Magnetic Stimulation Modulate Seizures in Epileptic Patients

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 protein- surfactant 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

helical content in its secondary structure, but with 1 Introduction little evident tertiary structure [17, 18]. It is important to elucidate the structure and stabilizing mechanism of the MG state, as an Cytochrome c (cyt c) plays an important role in intermediate between the native and denatured the biological electron transfer system and has been states, in order to understand the principles for extensively studied [1-3], including having its fully constructing a three-dimensional protein structure- resolved three-dimensional structure determined by X-ray small angle scattering studies have shown X-ray and nuclear magnetic resonance (NMR) [4- that the MG states of various proteins have a wide 6]. For cyt c, several probes (IR, UV-Vis, CD, range of structures from relatively disordered to fluorescence) can be used to monitor the structural highly [19-21]. This implies that the MG state is a changes needed to obtain the variety of states largely fluctuating ensemble of various energy accessible under different solution perturbations minimums. Moreover, the stability of the MG state (GuHCl, urea, pH, temperature, etc.) [7-15]. When is determined by a delicate balance of interactions, acidified, cyt c is denatured to a primarily random such as electrostatic repulsion between charged coil structure, destabilized due to the electrostatic residues and opposing forces such as hydrophobic repulsion between positively charged residue. A interaction. A significant influence of salts or molten globule (MG) state of cyt c can be achieved charges on the stability of the MG state reveals that by adding salt to this acid-denatured state, whereby the main driving force of the MG state is the the electrostatic repulsion is reduced, which is reduction of electrostatic repulsion between believed to drive the protein to become more charged residue, which favours unfolded compact [16]. That state is characterized by high conformations [17, 22, 23]. However, there is a lack of substantial evidence regarding the contribution of hydrophobic interactions to the the emission spectra on this instruments). Trp stability of the MG state. However such fluorescence for a--LA and h--LA forms was interactions have been suggested for the positive followed at 335 nm and 325 nm respectively [31]. heat capacity changes of the thermal unfolding of The Cu2+ and DTAB significantly affect the the MG of apomyoglobin [24, 25] and cyt c [26- fluorescence of free tryptophan under the 28]. In this article, the interaction between experimental conditions used. The temperature of alkaline and acid-denatured cyt c and dodecyl the cell compartments was kept constant at T=293 trimethylammonium bromide is studied. MG-like K by water circulations. states can be achieved at very low concentrations and above the CMC of dodecyl 2.2.2 Circular dichroism (CD) measurements trimethylammonium bromides at alkaline and acidic conditions respectively. Recently, other Far-UV experiments were performed on a Jasco- workers also showed the formation of MG-like 810 spectropolarimeter equipped with a Jasco 2- states of cyt c induced by low concentration n-alkyl syringe titrator. Spectra were recorded with protein sulfates [29]. Those authors suggested that concentrations 5 M in a 1-mm-path length quartz hydrophobic interactions play an important role in cuvette. A bandwidth of 1 nm and a response of 2 stabilizing the MG state. Another paper on cyt c in sec were used, with a scanning rate at 50 nm / min surfactant appeared, which focused on urea to obtain final spectra as an average of four scans. unfolding and refolding with SDS. In this present The instruments were calibrated with ammonium d- work, by comparing the results of different kinds of 10-camphorsulfonic acid [46]. The results are cationic surfactant-induced MG-like states, we expressed as the mean residue ellipticity [], which propose that the primary driving force for the is defined as [] = 100 x obsd / (lc), where obsd is formation of cyt c-dodecyl trimethylammonium the observed ellipticity in degrees, c is the bromides induced MG-like states is the reduction of concentration in residue mol.cm-3, and l is the electrostatic repulsion, although the hydrophobic length of the light path in cm. effect dose remain a factor. 3 Results and discussion 2 Materials and methods 3.1 Circular dichroism

2.1 Materials The far-UV CD spectra obtained in the titration of acid-denatured cyt c (5 M) with dodecyl Horse cytochrome c (type IV), in the oxidazed trimethylammonium bromide (DTAB) at pH 11 form, was purchased from Sigma and used without were recorded as shown in Figure 1. Three phases further purification. Dodecyl trimethylammonium can be found in this titration process, starting with bromide (DTAB) was also obtained from Sigma. the initial acid-denatured state, which can be All the pH values represent apparent pH meter viewed as dominantly a random coil showing a readings. 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 2.2 Methods ellipticity (negative) gain at both 222 nm and 207 nm, which is typical of -helix. This helix 2.2.1 Fluorescence measurements 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), Fluorescence measurements were made on Jasco with a stable MG-like state being formed at [SDS] / SP-6200 spectrofluorometer at an excitation [cyt c]  12. The inset curve marked in diamonds wavelength of 280 nm (while identical spectral line represents the titration of acid-denatured cyt c with shapes were observed over the excitation DTAB. A sigmoidal curve can be fit to this wavelength range (280-295) nm, it was ellipticity change, but the breadth of the transition experimentally preferable to use a 280 nm calls into question the possibility of a two-state excitation to reduce any light scattering problems in transition. For [DTAB] > 0.3 mM, the values of ellipticity at []222 increased. It is important to note 3.2Fluorescence spectra that cyt c in the presence of high concentrations of DTAB starts to unfold (data not shown). The Fluorescence spectra (Figure 2) arising from the helical contents in the unfolded state and the native single Trp in cyt c were also recorded for the same state of cyt c are 4 and 30% respectively, on the cyt-DTAB samples used in far-UV CD and Soret basis of the ellipticity values at 222 nm as absorption experiments. In the native state calculated by the method of Chen et al. [53]. The structure, Trp 59 is buried in the hydrophobic core helical content of the MG-like state of cyt c and almost no fluorescence is observed, induced by various concentrations of dodecyl presumably being quenched by Foster energy trimethylammonium bromide (DTAB), is 32.1% transfer to the heme group. However, when the according to this method. protein is denatures, the Trp residue becomes As a comparison, a titration of 5 M cyt c at pH 2 solvated and intense fluorescence results. As seen with positive charged surfactant DTAB was in Figure 2, even 0.05 mM of DTAB at pH 11 recorded at the same pH with far-UV CD, as shown induced a big decrease in the fluorescence of in Figure 2. Over the whole surfactant alkaline-denatured cyt c, and about 80% of the concentration range from 0 to 50 mM, no fluorescence was quenched for [DTAB] = 0.3 mM, precipitation is observed, which differs from what suggesting a hydrophobic collapse even at this low is observed at pH 11. The ellipticity change at 222 DTAB concentration. There is a blue shift of the nm is plotted as an inset in Figure 2B. In the DTAB peak (10 nm) accompanying this decreased case, ellipticity starts to increase at 6 mM, which is fluorescence intensity when [DTAB] is varied from the CMC for DTAB (data not shown) at pH 2 (A 0 to 0.3 mM. When [DTAB] is above 0.3 mM, an surface tension test shows that the CMC for DTAB increase in the fluorescence is observed and a small solution at pH 2 is around 6 mM). MG-like state is red shift occurs, especially at high DTAB obtained at [DTAB] 17 mM, much higher than for concentration (at pH 11), indicating that DTAB DTAB at pH 11, which offers charge neutralization starts to unfold the protein (data not shown). The as well as hydrophobic interactions. 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. 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. Fig. 1B, Fluorescence spectra of cytochrome c with [11] Y. Goto, Y. Hagihara, D. Hamada, M. (relative intensity) plotted against wavelength at various concentrations of DTAB concentration at pH = 11. The Hoshino and I. Nishii, Biochemistry, 32, 1993, arrow shows the addition of DTAB to cytochrome c. Dashed line is native state of cytochrome c at pH 7. 11878-11882.

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