useful, not least because most proteins have to be
flexible in order to function. We can therefore expect that
alternative states might provide an insight to function.14
They may also provide an insight into protein unfolding,
because they might identify parts of the ground state
structure that are of low stability and are hence most
likely to unfold first. We have recently developed a simple
technique, in which nonlinear amide proton temperature
dependence has been shown to provide information on
low-lying alternative states. In this article, I develop the
theory and provide the first detailed examination of a
protein using denaturant to probe higher energy alternative states. This methodology is based on “native-state”
hydrogen-exchange experiments, which also make use of
increasing denaturant concentrations.7–13 I have chosen to
work on horse cytochrome c, because its structure, function, and unfolding/refolding have been extensively investigated.10,15,16 I show that a large fraction of the protein
has access to low-lying alternative states that are characterized by local loss of structure over approximately five
amino acid residues. The alternative states are most dense
in the region known to unfold first, and least dense in the
region known to unfold last, which implies that the
unfolding pathway may start from the alternative states
identified here.
Grant sponsor: equipment grant from Wellcome Trust
*MPW is a member of the BBSRC-funded North of England
Structural Biology Centre (NESBIC).
Correspondence to: Mike P. Williamson, Dept. of Molecular Biology
and Biotechnology, University of Sheffield, Firth Court, Western
Bank, Sheffield S10 2TN, UK. E-mail: m.Williamson@sheffield.ac.uk
Received 3 January 2003; Accepted 4 March 2003
732
M. P. WILLIAMSON
where the enthalpy difference ⌬H and the entropy difference ⌬S are assumed to be temperature-independent. The
populations of the two states, p1 and p2, are then given by
THEORY
Theoretical Model for Curved Temperature
Dependence
The NMR chemical shift of an amide proton is most
strongly influenced by its hydrogen-bonding partner and
depends both on their relative distance and orientation.17,18 However, other functional groups also affect the
shift; consequently, for example, an amide proton in a
␤-sheet tends to resonate downfield from its random coil
position, whereas an amide proton in an ␣-helix tends to
resonate upfield from its random coil position.19 Nevertheless, the influence of the hydrogen-bonding partner is
great, implying that an increase in distance between H
and O, or an increased deviation from linearity of the
hydrogen bond, results in an upfield chemical shift change.
An increase in temperature results in an increase in the
thermal motion of the atoms in a hydrogen bond, and
therefore in general produces an increased mean separation and thus an upfield shift of the amide proton. The
change in separation is less when the hydrogen bond is
strong, implying that the gradient of the change in shift
with temperature provides a good measure of the presence,
and also the length and strength of an intramolecular
hydrogen bond.20 –22
Proteins expand in a linear manner with temperature.23,24 This linear expansion extends to hydrogen bonds,
which also increase in length proportional to the temperature increase.25 Cordier and Grzesiek25 have calculated
that hydrogen-bond lengths in ubiquitin increase by approximately 1.7 ⫻ 10⫺4/K, or approximately 1% over 60°.
With use of a simple r⫺3-based correlation with chemical
shift,17 this is calculated to give changes in chemical shift
of 1–2 ⫻ 10⫺3 ppm/K, which is approximately what is
observed for strongly hydrogen-bonded amides.21,22 The
change in chemical shift is calculated to be linear, to a good
approximation. In line with this, we21 and others20 have
shown that many amide protons do have a linear chemical
shift change with temperature. However, in a significant
fraction of cases, the temperature dependence is observed
to be curved. We have previously shown that the locations
of curved residues correlate well with known locations of
conformational exchange.6 In order to interpret the results
obtained on cytochrome c, I first present here a theoretical
justification of this observation.
We assume that two conformational states are accessible: state 1 (the ground state), with a linear chemical
shift temperature dependence given by
␦ 1 ⫽ ␦ 10 ⫹ g 1T
(1)
and a higher energy alternative state 2, characterized by
␦ 2 ⫽ ␦ 20 ⫹ g 2T ,
(2)
where g1 and g2 are the gradients of the temperature
dependence. We further assume that the two states are
separated by a free-energy difference ⌬G, given by
⌬G ⫽ ⌬H ⫺ T⌬S
(3)
p 2 /p 1 ⫽ exp(⫺⌬G/RT)
(4)
where R is the gas constant. This allows us to calculate the
temperature dependence of the chemical shift, which is
given simply by
␦ obs ⫽ ␦ 1p 1 ⫹ ␦ 2p 2
(5)
We have simulated the temperature dependence using
typical values for the above parameters. We hypothesize
that a common situation would be that in which the
alternative state has undergone a local structural rearrangement, and thus has a small number of amides
(between two and five) with different hydrogen-bonding
environments compared to the ground state, such that
they have different chemical shifts and/or temperature
coefficients. This could correspond, for example, to the
rearrangement of a loop, or the loss or addition of a helical
turn, and this model in turn produces simulated temperature dependence in agreement with experimental observations, as shown below.
The chemical shift of an HN in a hydrogen bond tends to
be downfield of its random coil position, except in ␣-helices,
where it is more often upfield. Thus, breaking a hydrogen
bond (accompanied by a change in conformation to a more
random coil–like structure) could result in either upfield or
downfield shifts, depending on the location of the hydrogen
bond, with the breaking of a hydrogen bond in a ␤-sheet
typically resulting in an upfield shift, and the breaking of a
hydrogen bond in an ␣-helix typically resulting in a
downfield shift. We therefore initially assume a groundstate shift of 8.5 ppm, with a typical hydrogen-bonded
temperature gradient of ⫺2 ppb/°,21 and an alternative
state shift of 8.0 ppm, with a typical non-hydrogen-bonded
gradient of ⫺7 ppb/°. We take the change in free energy per
hydrogen bond broken to be approximately 1 kcal/
mol.26 –28 The enthalpy of a hydrogen bond in water is
notoriously difficult to estimate, but is probably several
times greater than the free energy because of the wellknown enthalpy/entropy compensation effect.29,30 A reasonable guess can be taken from a study of Mulder et al.,31
who measured an excited state in the L99A mutant of T4
lysozyme, characterized by ⌬G ⫽ 2 kcal/mol, ⌬H ⫽ 7.1
kcal/mol, and T⌬S (25°C) ⫽ 5.1 kcal/mol. I have therefore
used these values as illustrative estimates.
The simulations [Fig. 1(a)] show that curvature is
expected, but that the amount of curvature decreases to
become insignificant above ⌬G ⫽ 5 kcal/mol, by which
point the population of the alternative state is less than
1%. We note that the free-energy limit of 5 kcal/mol is
almost independent of the model, because this limit is
related to the population of the nearest alternative state.
Because detection of curvature depends on shift change
multiplied by population [Eq. (5)], a very large chemical
shift change between states would allow the limit to be
raised slightly, and a small chemical shift difference would
force it to be lowered. However, the effect is unlikely to
733
ALTERNATIVE CONFORMATIONS IN CYTOCHROME c
lead to changes in the free-energy limit of more than 1
kcal/mol or so.
The amount of curvature is also decreased when the
difference in chemical shift between the two states is
reduced, the difference in temperature gradient is reduced, or the change in entropy is reduced without changing the overall free-energy difference [Fig. 1(b)], or when
additional alternative states with lower energy or gradient
differences are added to the ensemble [Fig. 1(c)]. All of the
curves discussed so far display convex curvature; concave
curvature is produced when the excited state has a lower
field shift than the ground state, as expected for many
␣-helical residues, as discussed above [Fig. 1(c), dashed
curve].
It is appropriate here to consider two other explanations
for the curved temperature dependence. The first is that
there is only a single folded state, with a linear temperature dependence, and that the curvature results from
chemical exchange of the amide proton with water. This is
unlikely for three reasons: (1) such a model would predict
only convex curvature (because the chemical shift of water
is always lower than that of the amide proton), and both
convex and concave are observed; (2) the proportion of
opened hydrogen bonds is generally expected to be less
than 0.01% even at high temperature, based on Englander’s data10; and (3) there is little correlation between the
locations of the curved residues seen here and the amide
exchange rates observed by Englander.*
The second explanation is that the excited state differs
from the ground state not in its entropy but in its heat
capacity. This would also produce a substantial temperature dependence of the relative free energy, and thus lead
to changes in relative populations and consequently to
nonlinear chemical shift changes. This is indeed perfectly
possible. However, we note that in order to see curvature, a
difference in chemical shift (or gradient) between the two
states is still required, implying that the two states
probably still differ in their hydrogen bonding. Thus,
although the thermodynamic origin of the curvature may
be different, the deduction (namely, an excited state
differing in its local hydrogen bonding) is still valid.
Effect of Added Denaturant
A number of recent researchers using amide exchange
rates to probe partially folded states of proteins have
shown that it can be very helpful to carry out experiments
at different concentrations of denaturant, such as guanidinium hydrochloride (GdmCl).7–13 A helpful way to understand these experiments is to note that the change in
unfolding free energy of a protein varies approximately
*There is some correlation, in that Englander found the highest
energy exchanges, corresponding to global unfolding, in the terminal
helices: In agreement with this, we find low-energy alternative states
(blue in Fig. 4) in a region he has identified as a low-energy subglobal
partially unfolded form. However, many other residues found by
Englander to have very high unfolding free energy at zero denaturant
concentration are here found to be curved, and many residues that he
found to have very low unfolding energy are here not curved. This is
not surprising, because the states probed by the two methods are very
different, as discussed further below.
linearly with denaturant (Fig. 2). The energy of alternative
states can also be expected to change linearly with denaturant. The scenario depicted in Figure 2 is one in which the
energy landscape is compressed but not otherwise altered
as denaturant concentration is increased; that is, the
unfolding pathway is maintained as the denaturant concentration is increased. This is by no means the only possibility, but it serves to illustrate the principle, which is that
increasing concentrations of denaturant will act to reduce
the energy difference between the ground state of the
folded protein and alternative states, and thus increase
the number of alternative conformations accessible within
the approximately 5 kcal/mol limit of our experiment. This
implies that some residues showing no curvature in the
absence of denaturant (because there are no sufficiently
different accessible alternative states) may start to become
curved at higher denaturant concentration, and also that
some initially curved residues may become less curved as
denaturant concentration is increased, if new states become accessible that have lower chemical shift difference
or chemical shift gradient difference [cf. Fig. 1(c)]. I
indicate below that both situations can be observed for
cytochrome c.
MATERIALS AND METHODS
Horse cytochrome c was purchased from Sigma and used
without further purification. It was degassed and reduced by
the addition of 7 mM sodium ascorbate, and all spectra were
run at pH 5.75 in 50 mM phosphate buffer containing 10%
D2O. Separate prepared solutions contained guanidinium
chloride at 0.2 and 0.4 M. All NMR spectra were run on a
Bruker DRX-500 NMR spectrometer. Chemical shifts were
referenced to internal trimethylsilylpropionate, whose chemical shift is independent of temperature.32 The temperature
calibration was checked with an ethylene glycol sample.
Homonuclear NOESY and TOCSY spectra were acquired for
each sample at 10° intervals in the range 20 – 80°C, with
mixing times of 100 ms. The spectra were assigned from
Wand et al.,33 and for almost every amino acid, a signal was
identified that could be uniquely assigned to a single amide
proton, whose chemical shift could be resolved and measured
in the directly detected dimension. In a small number of
residues for which no single, unique resonance could be
identified, no results are reported. Peaks were placed into a
database within FELIX (Accelrys, Inc.; San Diego, CA) and
dumped as ASCII files, which were subsequently processed
with the use of locally written UNIX routines. For each
proton, a straight line was fitted to the chemical shift
temperature dependence and subtracted from the experimental points to give a residuals plot, which was examined by eye
to determine whether it was curved.6,21 This proved a more
reliable and robust method than any computer-based method
I could devise.
RESULTS
Chemical shifts were measured for resolved amide protons, for temperatures in the range 280 –352 K, and at
GdmCl concentrations of 0, 0.2, and 0.4 M. Higher concentrations proved difficult, because the intense GdmCl signal
734
M. P. WILLIAMSON
Fig. 2. Schematic diagram of the free energy of unfolding of cytochrome c as a function of GdmCl concentration, for the ground state (solid
line) and an excited state (dashed line). For horse cytochrome c, global
unfolding occurs at 2.9 M GdmCl, with an unfolding free energy at 0
GdmCl of 6.9 kcal/mol.48,49 The excited state is illustrated 3 kcal/mol less
stable than the ground state at 0 GdmCl [cf. Fig.1(a), dashed curve].
Different protons may have excited states that range in behavior between
either remaining approximately parallel with the unfolded state, or merging with the unfolded state, depending on their relative hydrophobicities.
Nevertheless, the general principle, that the excited state becomes closer
in energy to the native state with an increase in denaturant, remains valid.
at low temperature started to obscure amide resonances in
the homonuclear two-dimensional (2D) spectra. A small
number of residues could not be assigned uniquely, namely,
G45 and K88, as well as (at one or more GdmCl concentration) E21, G41, E61, A83, G84, K86, and T89, and the four
proline residues 30, 44, 71, and 76.
A complete set of results is available on our website
(www.shef.ac.uk/⬃nmr/home.html) and is summarized in
Table I. Some examples are shown in Figure 3, for residues
showing a range of different curvatures. A number of
residues had unusual chemical shifts at the highest temperature measured, which was less than 15° below the
Fig. 1. (a) Simulations of the dependence of HN chemical shift
variation with temperature on difference in free energy between ground
state and higher energy alternative state. The calculation used, T⌬S
(25°C) ⫽ 5.1 kcal/mol [equivalent to ⌬S ⫽ 17.2 cal/mol K⫺1], and ⌬H ⫽
7.1 (solid), 8.1 (dashed), 9.1 (dot-dash), and 10.1 (dotted) kcal/mol,
corresponding to differences in free energy at 25°C of 2, 3, 4, and 5
kcal/mol, respectively. All calculations assumed shifts ␦1 ⫽ 8.5 ppm, ␦2 ⫽
8.0 ppm, and gradients g1 ⫽ ⫺2 ppb/K, g2 ⫽ ⫺7 ppb/K. In all calculations
shown, chemical shifts are calculated and fitted to a straight line, and the
linear dependence is subsequently subtracted, to leave only the residual
curvature around ␦ ⫽ 0. (b) Simulations of the dependence of HN
chemical shift variation with temperature on the nature of the excited
state. In solid is the “standard” excited state, as in part (a). The other
curves show a state with more similar chemical shift to the ground state
(dashed: ␦2 ⫽ 8.3 ppm); more similar gradient (dash-dot: g2 ⫽ ⫺4 ppb/K);
and more similar entropy (dotted: ⌬S ⫽ 10.2 cal/mol K⫺1 and ⌬H ⫽ 5
kcal/mol, leaving ⌬G still at 2 kcal/mol). (c) Simulations of the dependence
of HN chemical shift variation with temperature. The solid curve is the
“standard” excited state [as in panels (a) and (b)]. Curves in dash-dot and
dotted contain two excited states: the “standard” excited state plus one
with g2 ⫽ ⫺4 ppb/K (dash-dot) or one with T⌬S ⫽ 5.2 kcal/mol and ⌬H ⫽ 3
kcal/mol (dotted). The dashed curve has the characteristics of the excited
state and ground state exchanged; that is, the free energy and entropic
differences are the same as for the “standard” excited state, but ␦1 ⫽ 8.0
ppm, ␦2 ⫽ 8.5 ppm, g1 ⫽ ⫺7 ppb/K, g2 ⫽ ⫺2 ppb/K.
735
ALTERNATIVE CONFORMATIONS IN CYTOCHROME c
TABLE I. Residues in Horse Cytochrome c Showing Curved Amide Proton Temperature Dependence
Residue
GdmCl
1
2
0
0.2 M
0.4 M
Curvea
Shiftb
●
●
●
⫺
⫺
●
●
●
⫹
⫹
3
4
E
E
E
⫺
⫺
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
●
E
●
●
●
●
●
⫹
⫹
E
●
●
●
●
⫹
⫺
●
●
●
⫺
⫺
—
●
E
Residue 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
GdmCl
0
0.2 M
0.4 M
Curvea
Shiftb
—
E
—
—
●
●
⫺
⫺
E
●
E
●
●
⫹
⫹
E
⫹
⫹
—
●
E
●
●
⫺
⫹
●
— —
E
— —
— —
●
●
⫺
⫺
E
E
●
⫺
⫺
Residue 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84
GdmCl
0
0.2 M
0.4 M
Curvea
Shiftb
E
E
●
●
⫺
⫺
E
●
⫹
⫹
—
●
E
E
E
—
⫺
⫺
●
E
●
E
E
E
⫹
⫺
●
E
E
●
⫺
⫺
—
—
●
—
—
—
—
E
E
⫺
⫹
E
●
●
●
⫺
⫺
●
●
●
⫺
⫺
●
—
—
—
⫹
⫺
Residue 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
GdmCl
0
0.2 M
0.4 M
Curvea
Shiftb
—
— —
E
E
⫺
⫺
—
—
E
E
E
E
●
⫹
⫹
E
E
E
⫺
⫺
E
E
⫺
⫺
Curved dependences are indicated with a filled circle. Weak curvature is indicated with an open circle. Where no data are available, the residue is
marked with a dash. Many of these latter relate to proline residues.
a
A convex curvature (as in Fig. 1a) is marked ⫹, and a concave curvature⫺b Shifts less than random coil are indicated ⫺, and shifts greater than
random coil are marked ⫹. Some shifts, particularly of Gly41, are very close to their random coil values.
global denaturation temperature at zero GdmCl concentration. These shifts are therefore most likely due to incipient
cooperative global denaturation, and were omitted from
the fits. The locations of protons showing different consistent patterns of curvature are shown on the crystal
structure in Figure 4. Table I indicates whether the
curvature is concave or convex, and whether the chemical
shifts are lower or higher than random coil values. There is
remarkable agreement between these two measures, suggesting that the major determinant of whether curvature
is concave or convex is the difference in chemical shift from
the random coil value. This is in agreement with the
theoretical simulations [Fig. 1(c)], and further suggests
that the alternative state is in most cases in the direction
of the random coil state, and thus less hydrogen-bonded
and less structured than the major folded conformation.
DISCUSSION
Locations of Alternative States
The most striking feature of the results is how many
residues show curved amide proton temperature depen-
dence, and thus how many can access alternative states
that are within 5 kcal/mol of the ground state. From
Table I, the number of residues showing curvature in at
least one data set constitutes 43 out of 98 residues that
could be measured, or 44%. Because whether or not a
residue shows curved dependence is a somewhat subjective judgment, I prefer here to concentrate on residues
that show a consistent pattern of curvature [i.e., curved at
all three guanidinium concentrations measured; becoming
consistently more curved; or becoming consistently less
curved (Fig. 3)]. There are 25 residues of this type (Fig. 4).
It is not possible to draw conclusions about residues that
show no curvature, because lack of curvature could be
consistent either with an alternative state that has the
same chemical shift and gradient as the ground state, or
with lack of an alternative state. I also note that the
analysis discussed here relates only to amide proton shifts,
which are most sensitive to hydrogen bonding, and the
present analysis is thus primarily limited to changes in
amide hydrogen bonding. There may be many other
736
M. P. WILLIAMSON
Fig. 3. Experimental chemical shift changes for four representative amino acids in cytochrome c, at 0, 0.2 and 0.4 M GdmCl. Chemical shifts were
measured and fitted to a linear dependence, which was then subtracted, leaving only the residual difference from a linear dependence. The amino acid
residues shown are examples of consistently linear dependence (G6), curved dependence (M80), and residues that become more curved (G56) or less
curved (F82) with increasing guanidinium hydrochloride. The error bars give an indication of the approximate error in measured chemical shifts (⫿ 0.004
ppm).
changes in protein conformation that are not revealed by
this study.
As indicated in the simulations in Figure 1, residues
that become more curved with GdmCl (the green residues
in Fig. 4) most likely have an alternative state that
becomes either more similar in energy, or more different in
shift or gradient, as GdmCl concentration increases. The
most economic of these explanations is that an increase in
GdmCl does not change the nature of the alternative state
but brings it closer in energy to the ground state, by
contracting the unfolding energy landscape (Fig. 2). Thus,
in general, we may expect that the residues colored green
in Figure 4 have alternative states that are rather higher
in energy than the consistently curved residues, colored
red, in Figure 4. By contrast, residues that become less
curved (colored blue in Fig. 4) have more than one alternative state within 5 kcal/mol [Fig. 1(c)], or possibly have an
alternative state that becomes very close to the ground
state as GdmCl is increased. Thus, of the colored residues
in Figure 4, the green ones are those with ground states
most separate from any alternative state, whereas the
blue ones are those least separate.
Inspection of Figure 4 shows that the N- and C-terminal
helices form the most stable part of the structure. In
particular, the central parts of the helices, where they
make contact with each other, have no colored residues,
and thus show no indication of alternative states within 5
kcal/mol of the ground state. This fits well with numerous
studies of cytochrome c folding and unfolding,16,34,35 which
all indicate that these helices are the first to fold and last
to unfold, and therefore presumably form the most stable
core of the protein. The ends of these helices show some
“fraying,” as seen in many studies of protein dynamics. By
contrast, the part that shows the highest density of
alternative states contains the 60s helix and Met80. A
number of studies have suggested that this part of the
structure is one of the least stable parts,10 and that
dissociation of Met80 from heme accompanies the first step
ALTERNATIVE CONFORMATIONS IN CYTOCHROME c
737
Fig. 4. The structure of horse cytochrome c [Protein Data Bank (PDB) structure 1hrc50] showing amino
acids with consistent patterns of amide curvature. Residues that are curved at all GdmCl concentrations are
shown in red; residues that become more curved with increase in GdmCl in green; and residues that become
less curved in blue. The N- and C-termini, and the location of the heme ligand Met80, and the 60s helix, are
shown. The heme is shown in stick representation, with the central iron in brown.
in cytochrome c unfolding36,37 and the last step in folding.38 This result is of particular interest, because change
in ligation of Met80 is crucial to the function of cytochrome
c as an electron carrier.39 It may therefore be that local
fluctuations in a relatively stable scaffold allow the adjustments of the structure needed for correct function.
Number and Independence of States
In our earlier study of curved amide proton temperature
dependence,6 we suggested that the “cooperative units”
forming alternative states are of the order of five amino
acids in size. The experimental results presented here are
comparable to those shown in the simulations, being in the
midrange of the simulated curvatures. Simulations with
much larger or smaller energy differences produce curvatures that are too small or too large, respectively, compared to the experimental results. This implies that the
experimentally observed alternative states have energies
similar to those simulated. As discussed in the Theory
section, the parameters for the simulations were chosen to
match cooperative units of approximately five amino acids.
I therefore conclude, in agreement with our previous
results, that the alternative states within 5 kcal/mol of
the ground state are most likely on the order of five
residues in size. There are a large number of residues
involved in alternative states, and our results are not
consistent with their being accessed sequentially (i.e., the
first alternative state destabilizing adjacent residues and
facilitating their subsequent disruption); otherwise, many
residues would have shown a “blue” (Fig. 4) behavior. The
results therefore imply that cytochrome c has many smallscale (approximately five-residue) alternative states, differing in hydrogen bonding from the ground state, that are
accessed independently. These states cover most of the
protein (with the exception of the N- and C-terminal
helices) and are particularly dense close to Met80. These
data do not completely define the nature of these states,
but they suggest that the states are less structured, with a
cooperative breakage of up to five hydrogen bonds per
state. We suggest therefore that they could represent
738
M. P. WILLIAMSON
rearrangement of a loop, or loss of a helical turn, rather
than, for example, a more major change such as the
complete loss of a helix. Possibly the most significant
finding of this study is that there exist a large number of
accessible states, covering a high proportion of the structure of the protein, that are significantly populated under
equilibrium conditions: Proteins are not nearly as fixed in
their structure as we tend to think.
Relationship to Unfolding
This study characterizes alternative conformations close
to the ground state and has no necessary relevance to
unfolding. However, the correspondence between the sign
of the curvature and the chemical shift difference from
random coil (Table I), and the good match between the
locations of alternative states and the unfolding pathway
of cytochrome c, suggest that there is a connection, which
is not surprising, considering features that govern conformational stability also dictate folding.2 The alternative
states are good candidates for the earliest cooperative
unfolding events, which by inference can arise almost
anywhere in the protein (with the exception of the terminal helices). These results are of particular interest when
compared to those of Englander’s group on hydrogen
exchange in cytochrome c. In these studies,10,12 they
identified partially unfolded forms (PUFs) that are between 9 and 18 kcal/mol above the ground state, and
consist of approximately 15 residues each. One could
therefore imagine a sequence of unfolding events starting
from the fully folded state, in which local breathing
motions merge to form the small, low-energy alternative
states identified here, which then merge into the larger
PUFs identified by Englander, and lead on to global
unfolding. In general, the locations of these states match,
with the least stable part in the region of Met80, and the
most stable part at the terminal helices. Both models are
also hierarchic,2,40 – 43 in that they posit that tertiary
structure elements are lost first, and there are certain
secondary structures (in particular the terminal helices)
that are common intermediates in any folding/unfolding
pathway. However, there is an important difference between our alternative states and Englander’s PUFs, in
that his states are sequential (in the sense that the lowest
energy state is a subset of the next), whereas ours are not.†
This difference may lie partly in the nature of hydrogenexchange experiments, which lend themselves to sequential models.13 The high-energy states probed by amide
exchange experiments are clearly of higher energy and are
significantly more disrupted than the alternative states
studied here. Our model of independent alternative states
suggests rather that there are many competing unfolding
†
There are many other detailed differences. For example, in our
study, Leu68 is curved, whereas in the work of Bai et al.,10 its
hydrogen exchange has a linear dependence on denaturant, implying
that it is in the structural core and does not exchange except after
global denaturation. This discrepancy may be reconciled if the unfolding observed10 is of a larger scale and higher energy than seen here.
The implication of this would, however, be that their description of
“stable folding units” would no longer have a simple interpretation of a
region that is essentially unchanged up to global denaturation.
routes (“non-cooperative unfolding interactions”2) that may
merge at a key intermediate: In the case of cytochrome c,
all the unfolding routes appear to pass through a state in
which the N- and C-terminal helices remain intact and in
contact, whereas much of the rest of the structure is lost.
This model has much in common with the multiple folding
routes44 that are a feature of recent models.45 These
results provide experimental evidence that many structures are different from the ground state but close in
energy to it: In other words, the conformational ensemble
of cytochrome c is one of high structural entropy46 with a
large number of discrete local substates.47
ACKNOWLEDGMENTS
My thanks to Prof. M. Karplus and the referees for
helpful comments; to Jen Dawson, Beck Smith, and Richard Bingham for technical assistance; and to Jon Waltho,
Jeremy Craven, and Nicola Baxter for helpful discussions.
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