Post on 19-Mar-2018
Hydrophobic Clusters Affect Protein Stability
1
Clustering of large hydrophobes in the hydrophobic core of two-stranded α-helical
coiled-coils controls protein folding and stability.
Stanley C. Kwok, and Robert S. Hodges*
Dept. of Biochemistry and Molecular Genetics, Univ. of Colorado Health Sciences
Center, Denver, CO 80262, USA and Dept. of Biochemistry, Univ. of Alberta,
Edmonton, AB, T6G 2H7, Canada.
Running Title: Hydrophobic Clusters Affect Protein Stability
S.C.K. was supported by studentships from the National Science and Engineering
Research Council of Canada and Alberta Heritage Foundation for Medical research.
This research was supported in part by the Canadian Institutes of Health Research, the
Protein Engineering Network of Centers of Excellence, the University of Colorado
Health Sciences Center, and the National Institutes of Health (grant number; R01GM
61855) to R.S.H.
*To whom correspondence should be addressed. Tel. 303-315-8837. Fax. 303-315-1153.
E-mail: robert.hodges@uchsc.edu
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
2
Abstract: The de novo design and biophysical characterization of two 60-residue
peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core
clustering is described. Our goal was to investigate whether designing coiled-coils with
identical hydrophobicity but with different hydrophobic clustering of non-polar core
residues (each contained 6 Leu, 3 Ile and 7 Ala residues in the hydrophobic core) would
affect helical content and protein stability. The disulfide-bridged P3 and P2 differed
dramatically in α-helical structure in benign conditions. P3 with 3 hydrophobic clusters
was 98% α-helical while P2 was only 65% α-helical. The stability profiles of these two
analogs were compared, and the enthalpy and heat capacity changes upon denaturation
were determined by measuring the temperature dependence by circular dichroism spectra
and confirmed by differential scanning calorimetry. The results showed that P3
assembled into a stable α-helical two-stranded coiled-coil and exhibited a native protein-
like cooperative two-state transition in thermal melting, chemical denaturation and
calorimetry experiments. Although both peptides have identical inherent hydrophobicity
(the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil
positions), we found that the context-dependence of an additional hydrophobic cluster
dramatically increased stability of P3 (∆Tm ≈ 18 0C and ∆[Urea]1/2 ≈ 1.5M) compared to
P2. These results suggested that hydrophobic clustering significantly stabilized the
coiled-coil structure and may explain how long fibrous proteins like tropomyosin
maintains chain integrity while accommodating polar or charged residues in regions of
the protein hydrophobic core.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
3
INTRODUCTION
Understanding protein folding remains a challenging problem: how does information
encoded in the amino acid sequence translate into the three-dimensional structure
necessary for protein function? Although hydrophobic interactions are generally
accepted as the predominant source of free energy change that maintains the folded state,
this non-specific stabilization does not describe how the “hydrophobic collapse” guides
the formation of specific secondary structure (α-helices and β-sheets) in the final tertiary
and quaternary structure in the native protein. The concomitant model suggests that the
hydrophobic collapse restricts the conformation of the polypeptide chain into a “molten
globule” thus facilitating secondary structure folding in this limited conformational
context (1). Examples of hydrophobic interactions participating in the early events of
protein folding are observed via stopped flow fluorescence and nuclear magnetic
resonance (NMR) studies in apomyogloblin (2) and cytochrome c (3), illustrating the
importance of the packing of non-polar residues in stabilizing helix-helix interactions.
Recently, non-polar residues have also been observed to form non-native hydrophobic
clustering in denatured proteins (4,5), and the authors postulated that non-native
hydrophobic interactions can stabilize the long-range order of the protein scaffold, via an
intermediate not observed in the folded state, thus indirectly guiding the extended
polypeptide chain towards the correct native fold. Such an observation suggests that the
amino acid sequence encodes for structural characteristics other than that of the native
fold; in other words, the hydrophobic patterning in the sequence encodes the pathway that
ultimately leads to the native functional state. Considering that hydrophobic interactions
mediate protein folding both in the folded and the unfolded state, several questions arise:
1) how does a cluster of non-polar residues contribute to stability; 2) is the free energy
derived from the burial of hydrophobic residues simply a sum of the energy derived from
the removal of non-polar surface area from aqueous medium; 3) does hydrophobic
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
4
clustering enhance stability via favorable enthalpic, geometric packing and van der Waals
interactions?
The two-stranded α-helical coiled-coil is the simplest protein fold consisting of two
amphipathic α-helices wound around one another forming a left-handed super coil
stabilized by hydrophobic burial (6,7). All coiled-coils share a characteristic heptad
(seven-residue) repeat denoted as (abcdefg)n in which non-polar residues occupy the a
and d positions, forming an amphipathic surface where non-polar interactions allowed
assembly of two-, three- and higher oligomeric states (8). The quantitative contribution
of 20 amino acids in positions a and d and their effects on protein stability and
oligomerization state have been determined (9-11). In addition, the secondary structure
formation and hydrophobic collapse of coiled-coils are tightly coupled and cooperative
since single-stranded amphipathic α-helices are unstable in aqueous medium. This
hydrophobic surface where amphipathic α-helices interact via hydrophobic interactions
provides an ideal model to test the effects of hydrophobic clustering. We postulated that
hydrophobic clustering in the core of coiled-coils would have a significant influence on
secondary structure formation and protein stability. Here we present the de novo design
and characterization of two α-helical coiled-coils that have the same inherent
hydrophobicity, i.e., the identical hydrophobic core residues (6 Leu, 3 Ile and 7 Ala
residues), but with different clustering of large and small hydrophobic core residues (Fig.
1.). Their biophysical characteristics are compared by circular dichroism spectroscopy
(CD), analytical ultracentrifugation, and differential scanning calorimetry (DSC). The
results are discussed in the context of non-polar residue clustering enhancing protein
stability.
EXPERIMENTAL PROCEDURES
Peptide Synthesis and Purification
Peptides were synthesized by automated solid-phase methodology described previously
(12,13) by conventional t-butyloxycarbonyl (t-Boc) chemistry (reviewed in 14). The
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
5
peptides were synthesized on an Applied Biosystems model 430A peptide synthesizer as
described previously (15). Briefly, the polypeptide chain was assembled on
copoly(styrene, 1% divinylbenzene)-4-methylbenzhydrylamine-HCl (MBHA) resin, 100-
200 mesh, substitution 0.73 mmol amino groups per gram (Novabiochem, Switzerland).
The following side-chain protecting groups were used: benzyl (Thr, Ser), cyclohexyl
(Asp), 4-methylbenzyl (Cys), trityl (Asn), and tosyl (Arg). A peptide-resin core (1.0
gram of MBHA resin containing 0.6 mmol of peptide chain was swelled and washed
repeatedly with dichloromethane (DCM) and N,N-dimethylforamide (DMF) in a 25 ml
polypropylene solid-phase extraction reservoir. Activation reagent O-benzotriazol-1-yl-
1,1,3,3 tetramethyluronium hexafluorophosphate (HBTU, 0.45 M) was dissolved in
DMF/DCM/ dimethylsulfoxide (DMSO) (85:10:5 v/v/v) and reacted with excess t-Boc-
protected amino acid (1.1 equivalent to mmol of polypeptide chain) and excess
diisopropylethylamine (DIEA, 1.5 equivalent to mmol polypeptide chain bound to resin)
for 5 min. The activated amino acid ester (4.0 equivalent excess compared to mmol of
polypeptide chain bound to resin) was then coupled onto the solid-phase support by
agitation for an hour. Excess un-reacted amino acids were removed by three alternating
washes of DCM and DMF. Cleavage of the t-Boc and side-chain protecting groups and
the subsequent release of the completed peptides from the MBHA resin support was
achieved with hydrogen fluoride (HF) containing scavengers, 10% (v/v) anisole and 1%
(v/v) 1,2 ethanedithiol (EDT), magnetically stirred for 60 minutes in a reaction vessel
with the temperature-controlled at (-4oC) by immersion in a sodium chloride waterbath.
The peptide-resin was then washed three times with cold diethyl ether to remove
scavengers and amino acid protecting groups. Subsequent resin extraction with glacial
acetic acid and overnight lyopholization yielded the crude peptide.
Crude peptides were purified by reversed-phase chromatography (RPC, reviewed in 16)
on a Zorbax semi-preparative SB-C8 column (250 x 9.4 mm I.D., 5 µm particle size,
300Å pore size) by linear AB gradient elution (0.2% increasing acetonitrile/min), where
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
6
eluent A is 0.05% aqueous trifluoroacetic acid (TFA) and eluent B is 0.05% TFA in
acetonitrile. The purification was carried out at room temperature with a constant flow
rate of 2 ml/min. The purity and homogeneity of the peptide was verified by analytical
RPC on a Zorbax analytical 300 Å SB-C8 column (150 x 2.1 mm I.D., 5 µm particle size,
300Å pore size), by quantitative amino acid analysis (Beckman Model 6300 amino acid
analyzer), and by electrospray mass spectroscopy on a Fisons Quattro (Fisons, Pointe-
Claire, Quebec, Canada). Formation of the disulfide-bridged two-stranded homo-dimeric
coiled-coil was obtained by overnight stirring in a 100 mM NH4HCO3 buffer, pH 8.5, and
the desired product was purified by RPC (described above).
Analytical Ultracentrifugation Equilibrium Experiments
Sedimentation equilibrium analysis was performed on a Beckman XLA analytical
ultracentrifuge with absorbance optics at 274 nm for the detection of tyrosine. Samples
were first dialyzed exhaustively against an aqueous solution of 100 mM KCl, 50 mM
PO4, pH 7.0 (benign buffer) at 4 oC. A 100 µl aliquot was loaded into the 12 mm, Epon
cell (charcoal-filled), and the initial loading concentrations of peptide stock solutions
ranged from 50 – 500 µM in benign buffer. The samples were spun at 20 oC at 20,000,
25,000 and 35,000 rpm for 24 hours to achieve equilibrium, as demonstrated by
successive identical radial absorbance scans at 274 nm. The behavior of the peptide
species at equilibrium is described by the following equation:
Mbuoy = Μω (1 - νρ) (1)
where Mbuoy is the measured buoyant weight, Μω is the molecular weight in Daltons, ν is
the partial specific volume of the sample and ρ is the density of the buffer solution. The
partial specific volume of the sample and density of the buffer were calculated using
SednTerp (ver. 1.06, University of New Hampshire) using the weighted average of the
amino acid content. The peptide oligomerization behavior was determined by fitting the
sedimentation equilibrium data from different initial loading concentrations and rotor
speeds to various monomer-oligomer equilibrium schemes using WIN NonLIN (ver
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
7
1.035, University of Connecticut), a nonlinear least squares algorithm for equilibrium
ultracentrifugation analyses (17).
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectroscopy was performed on a Jasco-810 spectropolarimeter
with constant N2 flushing (Jasco Inc., Easton, MD.). A Lauda circulating water bath was
used to control the temperature of the optic cell chamber, where rectangular cells of 1
mm path length was used. The concentration of peptide stock solutions was determined
by absorbance at 275 nm in 6 M Urea (extinction coefficient, ε = 1420 cm-1·M-1, one
tyrosine per peptide chain). For wavelength scan analysis, a 5 mg/ml stock solution of
each peptide in 100 mM KCl, 50 mM PO4, pH 7.0 (benign buffer) was diluted and
scanned in the presence and absence of 50% trifluoroethanol (TFE). Mean residue molar
ellipticity was calculated using the equation:
[θ ] = θobs* mrw /10 l c (2)
where θobs is the observed ellipticity in millidegrees, mrw is the mean residue molecular
weight, l is the optical path length of the CD cell (cm), and c is the peptide concentration
(mg/mL). Each peptide spectrum was the average of eight wavelengths scans collected at
0.1 nm interval from 195 to 250 nm. The uncertainty in the molar ellipticity values was
+/-300 deg·cm-1·dmol-1. Protein stability measurements were monitored at wavelength
222 nm, indicative of secondary structure of α-helices, by thermal and chemical (urea)
denaturations (18).
Temperature-induced Denaturation Monitored by Circular Dichroism
For thermal melting experiments, data points were taken at 1 oC interval at a scan rate of
60 oC per hour. The temperature dependence of the mean residue ellipticity θ was fitted
to obtain fraction of the unfolded state, PU(t), using a non-linear least-squares algorithm
assuming a two-state unfolding reaction with pre-transitional (folded state, θ N(t)) and
post-translational (unfolded state, θ U(t)) baseline corrections (19,20):
θ (t) = [(1 - PU(t)) * θ N(t)] + [PU(t) * θ U(t)] (3)
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
8
where the pre- and post-transitional baselines are assumed to be linearly-dependent on
temperature, and with θ N(0) and θ U(0) as 0 oC intercepts, respectively:
θ N(t) = θ N(0) – m * T, (4)
and
θ U(t) = θ U(0) – m * T. (5)
The calculated fraction of the unfolded state, PU(t), is given by:
PU(t) = exp [(-∆GU(t) /RT)] / [1 + exp (-∆GU(t) /RT)], (6)
where ∆GU(t) is the apparent Gibbs free energy of folding described by the Gibbs-
Helmholtz equation:
∆GU(t) = ∆Ho (1 - T / T m) + ∆Cp (T - T m) - T ln ( T / T m ) (7)
where tm is the temperature midpoint of the thermal transition, ∆Ho is the apparent
enthalpy of unfolding and ∆Cp is the change in heat capacity change associated with
protein unfolding. Although ∆Cp is temperature dependent (21,22), but in the narrow
temperature range of our experiments (5 – 60 oC), this term is generally insensitive to
changes (23). These thermodynamic parameters were fitted using the program Igor Pro
(WaveMetrics, Inc.) with the protocol described in (20).
Chemical denaturation Monitored by Circular Dichroism
For chemical denaturation experiments, the stock peptide solution was diluted with
appropriate volumes of benign buffer and a stock solution of 10.0 M urea in benign
buffer to give a series of data points in increasing denaturant concentration. Data points
were left to equilibrate overnight before scanning and to ensure accuracy, selected data
points were rescanned to ascertain proper buffer equilibration. The data were fitted to a
linear extraction method (LEM) described previously in (15) to determine denaturation
midpoint, slope associated with the transition and the change in free energy associated
with the transition, ∆Gu. A two-state unfolding model was used to derive peptide
stability values from urea denaturation results:
ff = ([θ ]obs-[θ ]u)/([θ ]f-[θ ]u) (8)
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
9
where [θ ]f and [θ ]u represents the mean residue molar ellipticity for the fully folded and
unfolded species, respectively, and the [θ ]obs is the observed molar ellipticity at a given
denaturant concentration. The free energy of unfolding was derived from the equation:
∆GU = RT ln Ku (9)
where Ku is the equilibrium constant of the unfolding process. In the case of disulfide-
bridged peptides, where the unfolding process is concentration independent, Ku can be
simplified as:
Ku = (1- ff)/(ff) (10)
thus,
∆GU = RT ln (1- ff)/(ff). (11)
Estimates of the free energy of unfolding in the absence of denaturant, ∆GU(water) and
slope term m were obtained by linear extrapolation to zero:
∆GU = ∆GU(water) – m[denaturant] (12)
(24,25), where m is the slope associated with unfolding.
Differential Scanning Calorimetry
Excess heat versus temperature for the peptides was determined using a Microcal
differential scanning calorimeter (Microcal, Northampton, MA). Sample concentrations
ranged from 105 to 140 µM of coiled-coil dimer, and peptides were dissolved in 100 mM
KCl, 50 mM PO4, pH 7.0 buffer. The sample solutions and buffer were filtered and
degassed under vacuum and stored at 5 oC. Buffer scans were repeated until identical
baselines were achieved. The heating rate was 60 oC per hour and the cooling rate was
90 oC per hour, with the excess heat monitored from 5 to 70 oC. Each sample was heated
and cooled for three cycles to ensure folding reversibility. Data analyses were carried out
in Microcal Origin software (Microcal DSC, version 1.2a) using a two-state model with
change in heat capacity.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
10
RESULTS
Design of the α-helical Coiled-coils with Different Hydrophobic Clustering
The peptides used in this study were modeled on heptad sequences that had strong α-
helical potential and a heptad repeat (gabcdef)n where non-polar residues at positions a
and d facilitates coiled-coil formation. In the design of these hydrophobic clustered
peptides, we took advantage of the features of the successful α-helical coiled-coil models
in our laboratory (6,7,9-11), for example, complementary packing in the protein core
(26), balance of charged residues across the coiled-coil interface in heptad positions e and
g (27,28), and a flexible disulfide-bridge linkage (29). The coiled-coil sequences
consisted of eight heptads (56 residues) based on two repeating heptad sequences:
ExEAxKA and KxEAxEG where positions x are hydrophobic core positions occupied by
Ala, Ile or Leu in positions a or d (Fig 1.). We defined a hydrophobic cluster as a
consecutive string of three large non-polar residues (Ile or Leu) in the core positions of
the coiled-coil. In our coiled-coils, non-polar residues Leu, Ile, Leu in the consecutive d,
a, d heptad positions defined a stabilizing hydrophobic cluster. Our approach was to
design two proteins with identical inherent hydrophobicity, i.e., identical number and
character of non-polar residues in equivalent coiled-coil core positions but with a
different arrangement, i.e., P3 having three hydrophobic clusters and P2 having two
(Fig.1, rectangular boxes). The N-terminal hydrophobic cluster of P3 was disrupted by
an interchange of Ile at position 9 and Ala at position 16, both at heptad a positions, to
give P2. (Fig. 1, bottom). Therefore, the two analogs have identical inherent
hydrophobicity, but different clustering patterns. The hypothesis is that the hydrophobic
clusters are independent units that contribute to coiled-coil stability and folding when
separated along the coiled-coil chain by consecutive strings of Ala residues (Fig. 1, open
circles). Thus, this pattern of large and small non-polar core residues helped distinguish
the contribution of a hydrophobic cluster from inherent hydrophobicity. Inter-chain and
intra-chain ionic interactions were engineered by placing Lys and Glu at positions b, e
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
11
and g resulting in ionic stabilization due to inter-chain electrostatic attractions (i to i’ + 5
or g to e’) and intra-chain ionic attractions (i to i +3 or i to i + 4). To promote coiled-coil
formation, a C-terminal disulfide bridge, Gly-Gly-Cys-Tyr linker was introduced to
faciliatate the formation of a parallel and in-register coiled-coil, and the single Tyr
residue allows for protein concentration determination by UV spectroscopy. The
disulfide bridge was distant from the N-terminal hydrophobic cluster under investigation
(Fig. 1).
Secondary Structure Characterization by Circular Dichroism
Circular dichroism is a sensitive probe of secondary structural features, and this
technique was used to detect the difference in helical content between the two peptides.
Reduced P3 and P2 peptides were helical in benign condition (approximately 50%
α-helical), but in the helix-promoting environment of 50% TFE (30), significant helical
structure was induced in both peptides (Table 1). Though the amino acid sequences of
these peptide have a strong underlying helical propensity, potential stabilizing ionic
interactions and amphipathicity, in the reduced state, there is insufficient hydrophobic
stabilization to overcome the monomer-dimer equilibrium, at the concentration of ≈
200µM (Table 1), to form a fully folded coiled-coil. An interchain disulfide-bridge has
been shown to enhance coiled-coil folding and stability by eliminating the concentration-
dependent monomer-dimer equilibrium that prevented folding (7,31). In contrast to the
reduced peptides, both the disulfide-bridged two-stranded coiled-coils P3 and P2
exhibited more helical structure, and the P3 coiled-coil with three hydrophobic clusters
was fully folded (98% α-helical) at room temperature (Fig. 2, Table 1). The P2 coiled-
coil with two hydrophobic clusters was only 65 % folded at 20 oC in benign buffer
though more helicity was induced at 5 oC (Fig. 3, Panel A). P3 coiled-coil showed a
[θ ]222/208 = 1.02, indicative of a fully-folded coiled-coil (32) whereas that of P2 coiled-
coil is less than one (0.74) due to the presence of the single-stranded unfolded state (Fig.
2.). Thus without the presence of the third hydrophobic cluster, P2 did not fully fold in
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
12
benign condition. In 50% TFE, both the disulfide-bridged peptides showed nearly 100%
helical content, and the [θ ]222/208 ratios for P3 and P2 were respectively, 0.91 and 0.89,
indicative of the single-stranded α-helical conformation (32). The significant helix
induction for P2 in TFE, an increase of 35% helicity, showed the underlying high helical
propensity of this sequence. However, P2 remained partially unfolded in benign
condition because of insufficient hydrophobic stabilization in the hydrophobic core
(having only 2 clusters).
Sedimentation Equilibrium Analyses of Oligomeric States
The packing of hydrophobic core residues had been shown to affect the oligomerization
states of coiled-coils (9,10). To determine that P2 and P3 had the same oligomerization
state, sedimentation equilibrium analyses were carried out. Using different protein
concentrations (from 50 – 500 µM) and rotor speeds (20,000 – 35,000 rpm), we found
that the oligomerization behavior of coiled-coils P3 and P2 were best-fitted to that of a
single homogeneous species with molecular weights consistent with a two-stranded
coiled-coil (Fig. 2 and Table 1). Overall, sedimentation equilibrium analyses showed that
the interchange of hydrophobic residues Ile and Ala that distinguish P2 and P3 did not
change the overall oligomerization state, i.e, these coiled-coils are two-stranded and do
not assemble into high-order oligomerization states. Therefore we attributed the
difference in helicity to a difference in stability due to the presence of an additional
stabilizing hydrophobic cluster in peptide P3.
Comparison of Coiled-coil Stability by Thermal and Urea Denaturation
The P3 coiled-coil with three hydrophobic clusters exhibited a highly co-operative
unfolding similar to that of native proteins (Fig. 3). In contrast, P2 coiled-coil was only
partially folded with marginal stability. We determined the stability of these two analogs
using thermal and urea denaturation and found that P3 was significantly more stable than
P2, with an increase of thermal midpoint of more than 18 oC, and a corresponding
increase in urea denaturation midpoint of approximately 1.5 M (Fig. 3, Table 2). To
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
13
calculate the free energy difference between these two analogs, the linear extrapolation
method was used to evaluate the chemical denaturation data (Table 2) because it is
difficult to estimate thermodynamics parameters ∆H and ∆Cp from non-ideal thermal
melting curves (observed in P2). In addition, a more accurate protocol would be to
estimate the free energy change from chemical denaturation, because the slope around the
urea denaturation transition is reliable (26). The free energy contribution of the N-
terminal hydrophobic cluster in the P3 coiled-coil to stability was estimated to be 2.1
kcal•mol-1 per coiled-coil, and this difference in stability can explain why P2 coiled-coil
did not fully fold. The slope term m associated with the transition for P3 was
significantly higher than that of P2 (Table 2), and might describe the non-ideal behavior
of the poorly formed secondary structure of P2. The m slope value generally correlates
with the magnitude of changes in the non-polar accessible surface area between the
folded and the unfolded state (33).
Differential Scanning Calorimetry of Coiled-coils
In addition to the CD experiments, differential scanning Calorimetry (DSC) was also
used to measure the contribution of hydrophobic clusters to coiled-coil stability. This
technique is one of the most accurate methods for evaluating protein unfolding transitions
and analyses of enthalpy and heat capacity changes of temperature-induced processes
(34). P3, the three-clustered peptide, showed a cooperative transition with a defined pre-
and post-transitional baseline (Fig. 4), but the 2-clustered peptide, P2, did not exhibit any
measurable transition peak (results not shown), thus illustrating the enthalpic and entropic
disruption of the coiled-coil due to the loss of a hydrophobic cluster. Despite the fact that
these two coiled-coils have the potential to bury nearly identical hydrophobic surface
area, our results suggested that the two-state behavior of peptide unfolding/folding
required a minimum of three hydrophobic clusters to form a stably folded protein (Fig. 4,
∆Tm ≈ 42 0C). We then compared the thermodynamics parameter ∆H and Tm for P3
obtained from thermal melting and DSC experiments and found them to be in excellent
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
14
agreement (Table 2.). The estimated ∆Cp was 1.62 kcal•mol-1•
oC-1, which equated to
13.5 cal•mol-1•
oC-1 per residue; this value was a relative measurement of the difference in
exposed non-polar surface area between the folded and unfolded states (24, 36) and
agreed well with published values of those of small soluble proteins (35), typically in the
range of 8-15 cal•mol-1•
oC-1 per residue.
DISCUSSION
Hydrophobic interactions contribute significantly to protein stability because the burial of
non-polar surface area is thermodynamically favorable in aqueous solution, and this study
has shown that the hydrophobic stabilization is context dependent. In the coiled-coil
model, the additional cluster of three large non-polar residues in P3 enhances the folding
of secondary structure and protein stability when compared to P2 where this cluster is
missing. Both the three-clustered P3 and the two-clustered P2 peptides have the same
inherent hydrophobicity (6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core), yet
their folding and stability differ dramatically. P3 with three hydrophobic clusters is a
native protein-like two-stranded coiled-coil with a co-operative unfolding transition. In
contrast, P2 with two hydrophobic clusters is only partially folded and significantly less
stable compared to P3. Furthermore, P3 coiled-coil showed a single unfolding transition
in DSC, whereas the unfolding transition was not measurable with P2 under identical
experimental conditions. Thus the disruption of the hydrophobic cluster in P2 drastically
decreases enthalpy component of hydrophobic stabilization, which would affect the
packing of the two interacting α-helices. The deconvolution of the entropic and the
enthalpic components of hydrophobic-residue mutations, i.e., Leu to Ala mutation, is still
controversial; for example, Dũrr and Jelesarov found that the hydrophobic destabilization
of a Leu to Ala substitution is largely entropic at room temperature but becomes
increasingly enthalpic at higher temperature (37).
Historically, hydrophobic cluster analysis (HCA) structural prediction algorithm, based
on the principles that hydrophobic amino acids cluster together in native folded state, has
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
15
been employed to good effect in identifying proteins with little sequence homology, but
with similar overall protein topology (reviewed in 38). Although HCA does not predict
the hydrophobic effect or protein stability, our results clearly show that significant
stabilization can be achieved when hydrophobes cluster in the coiled-coil core.
Supporting the results from these prediction programs, experimental data on GCN4
coiled-coil folding suggested that the high-energy folding transition state is a
hydrophobic collapsed form that contained little secondary structure (39), and therefore,
the hydrophobic effect can be an early folding determinant of protein folding, whereas
formation of helical secondary structure occurs later.
Hydrophobic clustering may play a significant role in the structure and function of long
native coiled-coil proteins. In a recent study on the assembly of tropomyosin, a 284-
residue coiled-coil, Silva and co-workers observed that independent folding subdomains
with different susceptibility to pressure were evident along its length (40). When
subjected to increase pressure, tropomyosin melted in discrete cooperative blocks along
the molecule, and the unfolded domains were likely unstable sites along the coiled-coil
chain (40). We examined the hydrophobic clustering in the sequence of tropomyosin and
indeed found 9 hydrophobic clusters occupied by 3, 4, 5 consecutive large hydrophobes
(I, L, M, V, Y, F) in the core a and d positions, separated by destabilizing residues (A, S,
T, Q, D, E and K) in the hydrophobic core (Fig. 5) (41). The authors of that study
proposed that increasing osmotic pressure induced water molecules to infiltrate into the
less stable “pressure-sensitive” domains in hydrophobic core of tropomyosin, but the
more stable, “pressure-insensitive” domains maintain coiled-coil integrity. It is
interesting, that our clusters of large hydrophobes (Ile, Leu) and small hydrophobes (Ala)
could correspond to the “pressure-insensitive” and “pressure-sensitive” domains,
respectively. Hydrophobic clustering may be an important mechanism for long native
coiled-coil proteins to maintain chain integrity yet still accommodate the burial of polar
and charged residues to control stability and facilitate different biological functions.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
16
Regardless of the length of the coiled-coil, hydrophobic clusters can serve as “knots” to
keep the chain together while allowing flexible regions for function.
Hydrophobic clusters control protein stability, and is perhaps an evolutionary feature for
native proteins to incorporate structurally-important stabilizing regions, as well as less
stable and more flexible functional domains in a single protein fold. The destabilizing
cluster regions of a coiled-coil may be involved in conformation change that allows for
protein-protein interactions. For example, the less stable core regions of tropomyosin
could be more easily disrupted for interactions with other proteins (members of the
troponin complex and actin involved in muscle regulation) (42). Another example of the
importance of flexibility in coiled-coils is the so-called "spring-loaded" mechanism for
viral fusion by the hemagglutinin protein of influenza (43), which allows viral entry into
host cells. Conformational change in coiled-coil domains has been implicated, for
example, in kinesin, kinesin-like, dynein motor proteins, intermediate filament proteins
and tropomyosins (44-50) In addition to mediating protein interactions, hydrophobic
residues in the coiled-coil core have been known to control oligomerization state in de
novo designed coiled-coils (9-11) and maintain chain orientation in GCN4 by an Asn-Asn
pairing (51). Thus destabilizing clusters could also be involved in these roles. Lastly,
hydrophobic clusters are natural nucleation sites for protein folding, since they provide
the necessary hydrophobic stabilization for early folding intermediates (40). Further
investigations into the structural and functional roles of hydrophobic clustering will
improve our understanding of the mechanism of coiled-coil folding and the folding of
proteins in general.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
17
REFERENCES:
1. Dill, K.A. (1999) Protein Sci 8, 1166-1180.
2. Yao, J., Chung, J., Eliezer, D., Wright, P.E., & Dyson, H.J. (2001) Biochemistry 12,
3561-3571.
3. Colon, W., Elove, G.A., Wakem, L.P., Sherman, F., & Roder, H. (1996) Biochemistry
35, 5538-5549.
4. Shortle, D., & Ackerman, M.S. (2001) Science 293, 487-490.
5. Klein-Seetharaman, J., Oikawa, M., Grimshaw, S.B., Wirmer, J., Duchardt, E., Ueda,
T., Imoto, T., Smith, L.J., Dobson, C.M., & Schwalbe, H. (2002) Science 295, 1719-
1722.
6. Hodges, R.S., Zhou, N.E., Kay, C.M., & Semchuk, P.D. (1990) Pept Res. 3, 123-137.
7. Zhou, N.E., Zhu, B.-Y. & Kay, C.M., & Hodges, R.S. (1992) Biopolymers 32, 419-
426.
8. Lupas, A. (1996) Trends Biochem Sci. 21, 375-382.
9. Wagschal, K., Tripet, B., & Hodges, R.S. (1999a) J. Mol. Biol. 285, 785-803.
10. Wagschal, K., Tripet, B., Lavigne, P., Mant, C.T. & Hodges, R.S. (1999b)
Protein Sci. 11, 2312-2329.
11. Tripet, B., Wagschal, K., Lavigne, P., Mant, C.T., & Hodges, R.S. (2000) J. Mol.
Biol. 300, 377-402.
12. Hodges, R.S., Semchuk, P.D., Taneja, A.K., Kay, C.M., Parker, J.M., & Mant, C.T.
(1988) Pept Res. 1, 19-30.
13. Sereda, T.J., Mant, C.T., Quinn, A.M., & Hodges, R.S. (1993). J Chromatogr. 646,
17-30.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
18
14. Merrifield, B. (1997) Meth. Enzymol. 289, 3-13.
15. Kwok, S.C., Mant, C.T., & Hodges, R.S. (2002) Protein Sci. 11, 1519-1531.
16. Mant, C.T., Kondejewski, L.H., Cachia, P.J., Monera, O.D., & Hodges, R.S. (1997)
Meth. Enzymol. 289, 426-469.
17. Johnson, M.L., Correia, J.J., Yphantis, D.A., & Halvorson, H.R. (1981) Biophys J.
36, 575-588.
18. Cooper, T.M., & Woody, R.W. (1990) Biopolymers 30, 657-676.
19. Santoro, M.M., & Bolen, D.W. (1988) Biochemistry 21, 8063-8.
20. Lavigne, P., Kondejewski, L.H., Houston, M.E. Jr,, Sonnichsen, F.D., Lix, B., Sykes,
B.D., Hodges, R.S., & Kay, C.M. (1995) J. Mol. Biol. 254, 505-520.
21. Makhatadze, G.I., & Privalov, P.L. (1995). Adv. Protein. Chem. 47, 307-425.
22. Gomez, J., Hilser, V.J., Xie, D., & Freire, E. (1995) Proteins 22, 404-412.
23. Betz, S.F., Liebman, P.A., & DeGrado, W.F. (1997) Biochemistry 36, 2450-2458.
24. Pace, C.N. (1986) Meth. Enzymol. 131, 266-280.
25. Shortle, D. (1989) J. Biol. Chem. 264, 5315-5318.
26. Kellis, J.T. Jr., Nyberg, K., Fersht, A.R. (1989) Biochemistry 28, 4914-4922.
27. Kohn, W.D., Kay, C.M., & Hodges R.S. (1997) J Mol Biol. 267, 1039-1052
28. McClain, D.L., Binfet J.P., & Oakley, M.G. (2001) J Mol Biol. 313, 371-383.
29. O’Shea, E..K., Klemm, J.D., Kim, P.S., & Alber, T. (1991). Science 254, 539-544.
30. Sonnichsen, F.D., Van Eyk, J.E., Hodges, R.S., & Sykes, B.D. (1992) Biochemistry
37, 8790-8798.
31. Zhou, N.E., Kay, C.M., Sykes, B.D., & Hodges, R,S. (1993) Biochemistry 32, 6190-
6197.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
19
32. Lau, S.Y.M., Taneja, A.K., & Hodges, R.S. (1984) J. Biol. Chem. 317, 129-140.
33. Myers, J.K., Pace, C.N., & Scholtz, J.M. (1995). Prot. Sci. 4, 2138-2148.
34. Privalov, P.L., & Potekhin, S.A. (1986). Meth. Enzymol. 131, 4-51.
35. Makhatadze, G.I., & Privalov, P.L. (1990). J. Mol. Biol. 213 , 385-391.
36. Xie, D., & Freire E. (1994) J. Mol. Biol. 242, 62-80.
37. Dűrr, E., & Jelesarov, I. (2000) Biochemistry 39, 4472-4482.
38. Callebaut, I., Labesse, G., Durand, P., Poupon, A., Canard, L., Chomilier, J.,
Henrissat, B., & Mornon J.P. (1997) Cell Mol Life Sci. 53, 621-645.
39. Sosnick, T.R., Jackson, S., Wilk, R.R., Englander, S.W., & Degrado, W.F. (1996)
Proteins 24, 427-432.
40. Suarez, M.C., Lehrer, S.S., & Silva, J.C. (2001) Biochemistry 40, 1300-1307.
41. Sodek, J., Hodges, R.S., Smillie, L.B., Jurasek, L. (1972) Proc. Natl. Acad. Sci. U.
S. A. 69, 3800-3804.
42. Tripet, B., Van Eyk, J.E., Hodges, R.S. (1997) J Mol Biol 271, 728-750.
43. Carr, C.M., & Kim, P.S. (1993) Cell 73, 823-832.
44. Tripet, B., & Hodges, R.S. (2002) J Struct Biol 137, 220-235.
45. Chana, M., Tripet, B.P., Mant, C.T., Hodges, R.S. (2002) J Struct Biol 137, 206-
219.
46. Thomasin, A.S., Strelkov, S.V., Burkhard, P., Aebi, U., & Parry, D.A.D. (2002) J
Struct Biol 137, 128-145.
47. Araya, E., Berthier, C., Kim, E., Yeung, T., Wang, X., & Helfman, D.M. (2002) J
Struct Biol 137, 176-183.
48. Gee, M.A., & R.B. Vallee. (1998) Eur Biophys J 27, 466-473.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
20
49. Vallee, R.B., & Gee, M.A. (1998) Trends Cell Biol 8, 490-494
50. Gee, M.A., Heuser, J.E., & Vallee, R.B. (1997) Nature 390, 636-639
51. Gonzalez, L. Jr., Woolfson, D.N., & Alber, T. (1996) Nat Struct Biol 12, 1011-1018.
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
21
Fig. 1. Peptide nomenclature, sequences and schematics of the coiled-coil analogs used
in this study. Top panel shows the sequence of two peptides, P3 and P2 which contain 3
and 2 clusters, respectively. The heptad repeat is denoted as gabcdef which is shown
above the first heptad of the coiled-coil. Bottom panel shows a schematic representation
of the hydrophobic residues in positions a and d where large hydrophobes are shown by
dark circles (Leu, Ile) and small hydrophobes are shown by open circles (Ala). Three
continuous large hydrophobic residues in the core positions d, a, d constitute a
hydrophobic cluster (rectangular box).
Fig. 2. Biophysical characterization of the two-stranded disulfide-bridged coiled-coil
peptides: secondary structural content and oligomeric state. Panel A: Circular dichroism
spectra of cluster peptides in benign condition (50 mM PO4 (K2HPO4/KH2PO4), 100 mM
KCl, pH 7.0 at 20 oC). The concentration of P3 (•) and P2 (o) are 103 and 110 uM,
respectively. Panel B: Sedimentation equilibrium of cluster peptides in benign condition
at 20,000 rpm: the residuals are shown above the UV absorbance data which were best
fitted to a single-species (two-stranded disulfide-bridged coiled-coil model). Initial
peptide concentrations for P3 and P2 were 100 µM.
Fig. 3. Stability comparison of the P3 (3 cluster) and P2 (2 cluster) peptides. Panel A,
temperature melting of the coiled-coils monitored by circular dichroism spectroscopy at
222 nm. Experimental condition was 50mM PO4 (K2HPO4/KH2PO4), 100mM KCl, pH
7.0 buffer at a starting temperature of 5 oC. Panel B, urea denaturation experiments were
carried out at 20°C in a 50mM PO4 (K2HPO4/KH2PO4), 100 mM KCl, pH 7.0 buffer with
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Hydrophobic Clusters Affect Protein Stability
22
increasing concentrations of urea as denaturant. Peptide P3 (•) and peptide P2 (o) had
respective concentrations of 103 and 110 uM of disulfide-bridged two-stranded coiled-
coil. The ellipticity of peptide P3 was taken as fully-folded to determine fraction folded.
Fig. 4. Stability profile of the 3-clustered P3 peptide: Thermal melt of P3 peptide as a
function of temperature (•) overlaid on the differential scanning calorimetry profile (o).
Experiment condition was 50 mM PO4 (K2HPO4/KH2PO4), 100 mM KCl, pH 7.0 and
peptide concentration was 103 µM of the disulfide-bridged two-stranded peptide.
Fig. 5. Schematic representation of the hydrophobic core residues, in heptad positions a
and d, of the 284-residue coiled-coil protein tropomyosin. Large hydrophobic residues
are shown by dark circles (Leu, Ile, Val, Met, Phe, Tyr) and small nonpolar, polar and
charged residues are shown by open circles (Ala, Ser, Thr, Gln, Asp, Glu, and Lys)).
Three or more continuous large hydrophobic residues in the core positions constitute a
hydrophobic cluster (rectangular box).
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Table 1 23
P3
P2
Table 1. Biophysical Characterization of the Oxidized and Reduced Hydrophobic Cluster Peptides
PeptideName a
-35,100
-34,600
[θ ]222 (reduced) b
Benign(20° C)
50%TFE
- 34,200
- 22,800
-34,900
-35,200
[θ ]222 (oxidized) c
Benign(20° C)
50%TFE
- 18,800
- 16,500
a Peptides are named by the number of stabilizing hydrophobic clusters shown in Figure 1.b [θ ]222 (reduced) is the mean residue molar ellipticity measured at 222 nm in a reducing buffer 2 mM dithioerythritol (DTE), 50 mM PO4
(K2HPO4/KH2PO4), 100 mM KCl buffer, pH 7.0 in the absence (Benign) or presence of 50% trifluoroethanol (TFE, v/v) at 20 oC. Peptide concentrations were 206 uM and 220 uM (of monomer) for P3 and P2, respectively.
c [θ ]222 (oxidized) is the mean residue molar ellipticity measured at 222 nm in a non-reducing buffer of 50 mM PO4 (K2HPO4/KH2PO4), 100 mM KCl buffer, pH 7.0 in the absence (Benign) or presence of 50% trifluoroethanol (TFE, v/v) at 20 oC. Peptide concentrations were 103 uM and 110 uM (of dimer) for P3 and P2 respectively.
d % Helix was calculated from [θ ]222 based on the benign value divided by the value at 50% TFE multiply by a 100.e The helical ratio [θ ]222/208 was calculated by dividing the observed molar ellipticity value at 222 nm by that at 208 nm.f Apparent molecular weights were determined from sedimentation equilibrium analyses of disulfide-bridged peptides.g Oligomeric state was calculated by dividing the apparent molecular weight by the mass of the disulfide-bridged two-stranded peptide (theoretical MW: 12334).
14,800
13,700
1.2
1.1
Sedimentation Equilibrium
ApparentM.W. f
OligomerRatio g
% Helix (reduced) d
Benign(20° C)
53.6
47.7
% Helix (oxidized) d
Benign(20° C)
98.0
64.8
[θ ] 222/208 e
0.74
0.69
[θ ] 222/208 e
1.02
0.74
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Table 2 24
Table 2. Thermodynamics of Clustered Peptides and Stability Contribution of Hydrophobic Clustering
a Peptides are named by the number of stabilizing hydrophobic clusters shown in Figure 1.b Tm (CD) is the temperature at which there is a 50% decrease in molar ellipticity, [θ ]222, compared to the fully-folded peptide with 3 clusters as determined by circular dichroism spectroscopy.c [Urea]1/2 is the denaturation midpoint of the two-state unfolding of an α-helical coiled-coil to a random coil, i.e., the urea concentration (M) required to achieve a 50% decrease in molar ellipticity, [θ ]222, with a fully-folded coiled-coil taken as –34,200 at 20 oC. d m is the slope term defined by the equation ∆Gu = ∆G(H20) - m [denaturant], and ∆G(H20) is the free energy of denaturation in the absence of denaturant whereas ∆Gu is the free energy of unfolding at a given denaturant concentration.e ∆∆Gu is the free energy difference contributed by a stabilizing cluster: ∆G(H20) (3 Clusters – 2 Clusters).f Tm (DSC) is the temperature midpoint associated with the transition peak during differential scanning calorimetry. g ∆H (CD) is the change in enthalpy derived from the thermal denaturation experiment using CD spectroscopy.h ∆H (DSC) is the change in enthalpy derived from the calorimetry experiment.i ∆Cp is the change in heat capacity associated with the the denaturation transition observed in the differential scanning calorimetry experiment.
PeptideName a
Tm (melting) b(oC)
[Urea]1/2c
(M)m, slope d
(kcal•mol-1•M)∆G (H20)
d
(kcal•mol-1)
2.10
0.65
1.35
0.67
2.92
0.86
41.6
23.1
2.1
-
P3
P2
∆∆G (H20)e
(kcal•mol-1)
PeptideName a
42.3
Tm (CD) b(oC)
40.041.6
Tm (DSC) f(oC)
∆H (DSC) h(kcal•mol-1)
∆H (CD) g(kcal•mol-1)
∆Cp (DSC) i(kcal•mol-1 •C-1)
42.9 1.62P3
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Figure 1 25
Amino Acid SequencesPeptideName
P3P2
gabcdefAc-EAEALKA-EIEALKA-KAEAAEG-KAEALEG-KIEALEG-KAEAAEG-KAEALEG-EIEALKA-GGCY-amAc-EAEALKA-EAEALKA-KIEAAEG-KAEALEG-KIEALEG-KAEAAEG-KAEALEG-EIEALKA-GGCY-am
PeptideName
Schematic Representation of Hydrophobic residues at a and d positions
P3P2
a d a d a d a d a d a d a d a d
HydrophobicClusters
3 Clusters2 Clusters
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Figure 2 26
-32,000
-24,000
-16,000
-8,000
205 215 225 235
0
0.30
0.40
0.50
0.60
17.5 18 18.5 20.5 21 21.5
-0.02
0.00
0.02
17.6 18.0 18.4 20.6 21.0 21.4
radius2 / 2 (cm2) radius2 / 2 (cm2)Ab
sorb
ance
R
esid
uals
wavelength (nm)
P2P3
[θ ]
(deg●c
m2 ●
dmol
-1)
A B
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Figure 3 27
Temperature (oC)
Frac
tion
Fold
ed
[Urea] (M)
.
0
0.2
0.4
0.6
0.8
1
10 20 30 40 50 60 70 1 2 3 4
A B
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Figure 4 28
0
500
1000
1500
2000
2500
3000
3500
15 25 35 45 55 65 75
-35,000
-30,000
-25,000
-20,000
-15,000
-10,000
-5,000
0
[θ] 22
2(d
eg●c
m2 ●
dmol
-1)
Temperature (oC)
Cp
(cal●mol -1●
oC-1)
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Figure 5 29
1 99
193
100 190
284
by guest on May 19, 2018 http://www.jbc.org/ Downloaded from
Stanley C. Kwok and Robert S. Hodgescoiled-coils control protein folding and stability
-helicalαClustering of large hydrophobes in the hydrophobic core of two-stranded
published online July 3, 2003J. Biol. Chem.
10.1074/jbc.M305306200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on May 19, 2018
http://ww
w.jbc.org/
Dow
nloaded from