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PROTEINS: Structure, Function, and Genetics 30:435–441 (1998)
Proteolysis as a Probe of Thermal Unfolding
of Cytochrome C
Leyu Wang, Robert X. Chen, and Neville R. Kallenbach*
Department of Chemistry, New York University, New York, New York
Recent hydrogen exchange
experiments on native cytochrome c implicate
a sequential unfolding pathway in contrast to
a simple two-state process. We have studied the
heat-induced unfolding of this protein by using spectroscopic measurements to detect
changes in conformation and proteolytic enzyme digestion to identify regions of the protein
that are labile. Several spectroscopic profiles were
monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment
of Trp 59, and absorbance at 420 nm, the Soret
band of the heme. The apparent Tm values for
these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent
with population of an intermediate state in
unfolding. We find a single strong region of
cleavage at low temperature with retention of
structure in each fragment. Proteins 30:435–
441, 1998. r 1998 Wiley-Liss, Inc.
Key words: cytochrome c; thermal unfolding;
proteolysis; proteinase K; thermolysin
Many single-domain proteins can spontaneously
refold to their native active states after unfolding in
dilute solution. The mechanism of this reaction
remains a matter of considerable debate, although
there is an emerging consensus that globular proteins above a critical size fold via formation of
intermediate structured subdomains or specific nucleation regions.1–3
Recent theoretical studies by Srinivasan and Rose4
argue that important aspects of folding can be modeled as a hierarchical process, in which stable intermediates accumulate. Others have proposed formation of a
framework of sequential intermediates,2 based on various hypothetical subunits and mechanisms for interaction among subunits.5 What remains unclear at present
is the detailed mechanism of folding and the nature of
the barriers separating intermediates.6
The hypothesis that folding proceeds via a series of
distinct intermediates is strongly supported by the
recent analysis of peptide hydrogen exchange in
horse heart cytochrome c (cyt c), which reveals a
specific set of subdomains that unfold (or refold)
cooperatively within the native structural manifold.7
The intermediates in cyt c are identified as clusters
or groups of peptide NH that share a common
opening channel; four such sets have been identified
in the protein to date, each associated with a particular free energy and sensitivity to denaturing solvent.
Within each subdomain, clusters of NH groups exchange initially via a local process, which exhibits
minimal sensitivity to GuHCl. Others in the cluster
exchange only when released by a larger scale cooperative unfolding reaction, involving perhaps a dozen
NH groups or more. Because the pathway(s) by
which NH groups exchange from proteins is not yet
elucidated in detail, the issue we address here is
whether subdomains can be detected directly, by
using reactive probes of the backbone as an alternative to NH exchange. In a sense, NH exchange
represents a reactive probe of the peptide backbone
that perturbs the bond minimally, substituting D for
H or vice versa. Scission by a protease is a more
severely perturbing probe but one that allows facile
detection of reactive sites.
The native states of proteins tend to resist cleavage by proteases. Limited proteolysis has been used
to probe the surface accessibility of particular side
chains and the flexural dynamics of proteins.8–10
Proteinase K, an extremely stable protease with a
relatively broad cutting specificity,11 provides a probe
for conformational flexibility in proteins that is able
to monitor structural changes during protein unfolding. An enzyme, such as trypsin, on the other hand,
cleaves charged groups most likely to occur on the
protein surface and would thus be less sensitive as a
probe of local unfolding reactions.
Cyt c is a small stable globular protein that has
served as a model for numerous folding and unfolding studies.12 Horse heart cyt c contains 104 amino
acids, covalently linked to a heme group (Fig. 1). It
acts as a soluble electron carrier in oxidative phosphorylation, transferring electrons from cytochrome
reductase to the terminal oxidase complex. Recent
Contract grant sponsor: National Institutes of Health; Contract grant number: GM40746.
*Correspondence to: Neville R. Kallenbach, Department of
Chemistry, New York University, New York, NY 10003.
Received 29 August 1997; Accepted 4 November 1997
experiments on the solvent-induced unfolding of
native cyt c have been interpreted in terms of a
sequential, progressive unfolding model, in contrast
to a simple all-or-none or fully cooperative unfolding
process. Figure 1 illustrates the structure of cyt c in
ribbon form, with regions of the protein colored red,
yellow, green, and blue to indicate the sequentially
unfolding subdomains in the molecule identified by
hydrogen exchange rate measurements.7
Determination of the rates of exchange of NH
peptide hydrogens of the backbone of cyt c as a
function of added denaturant suggests that the
protein unfolds in discrete cooperative subdomains,
Fig. 1. Structure model of cytochrome c, colored to indicate the
subdomains in the molecule that exchange cooperatively with
different free energies, according to Bai et al.7 The lowest energy
opening set of groups is red, with yellow, green, and blue
representing higher energy opening sets of groups, respectively.
The blue set of NH groups merge at higher guanidine concentration into an opening corresponding to unfolding of the entire
Fig. 2. Schematic illustration of the sequential, cooperative
unfolding and refolding reaction identified in cytochrome c, according to Bai et al.7 On the left, the native protein includes NH groups
from four distinct regions (represented as blocks) of the molecule
shown in Figure 1. The lowest energy opening reaction includes
NH groups from the loop colored in red. Once the red tier has
exchanged, members of the yellow group unfold, followed by the
identified by the different colors shown in Figure 1.
As urea or guanidinium chloride is added, the first
opening reaction detected occurs in the region colored red, the second is in the domain indicated by
yellow, followed by the green and blue regions.
Exchange of NH in the latter subdomain corresponds
to complete unfolding or denaturation of the protein
(see diagram in Fig. 2). A corresponding hierarchy of
states can be detected in thermal unfolding of the
protein, implying that these subdomains represent
intrinsic units in the folding or unfolding of the
HD exchange measurements monitored by NMR
allow determining the extent of unfolding within
each subdomain indirectly, from the protection factors by which exchange of the slowest NH within
that subdomain are retarded relative to unstructured NH of the same sequence. The opening reactions responsible for release of peptide NH for solvent exchange have not been characterized; hence,
the picture of unfolding that emerges from NH
exchange rates does not tell us what fluctuations are
The question we address here is, can a stepwise
unfolding reaction be monitored more directly by
using protease digestion as a reactive probe, for
example? Several studies indicate that the accessibility of certain proteases to target sites in a protein is
determined by fluctuational opening reactions or
local dynamics.8 In this work we describe traditional
spectroscopic measurements of thermal unfolding of
cyt c as well as monitor the ability of proteinase K
and thermolysin to digest cyt c at various temperatures. These enzymes retain activity up to 100°C.
The pattern of cutting is consistent with the presence of an intermediate in the opening process as cyt
c unfolds, the initial site of attack lying within the
yellow subdomain of the molecule (Fig. 2).
Proteins and Enzymes
Horse heart cyt c was purchased from Sigma (St.
Louis, MO) and used without further purification.
Proteinase K was purchased from USB. Thermolysin
two green domains and finally by the blue group, including the
ends of the chain. Once the blue tier has exchanged, the protein is
fully unfolded as shown on the right. Refolding is thought to begin
with cooperative formation of the blue set, progressing stepwise to
green, yellow, and red, generating the native state. The three
intermediates in the center of the figure probably correspond to
molten globule states of the protein.
TABLE I. Molecular Weight of Fragments of Horse
Heart Cytochrome c Produced by Limited
Proteolysis With Proteinase k as a Function
of Temperature in Figure 6
Temperature (°C)
On ice
Room temperature
Molecular weight of
fragments (kD)†
5.8, 5.4
5.8, 5.4
5.8, 5.4
5.8, 5.4, 4.4, 3.2
5.8, 5.0, 4.4, 3.2
5.8, 5.0, 4.4, 3.2
5.8, 5.0, 4.4, 3.2
9.7, 7.0, 5.8, 5.0, 4.4, 3.9, 3.2, 2.9
Same as 80°C
Same as 80°C
molecular weight of the fragments was got from its
mobility on SDS-PAGE compared with the protein markers:
insulin (a and b chain, 2,900), bovine trypsin inhibitor (5800),
lysozyme (14,600), b-lactoglobin (18,500), carbonic anhydrase
(28,860), ovalbumin (44,000). The molecular weight of cytochrome c (12.4 kD) estimated from the gel was 13.4 kD.
Fig. 3. SDS-PAGE of the proteolysis of cytochrome c by
proteinase K. The protein was treated on ice and at room
temperature with proteinase K at 1:50, 1:25, 1:10, 1:5 ratios (w/w)
of protease to cytochrome c in 20 mM Tris-HCl buffer, pH 5 7.5.
The reaction time is 24 hours, and the reaction was stopped by
TABLE II. Molecular Weight of Fragments
of Horse Heart Cytochrome c Produced by
Limited Proteolysis With Thermolysin as a
Function of Temperature in Figure 7
Temperature (°C)
On ice
Room temperature
Molecular weight of
fragments (kD)†
No cut
5.5, 4.3
5.5, 4.3
5.5, 4.3
5.5, 4.3
No significant fragment
No significant fragment
9.7, 6.7, 5.5, 4.3, 3.1
Same as 80°C
molecular weight of cytochrome c (12.4 kD)
estimated from the gel was 13.0 kD.
was purchased from Sigma. All other reagents were
highest purity grade.
Limited Proteolysis and Electrophoresis
Digestion of cyt c was carried in 20 mM Tris-HCl,
pH 5 7.5, with an enzyme/protein ratio at 1:190,
1:50, 1:25, 1:10, and 1:5 for proteinase K, with an
enzyme/protein ratio at 1:200 for thermolysin. Before mixing protease with cyt c, the cyt c solution was
equilibrated at each temperature in a water bath for
3–5 minutes. The proteolysis reaction was stopped
by addition of PMSF for proteinase K with EDTA for
thermolysin. SDS-PAGE was used to monitor the
digestion. We used the high-resolution tricine buffer
electrophoresis system.13 Gels were stained with
Coommassie Brilliant Blue R-250. Mixtures of proteins of known molecular mass were used as a
calibration standard in SDS-PAGE.
Fig. 4. SDS-PAGE of the proteolysis of cyt c by proteinase k at
1:50 enzyme/protein ratio at 37°C. Lane 1: protein markers; lane
2: proteinase k control; lane 3: cyt c control; lane 4: 4 min; lane 5:
8 min; lane 6: 16 min; lane 7: 32 min; lane 8: 1 hour; lane 9: 2
hours; lane 10: 3 hours; lane 11: 4 hours.
Transblotting and N-Terminal Sequencing
The protein fragments were transferred to PVDF
membrane by using the MilliBlot-Graphite Electroblotter System. The membrane then was stained with
Coommassie Brilliant Blue R-250. The fragment
bands were cut and submitted to a protein-sequencing analyzer for N-terminal sequencing.
Thermal Unfolding Measurements
Both 20 µM cyt c in 20 mM Tris-HCl buffer
(pH 5 7.5) containing 20 mM NaCl were used in
melting curve measurement. CD versus temperature was performed at 222 nm on AVIV 60 DS
instrument. A cell of 1-mm length was used for the
far UV studies, with an equilibration time of 1
minute (1 degree one point). The corresponding
absorption versus temperature profiles were moni-
Fig. 5. SDS-PAGE of the proteolysis of cyt c by proteinase k at
1:190 enzyme/protein ratio at 60°C. Lane 1: 32 min; lane 2: 16
min; lane 3: 15 min; lane 4: 10 min; lane 5: 8 min; lane 6: 5 min;
lane 7: 4 min; lane 8: 2 min; lane 9: 1 min; lane 10: cyt c control;
lane 11: protein markers.
Fig. 7. SDS-PAGE of the proteolysis of cyt c by thermolysin at
1:200 enzyme/protein ratio as a function of temperatures. Lane 1:
cyt c control; lane 2: cyt c control; lane 3: 40 min on ice; lane 4: 40
min at room temperature; lane 5: 20 min at 30°C; lane 6: 10 min at
40°C; lane 7: 5 min at 50°C; lane 8: 5 min at 60°C; lane 9: 5 min
70°C; lane 10: 5 min at 80°C; lane 11: 5 min at 90°C.
ments with those of a set of markers. The apparent
molecular weight of protein markers was used to
generate a curvilinear line relating log molecular
weight of the protein to the migration distance on the
separating gel using a least-squares analysis. The
molecular weights of the fragments in Figures 6 and
7 are listed in Tables I and II, respectively. According
to this method, the apparent molecular weight of cyt
c is 13.4 kD in Figure 6 and 13.0 kD in Figure 7,
5–7% higher than its formula molecular weight of
12.4 kD.
Limited Proteolysis
Fig. 6. SDS-PAGE of the proteolysis of cyt c by proteinase k at
1:50 enzyme/protein ratio as a function of temperatures. Lane 1: 4
min at 90°C; lane 2: 3 min at 80°C; lane 3: 3 min at 70°C; lane 4: 4
min at 100°C; lane 5: 4 min at 60°C; lane 6: 8 min at 50°C; lane 7:
16 min at 40°C; lane 8: 16 min at 30°C; lane 9: 32 min at room
temperature; lane 10: 32 min on ice; lane 11: protein markers;
lane 13: proteinase k control; lane 14: cyt c control.
tored at 280 nm and 420 nm by using a Perkin Elmer
552 UV-VIS spectrophotometer. A 10-mm path cell
was used, and the equilibrium time was 2 minutes (2
degree per time point).
Calibration of Fragment Sizes
Estimates of fragment molecular weight were
made by comparing the relative mobility of frag-
Figure 6 shows the results of cutting by proteinase
K as a function of temperature; the sizes of the
fragments are listed in Table I. The catalytic activity
of proteinase K drops as a function of temperature,
so that at 60°C the enzyme is about 80% as active as
at 37°C.14 Cyt c becomes increasingly labile to protease as the temperature increases, and above 80°C,
the protein can be cut in each of the colored regions
shown in Figure 1. We also conclude that the increased scission does not result from a change in
activity of proteinase K, but from conformational
changes in the protein, by comparing Figures 3, 4,
and 5. At temperatures below room temperature,
two fragments result from the digestion, regardless
of length of exposure to the enzyme. The N-terminal
sequence of band II is TDANKNKGITWK, which
results from breaking the peptide bond between
amino acid Tyr 48 and Thr 49. The N-terminus of
band III is ANKGITWKE, which comes from cutting
between amino acid Asp 50 and Ala 51. Because the
N-terminal of cyt c itself is acetylated, we cannot see
Fig. 8. Unfolding profiles of cytochrome c as a function of
increasing temperature. The protein concentration is 20 µM in 20
mM Tris-HCl buffer (pH 5 7.5) containing 20 mM NaCl. A: CD
measurement at 222 nm. B: Van’t Hoff plot of data in A: ln Ku as
function of 1/T. C. Absorption at 280 nm (triangle) and 420 nm. D.
Van’t Hoff plot of data in C: ln Ku as function of 1/T. The data in A
and C were initialized by using the corresponding value at 10°C.
the corresponding N-terminal fragments by this
CD and Absorption Unfolding Profiles
The CD profile shown in Figure 8 indicates that
cyt c loses its helical structure at pH 7.5 when the
temperature exceeds 80°C. The Tm of the protein is
close to 83°C measured by [u] 222. By contrast, the
values of A280 and A420 show that a conformational
change sets in at temperatures as low as 40°C; the
Tm detected by A280 is 67.1°C, whereas that for A420 is
68.3°C. The absorption at 280 nm decreases by about
15% from 20°C to 80°C, which indicates there is a
significant change in the neighborhood of the chromophore, the side chain of Trp59. Thus, the loop region
of the cyt c molecule opens before the helical structure in the molecule unfolds significantly. It is known
that a change in heme ligation occurs on thermal
unfolding of cyt c, detected by a change in heme
absorbance at 695 nm.
From the above analysis, we conclude that the
thermal unfolding of cyt c at pH 7.5 is not a twostate, or concerted, reaction as it is at pH 5. The loop
region of cyt c appears to unfold before the bulk of the
helical structure in the molecule. Proteinase K shows
a wide temperature range in its activity, remaining
active at 100°C. This makes it possible to discriminate structured subdomains as folding and unfolding progresses, even under conditions in which U is
stable. Below the major unfolding reaction, specific
regions of proteolysis are seen, the fragments showing resistance to further proteolysis. Digestion at
elevated temperature reveals several fragments with
proteinase K and thermolysins (Figs. 6 and 7).
The thermal unfolding profiles (Fig. 8) show that
cyt c does not unfold in a two-state process. Instead,
intermediates of some kind must be involved, because the profiles at 222 and 420 nm are not superimposable. Earlier studies had revealed this effect,
without fully explaining it. As the protein unfolds,
side chains other than the normal ones can interact
with the heme,15 and one picture of the unfolding of
the protein consistent with the spectroscopic data is
that the heme cavity undergoes rearrangements at
temperatures below the unfolding of the secondary
structure.16,17 The fragmentation data show that
proteinase K initially attacks a region in the molecule located in the sequence within the subdomain
indicated by yellow in the sketch in Figure 1. The
resistance to proteolysis of each resulting fragment
suggests that the fragmented molecule retains substantial structure. Subsequent cleavage proceeds
from the fragments resulting from this initial event,
consistent with a sequential opening mechanism.
The scission data are consistent with the results of
reconstitution experiments, which indicate that fragments with chain length greater than that of the
native protein reassemble to form a native-like core
if the cut site lies within the region we have identified.18–20 In addition, Fontana and his co-workers21
have reported that thermolysin cleaves cyt c selectively at the Gly56-Ile57 bond in the presence of
TFE. This bond lies within the yellow subdomain,
indicating that even in the distorted state induced by
TFE, containing additional helical structure, a sequential unfolding mechanism dominates.
Do these results support the detailed unfolding
pathway delineated in cyt c by HX analysis?7 Initial
scission is detected here within the yellow subdomain, rather than the predicted red sequence (70–85
in the sequence, Fig. 2). However, this might reflect a
lack of target sequences for the enzyme within the
red subdomain relative to yellow. Despite its broad
specificity, proteinase K has sequence preferences.
Cuts in the red domain certainly are detected in the
unfolded protein, however, so the issue is not settled.
The red subdomain is considerably smaller in size
than yellow. The breakdown of the fragments released following initial cutting is consistent with
subsequent opening at sites within the blue and
green domains shown in Figure 2, although we have
not proven this as yet. Overall, our observations are
consistent with a sequential unfolding mechanism,
centered on the loop within the subdomain colored
yellow, but do not yet establish the process defined by
HX experiments in detail. The nature of the breathing reactions that release NH hydrogens for exchange might differ fundamentally from those that
expose segments of the backbone to proteolysis in
principle. The agreement seen thus suggests a common pathway for at least the cooperatively exposed
NH groups in the yellow subdomain. Additional
reactive probes might reveal further details of the
opening pathway in cyt c.
L.W. is a recipient of a Henry Mitchell MacCracken Fellowship at NYU. We thank Dr. Martine
Cadene for help in N-terminal sequencing at NYU
medical center and Angelo Fontana (CRIBI, University of Padua, Padua, Italy) for helpful discussion.
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