Protein stability: Determination of structure and stability of the transmembrane protein Mce4A from M. tuberculosis in membrane-like environment

Accepted Manuscript Protein stability: Determination of structure and stability of the transmembrane protein Mce4A from M. tuberculosis in membrane-like environment

Shagufta Khan, Parvez Khan, Md. Imtaiyaz Hassan, Faizan Ahmad, Asimul Islam PII: DOI: Reference:

S0141-8130(18)30656-1 https://doi.org/10.1016/j.ijbiomac.2018.12.183 BIOMAC 11342

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

7 February 2018 8 December 2018 21 December 2018

Please cite this article as: Shagufta Khan, Parvez Khan, Md. Imtaiyaz Hassan, Faizan Ahmad, Asimul Islam , Protein stability: Determination of structure and stability of the transmembrane protein Mce4A from M. tuberculosis in membrane-like environment. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.183

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ACCEPTED MANUSCRIPT Running head: Thermodynamic stability of Mce4A Protein Stability: Determination of structure and stability of the transmembrane protein Mce4A from M. tuberculosis in membrane-like environment Shagufta Khan1, Parvez Khan1, Md. Imtaiyaz Hassan1, Faizan Ahmad1 and Asimul Islam1* Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia,

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New Delhi-110025, India

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*To whom correspondence should be addressed

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Dr. Asimul Islam Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an obligate pathogen that causes 10.4 million new infections worldwide, out of which about 1.4 million die every year. SDS is routinely used to mimic the native hydrophobic environment of phospholipid bilayer. Here, we report structure and stability of a mammalian cell entry protein from M. tuberculosis (Mce4A) in the

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absence and presence of SDS. The far-UV circular dichroism (CD) measurements suggested that SDS induces -helical structure in Mce4A. Stability of the protein in the absence and presence of SDS was measured from the analysis of the urea-induced denaturation curves of three physical properties (CD, intrinsic fluorescence and near-

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UV absorption). These measurements led to the conclusion that SDS stabilizes Mce4A. Binding of SDS with Mce4A was measured in isothermal titration

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calorimeter, which led to the conclusion that there is strong binding of SDS with Mce4A. We propose that the membrane associated Mce4A is more structured and

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more stable.

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Keywords: Mce4A, SDS, CMC, Urea denaturation, Protein stability

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Introduction Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an obligate pathogen that causes 10.4 million new infections worldwide, out of which about 1.4 million die every year (WHO Report, 2016) [1]. According to the WHO Report 2016,

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there is an increase in TB cases during 2013–2015, and this increase was mostly due to increase in TB cases in India. TB remained as one of the top 10 causes of death worldwide in recent years. Although, there are several strategies (DOTS and BCG) available for treatment of tuberculosis, the emergence of multidrug resistant (MDR)

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and extreme drug resistant (XDR) of M. tuberculosis have given serious challenges to control and treatment of TB [2-4]. Hence, there is an urgent need of development of

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new strategies for the treatment of the disease. So, designing of new drugs requires better understanding of the pathogenesis of M. tuberculosis.

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Mce4A, a MCE-family protein of 43 kDa molecular mass, is encoded by mce4 operon of M. tuberculosis [5, 6]. It plays major role in cell invasion and helps in the latent

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infection of TB by utilizing host’s cholesterol for carbon and energy source like

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Rhodococcus [7, 8]. In latent infection of TB at day 20, differential expression of mce4 was observed in vivo (in lung tissues of rabbits and spleens of guinea pigs) and

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in vitro (broth culture) conditions [6]. There is not much information available regarding structure and stability of Mce4A [5]. Even mechanism of utilization of cholesterol from host by the protein is poorly understood. Thus, there is a need for better understanding of Mce4A-host interaction. The structural and biophysical characterization of Mce4A is needed for designing putative drugs which can control the utilization of cholesterol by the host with help of this protein.

ACCEPTED MANUSCRIPT Sodium dodecyl sulphate (SDS), an anionic detergent with a 12-carbon hydrophobic tail and a negatively charged sulphate hydrophilic head group, is used in the studies of membrane-associated proteins. Interaction of SDS with proteins has great importance because it mimics the native hydrophobic environment of the phospholipids bilayer in vivo [12, 13]. Many studies were performed on interactions of ionic surfactants and

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proteins [14-16]. SDS binds to proteins [17-21] and disrupts either hydrophobic or electrostatic or both interactions within the protein [22-26]. However, there are reports of increase in helical content of globular proteins such as ovalbumin, carbonic anhydrase, -lactalbumin, pepsin and fetulin, etc. in the presence of SDS [26-30].

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In the present study, we aimed to monitor the effect of sub-micellar concentrations of SDS on structure and stability of Mce4A which has 23 amino acids long trans-

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membrane region [31, 32], for SDS is expected to provide native milieu. We report that there is an increase in -helical content of the protein in the presence of SDS, as

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revealed by the far-UV circular dichroism (CD). Thermodynamic stability of Mce4A

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in the absence and presence of SDS was also determined from the analysis of reversible two-state urea-induced unfolding curves of CD, intrinsic fluorescence and

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absorption. Furthermore, binding of SDS with Mce4A was measured in isothermal titration calorimeter, which showed strong binding of the detergent to the protein.

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SDS enhances stability of the protein both in term of Cm (midpoint of denaturation) and GD0 (Gibbs free energy of stabilization). Materials and Methods Materials All chemicals used were of molecular biology grade. Luria-Bertani broth, NaCl, SDS and imidazole were procured from Merck (Darmstadt, Germany). Sodium cacodylate trihydrate, kanamycin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were

ACCEPTED MANUSCRIPT obtained from Sigma (Saint Louis, MO, USA). Plasmid pET-28a from Novagen, Wisconsin (USA) was used as expression vector. Urea was purchased from MP Biomedicals, (France). Syringe filters of 0.22 μ were purchased from Millipore Corporation (USA). Expression and purification of Mce4A

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Mce4A was cloned into pET28a, expressed and purified from Escherichia coli BL21 (DE3) as described elsewhere [5, 33]. Purity of this protein was established through SDS-PAGE [34] and Western blotting experiments [5]. The purified protein has the molecular mass of 43 kDa. Protein concentration was determined by absorbance

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spectra measurements using its molar absorption coefficient () of 16,515 M-1cm-1 at 280 nm [35].

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Sample preparation

The urea-induced denaturation was studied at pH 6.0 and 25 ± 0.1 °C. Stock solution

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of urea (10.0 M) in 20 mM cacodylate buffer of pH 6.0 was prepared. Always freshly

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prepared urea solution was used in order to avoid formation of cyanate ions [36]. The concentration of urea was determined by measuring refractive indices of urea solution

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and its blank with the help of refractometer [37]. The protein solutions with different concentrations of urea were made in 1-ml volumetric flask and incubated overnight at

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25 °C for denaturation as well as renaturation experiments. Urea was added in successive samples with gradual increase in concentration. A similar procedure was employed in preparing the solution for renaturation experiments with the exception that the protein was first denatured by adding required (3.0 and 4.0 M) concentration of urea solution and then diluted with the same buffer [38]. Mce4A protein concentration used in all the measurements was in the range 0.30-0.50 mg ml−1. Critical micellar concentration (CMC) measurements

ACCEPTED MANUSCRIPT The CMC of SDS was determined by isothermal titration calorimetry (ITC) at pH 6.0 and 25 ± 0.1 °C. For CMC determination by ITC, concentrated surfactant solution of SDS (40 mM) was titrated into 20 mM MES buffers of pH 6.0 in a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA). Before ITC experiment, all the samples were degassed properly on a thermovac. The CMC value corresponds to the

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SDS concentration, at which breakpoint was observed in the plot between enthalpy change and SDS concentration.

Determination of thermodynamic parameters of SDS and Mce4A interaction The binding of SDS to Mce4A was monitored by using a VP-ITC titration

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microcalorimeter (MicroCal Inc., Northampton, MA) at pH 6.0 and 25 ± 0.1 °C. Before ITC experiment, all the samples were degassed properly on a thermovac. The

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sample and reference cells of the calorimeter were loaded with protein solution (15 μM) and the buffer, respectively, then several injections of 10 μL of SDS solution (20

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mM) were made into the sample cell having the protein. Each injection was completed over 20 s with an interval of 180 s between successive injections. The

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reference power and stirring speed were set at 18 μcal s-1 and 372 rpm, respectively.

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The heat of dilution for the protein and the heat of micellization of SDS were subtracted from the integrated data before curve fitting.

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Circular dichroism (CD) measurements The far-UV CD measurements of Mce4A were performed in Jasco spectropolarimeter (model J-1500) equipped with Peltier type temperature controller (PTC-517) to measure the structure and stability of the protein. Far-UV CD spectra were recorded in wavelength region of 250–200 nm using cuvette of 0.1 cm path length. The raw CD data at wavelength λ were expressed in terms of mean residue ellipticity, [] (deg cm2 dmol-1) using the relation,

ACCEPTED MANUSCRIPT [] =M0  /10 l c

[1]

where is the observed ellipticity in millidegrees at wavelength λ, M0 is the mean residue weight of the protein, c is concentration in mg ml-1 and l is the path length of the cell in centimeter. Each observed [θ]of the protein was corrected for the solvent contribution. All spectral measurements were performed in triplicate at pH 6.0 and 25

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± 0.1 °C. Fluorescence spectra measurements

Fluorescence studies were performed in Jasco spectrofluorometer (Model FP-6200) equipped with an external thermostated water bath using a 5 mm quartz cell. Both

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excitation and emission slit widths were set at 5 nm. Spectral measurements were done at pH 6.0 and 25 ± 0.1 °C. For measuring the intrinsic fluorescence of tyrosine

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residue, the protein sample was excited at a wavelength of 274 nm. The emission spectrum was measured in the range of 300–320 nm. The fluorescence intensity at

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306 nm (F306) was plotted as a function of urea concentration. All spectral

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measurements were performed in triplicate at pH 6.0 and 25 ± 0.1 °C. Absorption spectra measurements

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Absorption spectral measurements of Mce4A were carried out in Jasco UV/visible spectrophotometer (Jasco V-660) equipped with Peltier-type temperature controller

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(ETCS-761). All measurements were performed in 340–240 nm wavelength range, using 1 cm path length cuvette. All spectral measurements were performed in triplicate at pH 6.0 and 25 ± 0.1 °C. Analysis of transition curves The transition curves (plot of each spectral property y ([θ]222, F306 andε287) versus [u], the molar concentration of urea, were analyzed for estimating the stability parameters. This evaluation is based upon a least-square method, which is used to fit

ACCEPTED MANUSCRIPT the complete denaturation curve achieved at constant pH and temperature (K) according to the relation: y[u]= {yN[u] + yD[u]Exp-(GD0 – m[u])/RT}/ {1 + Exp-(GD0 – m[u])/RT)}(2) where y[u] is the observed optical property at urea concentration [u]; yN [u] and yD[u], respectively are optical properties of the native and denatured protein molecules under

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the same experimental conditions in which y[u] was measured; ∆GD0 is the value of Gibbs free energy change (∆GD) in the absence of the denaturant; m is the slope (∂G/∂[u]); R is the universal gas constant; and T is temperature in Kelvin. It should be noted that the Equation (2) assumes that a plot of ∆GD versus [u] is linear, and the

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dependencies of yN and yD on [u] are also linear (i.e., yN[u] =aN + bN[u], and yD[u] = aD + bD[u], where a and b are [u]- independent parameters, and subscripts N and D

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represent these parameters for the native and denatured protein molecules, respectively). The analysis of the denaturation curves of different optical properties

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according to Equation (2) gave values of ∆GD0, m and Cm (= GD0 /m). Values of fD as

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a function of [u] were determined from denaturation curves of []222, F306 and 287, using the following relation [39],

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fD = {y[u] – (aN + bN[u])}/{(aD – aN) + (bD – bN[u])}

(3)

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Result and Discussion

Bacillus Calmette Guerin (BCG), attenuated strain of Mycobacterium bovis, is the only vaccine available against M. tuberculosis infection. However, this vaccine is only effective in preventing tubercular meningitis or milliary tuberculosis in infants, but usually ineffective in protecting adults from pulmonary tuberculosis and thus being unable to halt the transmission chain among them [40]. Thus, the development of more potent and effective vaccines as well as drugs, against tuberculosis is one of the major goals to improve global health. For that, the understanding of host-pathogen

ACCEPTED MANUSCRIPT interaction and the active involvement of proteins should be addressed. As we know that Mce4A is a mammalian cell entry protein and helps in the entry of the Mtb into the host, it is a very good candidate for drug target [5]. Mce4A helps in the long term survival of Mtb in the host by utilizing cholesterol as source of energy and carbon, and it prolongs TB [8]. In order to target Mce4A, it is required to know the structural

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and thermodynamic stability of the protein. Therefore, here we have performed structural characterization of the protein in absence and presence of different concentrations of SDS. We have also measured urea-induced denaturation in absence and presence of SDS to give insight into the stability of this protein, which shall

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further help in designing of inhibitors against Mce4A. It is worth noting that SDS

amino acid residues [31, 32].

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provides membrane-like environment as Mce4A has trans-membrane region of 23

In order to estimate secondary structure and measure stability of Mce4A, we

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expressed and purified the Mce4A [5]. The solutions were prepared at pH 6.0 as Mce4A was found to be stable over the wide range of pH 5.5 ≤ pH ≤ 11.5 [5].

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Moreover, the primary site of infection of Mycobacterium tuberculosis is macrophage

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having cellular pH in the range 5.5 - 6.0, so this pH range has a physiological relevance [41]. The stability of Mce4A was measured by the chemical denaturation

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method in which urea was used as a denaturing agent. CD, fluorescence and absorption spectroscopies were exploited to monitor the thermodynamic stability of the protein. In this paper, we discuss the structural and thermodynamic stability of Mce4A in the absence and presence of SDS. Critical micelle concentration determination Understanding properties of different lipids, micelles and membrane bilayers continues to provide vital information. SDS has tendency to form micelle. In order to

ACCEPTED MANUSCRIPT understand the mode of interaction of monomeric SDS with Mce4A, it is needed to determine the CMC value because monomeric and micellar forms of SDS interact differently with proteins [42]. CMC of SDS was determined by ITC experiments, for ITC is one of the most sensitive techniques for the direct measurements of thermodynamic changes in the course of binding as well as micellization [43]. To

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determine CMC value, concentrated surfactant solutions of SDS were titrated into 20 mM MES buffer of pH 6.0 at 25 ± 0.1 °C. In the isothermal titration, successive injection of concentrated SDS into the buffer provides a titration profile with endothermic peaks (Fig. 1). These endothermic peaks show the dispersion of the

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concentrated micelle solution (demicellization) into the buffer [44]. As the concentration of SDS was increased, the process of demicellization comes to a pause,

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and the enthalpy of the reaction approached towards zero, since critical micelle concentration (CMC) defined as a broad threshold of monomer concentration which is

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not seen here (Fig. 1) as a sharp “phase boundary” [45]. Thus, the CMC value of SDS at pH 6.0 was 2.43 mM which is in agreement with the earlier report [46]. From these

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experiments we can assume that SDS remains completely in monomeric form at or

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below 2.0 mM. This sub-critical micellar concentration (sub-CMC) of SDS (2.0 mM) at pH 6.0 was used to monitor the effect of monomeric SDS on structure and stability

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the protein.

Thermodynamic parameters of interaction between SDS and Mce4A The thermodynamic parameters and binding affinity of SDS to Mce4A were performed using ITC. The ITC binding isotherm of SDS with Mce4A at pH 6.0 and 25 ± 0.1 °C was represented in Fig 2. In this figure each peak in the top panel represents a single injection of the SDS into the Mce4A solution. An integrated plot of the amount of heat liberated per injection as a function of the molar ratio of the

ACCEPTED MANUSCRIPT SDS to protein was represented in the bottom panel. These isotherms were best fitted for the lowest χ2 using a single binding site model. The binding and thermodynamic parameters of SDS with Mce4A are summarized in Table 1. It can be seen in this table that at pH 6.0, SDS binds to Mce4A with high affinity (Ka ∼ 104) along with several SDS molecules interacting with the single binding site. Furthermore, from the

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observed thermodynamic parameters, enthalpy changes (ΔH°), and entropy changes (ΔS°), the forces involved in binding of SDS to proteins can be predicted [47]. It can be seen from Table 1 that positive values of ΔH° and ΔS° are indicative of hydrophobic interaction or conformational alterations [26]. Furthermore, the value of

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ΔG0 is negative, so the interactions of SDS and Mce4A were spontaneous under these conditions. Thus, it may be concluded that SDS monomers bind to Mce4A

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predominantly by hydrophobic interaction and bring about conformational changes in the structure of the protein. In order to quantify the change in conformation or to

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know whether this binding is increasing or decreasing the structure of protein, far-UV CD measurements were carried out in the absence and presence of various

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concentrations of SDS. The range of the concentration of the SDS was always sub-

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CMC, in order to maintain monomeric population of SDS as mentioned earlier. Far-UV CD measurements

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A variety of roles are increasingly being identified for both specific and non-specific interactions between membrane proteins and detergents in folding, stability and function [48-51]. The incorporation of lipids in detergent micelles frequently increases protein stability and folding. Although success in membrane protein research can be explained by both judicious and severe choices of detergents and lipids in vitro, the majority of the investigations of protein spectral analysis have been directed toward estimating the fractional contents of the major secondary structures in

ACCEPTED MANUSCRIPT proteins. Far-UV CD is an excellent technique to estimate change in secondary structure of protein [52-54] . Although the structural information obtained from CD is limited compared to that obtained by X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy, it is a valuable adjunct to these techniques and offers a number of advantages. These advantages include the ability to explore a wide range

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of solution conditions and temperatures, rapid data collection, and the consumption of relatively small amounts of sample material. It should be noted that crystal or NMR structure is not available for this protein as Mce4A has a tendency for aggregation. To see the effect of SDS on the secondary structure of the protein, far-UV CD

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measurements were performed in the presence of different sub-CMC concentrations of SDS. The effect of SDS on the secondary structure of the protein is shown in Fig.

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3. It was observed that at low concentrations of SDS (0.0– 0.1 mM), there is no significant change in the secondary structure of the protein while as we increase the

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concentration of SDS from 0.1 mM, there is an increase in helical contents of the protein. It should be noted that SDS is an ionic detergent which denatures water

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soluble proteins by completely perturbing secondary and tertiary of proteins [55, 56].

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However, membrane proteins like bR [57], Escherichia coli diacyl glycerol kinase [58], and Streptococcus lividans potassium channel [59] are susceptible to changes in

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the properties of their surroundings, the native membrane lipids in vivo, or detergent and lipids in vitro.

The far-UV CD spectrum of the native Mce4A showed that it is an α + β protein having the two negative peaks near 208 and 222 nm [5]. It is seen in Figure 3 that the far-UV CD negative peaks at 208 and 222 nm are enhanced with increasing concentration of SDS, which is the indication of increase in the helix content of Mce4A. Using the procedure of Greenfield [60] we estimated the percentage of α-

ACCEPTED MANUSCRIPT helix (Table 2). It is seen in Table 2 that there is an increase in the secondary structure (-helix) of the protein from 31 % to 41.8 % in the presence SDS which mimics the membrane environment for Mce4A, a trans-membrane protein having 23 amino acid residues long region buried in the membrane [31, 32]. Membrane and detergent environments tend to be hydrophobic and/or amphipathic, so they have different

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physical properties than aqueous solutions, producing different spectral characteristics for proteins embedded in them [61].

There are several studies that suggest that SDS induces -helix formation in a number of proteins [25, 26, 28, 62]. Jirgensons and Capetillo have proposed that SDS binding

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to proteins occurs both by ionic and hydrophobic interactions [25]. The binding of SDS by ribonuclease caused increase in the α-helical content of the protein [25, 28].

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To understand whether an increase in secondary structure of Mce4A contributes to the stability of the protein, we performed urea-induced denaturation of the protein in the

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absence and presence of 2.0 mM SDS at pH 6.0 and 25 ± 0.1 ºC. Here, we chose 2.0

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mM of SDS concentration because it is the sub-critical micellar concentration (subCMC) of SDS at pH 6.0, observed by ITC measurements (Fig. 1). Fig. 4 (A and B)

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shows representative urea-induced CD spectra of Mce4A in the absence and presence of 2.0 mM SDS. []222 was used as probe for monitoring the change in peptide

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backbone conformation which shows the change in the secondary structure. Ureainduced denaturation profiles (i.e., plots []222 as a function of [u], the molar urea concentration) of the protein in the absence and presence of 2.0 mM SDS are shown in Fig. 5A and 5B, respectively. It is seen from these figures that (i) there is no change in the secondary structure of Mce4A up to 2.25 M urea in the absence of SDS while in the presence of SDS, the range of molar concentration of urea increases from 2.25 to 2.75, (ii) loss of secondary structure occurs in the urea concentration range 2.5

ACCEPTED MANUSCRIPT - 6.25 M in the absence of SDS while the transition region in the presence of SDS lies in molar range of 3.00-6.50 M urea, and (iii) above 6.50 M urea denaturation is complete in the absence and presence of SDS. Fluorescence spectra measurements Mce4A contains 11 tyrosine residues and no tryptophan residue. This fact was

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exploited while performing intrinsic fluorescence measurements to investigate the thermodynamic stability of Mce4A as function of urea concentration in absence and presence of 2.0 mM SDS. Fig. 4 (C and D) shows representative emission spectra of Mce4A recorded between 300-320 nm. The change in the environment of tyrosine

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was monitored by observing change in the emission spectrum of Mce4A upon addition of increasing concentration of urea (Figs. 4C and D). It is observed that the

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wavelength at which maximum emission occurs (max) is 306 nm. It is seen in Fig. 4 (C and D) that in the presence of denaturing urea concentration, Mce4A undergoes

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unfolding, and max of Tyr residues was shifted from 306 nm to 307 nm (red shift)

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along with an increase in fluorescence intensity both in the absence and presence of SDS. It should be worth mentioning that ribonuclease A which is also devoid of

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tryptophan, shows an increase in fluorescence intensity in the similar wavelength region upon urea-induced denaturation with almost no red shift [63]. Figs. 5C and 5D

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show plots of fluorescence emission maxima at 306 nm (F306) versus [u] in the absence and presence of 2.0 mM SDS, respectively. These transition curves show that Mce4A undergoes cooperative denaturation induced by urea. Absorption spectra measurements To see the effect of urea on the tertiary structure of Mce4A, absorption spectra measurements were performed in 340–240 nm range. Effect of urea on absorption spectra of Mce4A in the absence and presence of 2.0 mM SDS are shown in Figs. 4E

ACCEPTED MANUSCRIPT and 4F, respectively. These absorption measurements were used to estimate ε287, the difference in molar extinction coefficient at 287 nm of the protein in the absence and the presence of urea. Plots of ε287 versus [u] in the absence and presence of SDS are shown in Figs. 5E and 5F, respectively. It is seen in Figs. 4E and 4F that as the

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concentration of urea was increased, there was decrease in absorbance, and the absorbance maxima shifted to lower wavelength (blue shift) on denaturation. UVabsorption maxima of proteins undergo shifts to shorter wave lengths upon protein denaturation and there is a decrease in absorbance. For tryptophan free proteins like ribonuclease A in 8 M urea, there was absorption spectrum shift towards shorter

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wavelength [64]. In understanding of this effect, attention has been focused

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particularly to the tyrosyl side chains, as all changes in absorption produced by the unfolding of the protein is due to changes in the environment of tyrosine [65, 66]. It should be kept in mind that the phenylalanine shows maximum absorption at 257 nm

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while there is no absorption of phenylalanine beyond 270 nm.

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Analysis of transition curves

The analysis of transition curves (Fig. 5) for determination of stability parameters,

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GD0, m and Cm according to Equation (2), assumes that urea-induced denaturation of

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Mce4A follow a two-state mechanism. A coincidence of normalized sigmoidal curves of all three probes is used to test for a two-state behavior of protein denaturation [15, 37]. Fig. 6 shows normalized transition curves of the urea-induced denaturation of Mce4A (i.e., plot of fD, the fraction of the denatured molecule versus [u]). Fig. 6 shows the coincidence of all normalized transition curves of different optical properties of the protein, which suggests that urea-induced denaturation of Mce4A, is a two-state process. It should be noted that the urea-induced denaturation of the protein was reversible both in the absence and presence of SDS (Fig. S1).

ACCEPTED MANUSCRIPT The values of stability parameters are given in Table 3. Furthermore, identical values of GD0, m and Cm (Table 3) obtained from the analysis of denaturation curves of different properties (Δε287, [θ]222 and F306) for the protein in the absence and presence of SDS, further support assumption that urea induces a two-state transition [39]. The

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thermodynamic stability of Mce4A in terms of GD0 and Cm, was increased in the presence of 2.0 mM SDS. Furthermore, SDS has a net stabilizing effect below its CMC, in case of bovine serum albumin, there was increase in H and Tm upon thermal denaturation [67].

Thermodynamic studies are an important complement to structural studies in drug

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designs [68]. The most effective drug design and development platform is an

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integrated process that uses all available information from structural, thermodynamic and biological studies [69]. Recent drug discovery efforts have been dominated by concept based on structure. Only structural data, even with the most complex

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computational methods, cannot fully define the driving force of the binding

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interaction or even cannot accurately predict the binding affinity. Thermodynamics provides quantitative data that can be used to explain the driving forces to evaluate

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Conclusions

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and understand the effects of substituent changes on binding affinity [70].

The mode of interaction of anionic detergent, SDS with Mce4A in subcritical micellar concentrations was hydrophobic in nature at pH 6.0 and 25 ± 0.1 ºC. With increase in concentration of SDS, an increase of α-helical structure in Mce4A was observed. The urea-induced denaturation studies gave an insight of the folding-unfolding mechanism of Mce4A. From spectroscopic studies, it was observed that urea-induced denaturation of Mce4A was reversible and two-state process. In the presence of 2.0

ACCEPTED MANUSCRIPT mM SDS (sub-CMC concentration), Mce4A shows higher values of GD0 as well as Cm. Acknowledgements This work was supported by grants from the Indian Council of Medical Research (ICMR) BIC/12(16)/2014, FIST Program (SR/FST/LSI-541/2012), Council of

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Scientific and Industrial Research (CSIR), India (37(1603-04)/13/EMR-II). SK is thankful to Maulana Azad National Fellowship, University Grants Commission (Government of India) for providing fellowship. FA is grateful to Indian National Science Academy for the award of Senior Scientist Position. AI, MIH and FA thank to the Department of Science and Technology and ICMR, India;

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Conflict of Interest: Authors declare there is no conflict of interest.

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Table 1 Thermodynamic parameters of interaction between Mce4A and SDS, derived from ITC measurements at pH 6.0 and 25 ± 0.1 °Ca. 4

No. of SDS molecule

Ka x 10

176 ± 3.62

(M ) 1.13 ± 0.25

∆S -1

-1

-1

-1

Kd

∆G

(cal mol deg )

(µM)

(kcal mol )

65.69 ± 2.42

18.8 ± 0.85

0.09 ± 0.01

-5.54 ± 0.25

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-1

(cal mol )

A ‘±’ represents an error from the mean of errors of triplicate measurements.

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∆H

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Table 2 Effect of SDS on the helical content of Mce4A % 

0.00

31.3

0.10

32.3

0.50

36.3

0.75

39.7

1.00

38.3

1.25

40.2

1.50

41.2

2.00

41.8

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[SDS], mM

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Table 3 Thermodynamic parameters obtained from the urea-induced denaturation of Mce4A at pH 6.0 and 25 ± 0.1 °Ca. GD0, kcal mol-1

Cm, M

0 mM SDS

2 mM SDS

0 mM SDS

2 mM SDS

0 mM SDS

2 mM SDS

[]222

4.71 ± 0.15

5.21 ± 0.15

1.14 ± 0.03

1.10 ± 0.05

4.13 ± 0.16

4.73 ± 0.25

287

4.67 ± 0.20

5.23 ± 0.14

1.12 ± 0.06

F306

4.62 ± 0.15

5.21 ± 0.18

1.13 ± 0.04

Average

4.67 ± 0.17

5.22 ± 0.16

1.13 ± 0.04

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Probe

m, kcal mol-1 M-1

4.17 ± 0.25

4.43 ± 0.27

1.17 ± 0.06

4.10 ± 0.24

4.45 ± 0.28

1.15 ± 0.06

4.13 ± 0.22

4.54 ±0.27

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A ‘±’ represents an error from the mean of errors of triplicate measurements.

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1.18 ± 0.07

ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Determination of enthalpy changes by isothermal titration calorimetry to predict critical micellar concentration of SDS at pH 6.0 and 25 ± 0.1 °C. The isothermal calorimetric enthalpy changes (upper panel) and resulting binding isotherms (lower panel) are shown for titration.

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Fig. 2 Isothermal titration binding isotherms of Mce4A with SDS at pH 6.0 and 25 ± 0.1 °C. The isothermal calorimetric enthalpy changes (upper panel) and resulting binding isotherms (lower panel) are shown for titration.

Fig. 3 Far-UV CD spectra of Mce4A in the presence of SDS (0−2.0 mM) at pH 6.0

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and 25 ± 0.1 °C.

Fig. 4 Urea-induced denaturation (0.0 −8.0 M) of Mce4A at pH 6.0 and 25 ± 0.1 °C.

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(A) Far-UV CD spectra of Mce4A in the absence of SDS, and (B) in the presence of 2.0 mM SDS. (C) Fluoresence emission spectra of Mce4A in the absence of SDS, and

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(D) in the presence of 2.0 mM SDS. (E) Absorption spectra of Mce4A in the absence of SDS, and (F) in the presence of 2.0 mM SDS.

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Fig. 5 Urea-induced denaturation curves (plots of [θ]222, F306, and 287 versus [u]) of

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Mec4A at pH 6.0 and 25 ± 0.1 °C. Panels A, C, and E show urea-induced denaruration profile in the absence of SDS. Panels B, D, and F show urea-induced

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denaturation profile in the presence of 2.0 mM SDS. Fig. 6 Normalized transition curves of [θ]222 , Δε287 and F306 versus [u] at pH 6.0 and 25 ± 0.1 °C. Panel A shows the normalized urea-induced denaturation plots of Mce4A in the absence of SDS and Panel B represents the normalized urea-induced denaturation plots of Mce4A in the presence of 2.0 mM SDS. Fig. S1 Spectra showing reversibility of urea-induced denaturation of Mce4A using far-UV circular dichroism (A & B), fluorescence spectra (C & D) and near-UV

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presence of 2.0 mM SDS.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6