Effect of dodecyl trimethylammonium bromide on folding and stability
of alkaline and acid-denatured cytochrome c
JAMSHID CHAMANI
Department of Biology, Faculty of Science,
Azad University of Mashhad, Mashhad,
IRAN.
.
Abstract: The molten globule (MG) state can be an intermediate in the protein folding pathway; thus, its
detailed description can help understanding protein folding. Dodecyl trimethylammonium bromide cationic
surfactant that is commonly used to mimic hydrophobic binding environments such as cell membranes, are
known to denature some native state proteins, including horse cytochrome c (cyt c). In this article, refolding of
alkaline and acid-denatured cyt c are studied under the influence of dodecyl trimethylammonium bromides to
form MG-like states at both low concentration (pH 11) and above the critical micelle concentration (pH 2)
using ultraviolet and visible absorption, fluorescence and circular dichroism (CD). The addition of dodecyl
trimethylammonium bromide to the unfolded state of cyt c at alkaline and acidic condition appears to support
the stabilized form of the MG state. Based on the results obtained, the merits of two models of the proteinsurfactant structure are discussed for various dodecyl trimethylammonium bromide concentration in inducing
of MG state at two different pH conditions. Therefore, hydrophobic interactions play a dominant role in
stabilizing the MG state.
Keywords: cytochrome c; cationic surfactants; molten globule-like state; protein folding, hydrophobic
interaction
1 Introduction
Cytochrome c (cyt c) plays an important role in
the biological electron transfer system and has been
extensively studied [1-3], including having its fully
resolved three-dimensional structure determined by
X-ray and nuclear magnetic resonance (NMR) [46]. For cyt c, several probes (IR, UV-Vis, CD,
fluorescence) can be used to monitor the structural
changes needed to obtain the variety of states
accessible under different solution perturbations
(GuHCl, urea, pH, temperature, etc.) [7-15]. When
acidified, cyt c is denatured to a primarily random
coil structure, destabilized due to the electrostatic
repulsion between positively charged residue. A
molten globule (MG) state of cyt c can be achieved
by adding salt to this acid-denatured state, whereby
the electrostatic repulsion is reduced, which is
believed to drive the protein to become more
compact [16]. That state is characterized by high
helical content in its secondary structure, but with
little evident tertiary structure [17, 18].
It is important to elucidate the structure and
stabilizing mechanism of the MG state, as an
intermediate between the native and denatured
states, in order to understand the principles for
constructing a three-dimensional protein structureX-ray small angle scattering studies have shown
that the MG states of various proteins have a wide
range of structures from relatively disordered to
highly [19-21]. This implies that the MG state is a
largely fluctuating ensemble of various energy
minimums. Moreover, the stability of the MG state
is determined by a delicate balance of interactions,
such as electrostatic repulsion between charged
residues and opposing forces such as hydrophobic
interaction. A significant influence of salts or
charges on the stability of the MG state reveals that
the main driving force of the MG state is the
reduction of electrostatic repulsion between
charged residue, which favours unfolded
conformations [17, 22, 23]. However, there is a
lack of substantial evidence regarding the
contribution of hydrophobic interactions to the
stability of the MG state. However such
interactions have been suggested for the positive
heat capacity changes of the thermal unfolding of
the MG of apomyoglobin [24, 25] and cyt c [2628].
In this article, the interaction between
alkaline and acid-denatured cyt c and dodecyl
trimethylammonium bromide is studied. MG-like
states can be achieved at very low concentrations
and
above
the
CMC
of
dodecyl
trimethylammonium bromides at alkaline and
acidic conditions respectively. Recently, other
workers also showed the formation of MG-like
states of cyt c induced by low concentration n-alkyl
sulfates [29]. Those authors suggested that
hydrophobic interactions play an important role in
stabilizing the MG state. Another paper on cyt c in
surfactant appeared, which focused on urea
unfolding and refolding with SDS. In this present
work, by comparing the results of different kinds of
cationic surfactant-induced MG-like states, we
propose that the primary driving force for the
formation of cyt c-dodecyl trimethylammonium
bromides induced MG-like states is the reduction
of electrostatic repulsion, although the hydrophobic
effect dose remain a factor.
2 Materials and methods
wavelength range (280-295) nm, it was
experimentally preferable to use a 280 nm
excitation to reduce any light scattering problems
in the emission spectra on this instruments). Trp
fluorescence for a--LA and h--LA forms was
followed at 335 nm and 325 nm respectively [31].
The Cu2+ and DTAB significantly affect the
fluorescence of free tryptophan under the
experimental conditions used. The temperature of
the cell compartments was kept constant at T=293
K by water circulations.
2.2.2 Circular dichroism (CD) measurements
Far-UV experiments were performed on a Jasco810 spectropolarimeter equipped with a Jasco 2syringe titrator. Spectra were recorded with protein
concentrations 5 M in a 1-mm-path length quartz
cuvette. A bandwidth of 1 nm and a response of 2
sec were used, with a scanning rate at 50 nm / min
to obtain final spectra as an average of four scans.
The instruments were calibrated with ammonium
d-10-camphorsulfonic acid [46]. The results are
expressed as the mean residue ellipticity [], which
is defined as [] = 100 x obsd / (lc), where obsd is
the observed ellipticity in degrees, c is the
concentration in residue mol.cm-3, and l is the
length of the light path in cm.
3 Results and discussion
3.1 Circular dichroism
2.1 Materials
Horse cytochrome c (type IV), in the oxidazed
form, was purchased from Sigma and used without
further purification. Dodecyl trimethylammonium
bromide (DTAB) was also obtained from Sigma.
All the pH values represent apparent pH meter
readings.
2.2 Methods
2.2.1 Fluorescence measurements
Fluorescence measurements were made on Jasco
SP-6200 spectrofluorometer at an excitation
wavelength of 280 nm (while identical spectral line
shapes were observed over the excitation
The far-UV CD spectra obtained in the titration
of acid-denatured cyt c (5 M) with dodecyl
trimethylammonium bromide (DTAB) at pH 11
were recorded as shown in Figure 1. Three phases
can be found in this titration process, starting with
the initial acid-denatured state, which can be
viewed as dominantly a random coil showing a
negative peak at 195 nm and a broad negative
band at 200-220 nm. For [DTAB] < 0.3 mM
(Figure 1), formation of secondary structure from
the acid-denatured state was observed as an
ellipticity (negative) gain at both 222 nm and 207
nm, which is typical of -helix. This helix
formation is almost linearly dependent on the
concentration of DTAB up to 0.3 mM, as can be
seen from the ellipticity change at 222 nm (inset),
with a stable MG-like state being formed at [SDS] /
[cyt c]  12. The inset curve marked in diamonds
represents the titration of acid-denatured cyt c with
DTAB. A sigmoidal curve can be fit to this
ellipticity change, but the breadth of the transition
calls into question the possibility of a two-state
transition. For [DTAB] > 0.3 mM, the values of
ellipticity at []222 increased. It is important to note
that cyt c in the presence of high concentrations of
DTAB starts to unfold (data not shown). The
helical contents in the unfolded state and the native
state of cyt c are 4 and 30% respectively, on the
basis of the ellipticity values at 222 nm as
calculated by the method of Chen et al. [53]. The
helical content of the MG-like state of cyt c
induced by various concentrations of dodecyl
trimethylammonium bromide (DTAB), is 32.1%
according to this method.
As a comparison, a titration of 5 M cyt c at pH
2 with positive charged surfactant DTAB was
recorded at the same pH with far-UV CD, as shown
in Figure 2. Over the whole surfactant
concentration range from 0 to 50 mM, no
precipitation is observed, which differs from what
is observed at pH 11. The ellipticity change at 222
nm is plotted as an inset in Figure 2B. In the DTAB
case, ellipticity starts to increase at 6 mM, which is
the CMC for DTAB (data not shown) at pH 2 (A
surface tension test shows that the CMC for DTAB
solution at pH 2 is around 6 mM). MG-like state is
obtained at [DTAB] 17 mM, much higher than for
DTAB at pH 11, which offers charge neutralization
as well as hydrophobic interactions.
Fig. 1A, Circular dichroism spectra of cytochrome c with
(relative intensity) plotted against wavelength at various
concentrations of DTAB concentration at pH = 11. The
arrow shows the addition of DTAB to cytochrome c.
3.2Fluorescence spectra
Fluorescence spectra (Figure 2) arising from the
single Trp in cyt c were also recorded for the same
cyt-DTAB samples used in far-UV CD and Soret
absorption experiments. In the native state
structure, Trp 59 is buried in the hydrophobic core
and almost no fluorescence is observed,
presumably being quenched by Foster energy
transfer to the heme group. However, when the
protein is denatures, the Trp residue becomes
solvated and intense fluorescence results. As seen
in Figure 2, even 0.05 mM of DTAB at pH 11
induced a big decrease in the fluorescence of
alkaline-denatured cyt c, and about 80% of the
fluorescence was quenched for [DTAB] = 0.3 mM,
suggesting a hydrophobic collapse even at this low
DTAB concentration. There is a blue shift of the
peak (10 nm) accompanying this decreased
fluorescence intensity when [DTAB] is varied from
0 to 0.3 mM. When [DTAB] is above 0.3 mM, an
increase in the fluorescence is observed and a small
red shift occurs, especially at high DTAB
concentration (at pH 11), indicating that DTAB
starts to unfold the protein (data not shown). The
inset of Figure 2 shows the sigmoidal curves for
the alkaline-denatured cyt c to the MG state upon
the addition of DTAB, TTAB and HTAB at low
concentrations (pH 11). The obtained results show
the hydrophobic chain effect to the decrease of
inflection points, which agree well with UV-Vis
and CD results. According to Figure 2, the addition
of high concentrations of DTAB (in the 5-17 mM
range) to the acid-unfolded state of cyt c at pH 2
causes a decrease in the fluorescence intensity,
suggesting that a structural change at the heme
occurs within this region.
Fig. 1B, Fluorescence spectra of cytochrome c with
(relative intensity) plotted against wavelength at various
concentrations of DTAB concentration at pH = 11. The
arrow shows the addition of DTAB to cytochrome c.
Dashed line is native state of cytochrome c at pH 7.
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