Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles

Journal of Photochemistry and Photobiology B: Biology 149 (2015) 1–8

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Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles Upendra Kumar Singh a, Neeraj Dohare a, Prabhash Mishra a, Prashant Singh b, Himadri B. Bohidar c, Rajan Patel a,⇑ a b c

Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India Department of Chemistry, A.R.S.D. College, University of Delhi, Delhi 110021, India Polymer and Biophysics Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, India

a r t i c l e

i n f o

Article history: Received 9 March 2015 Accepted 22 April 2015 Available online 16 May 2015 Keywords: Gramicidin Ionic liquid Lipid vesicles Red-edge excitation Dynamic surface tension

a b s t r a c t The present work is focused on the interaction between membrane bound gramicidin and 1-butyl-1methyl-2-oxopyrrolidinium bromide (BMOP) ionic liquid. Ionic liquids (ILs) are solvents that are often liquid at room temperature and composed of organic cation and appropriate anion. The gramicidin peptide forms prototypical ion channels for cations, which have been extensively used to study the organization, dynamics, and function of membrane spanning channels. The interaction was studied by circular dichroism, steady state, time-resolved fluorescence spectroscopy in combination with dynamic surface tension and field emission scanning electron microscopic methods (FESEM). The results obtained from circular dichroism shows that the BMOP interacts with the channel form of gramicidin in lipid vesicle without any considerable effect on its conformation. The Red-edge excitation shift (REES) also supported the above findings. In addition, the fluorescence studies suggested that BMOP makes ground state complex with ion channel, which was further supported by time resolved measurements. Furthermore, dynamic surface tension analysis shows the faster adsorption of BMOP with membrane bound gramicidin at the air–water interface. Additionally, FESEM results indicated that BMOP forms a film around the membrane bound gramicidin at higher concentration. These results are potentially useful to analyze the effect of ionic liquids on the behaviour of membrane proteins. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Gramicidin is a linear peptide occurring as a natural product from bacillus brevis. It is known to form ion channels in synthetic as well as natural membranes [1]. The specificity of gramicidin lies in forming prototypical ion channels which are selective for monovalent cations. Study of these prototypical ion channels is employed to obtain a plethora of information related to the structure and function of complex membrane spanning channels [2–4]. Gramicidin channels are referred as an excellent prototype for transmembrane channels due to its small size, easy availability, and the relative ease for chemical modification. These feature’s states gramicidin as unique among small membrane-active peptides and thus provide a platform to be utilized for revealing the factors responsible for folding of membrane proteins [5–7]. Gramicidin channels are ion-selective owing to its unique sequence of alternating L- and D-chirality, that renders it ⇑ Corresponding author. Tel.: +91 8860634100; fax: +91 11 26983409. E-mail addresses: [email protected], [email protected] (R. Patel). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.011 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

environment sensitive [8]. The combinations of dihedral angle generated in the conformation space by various gramicidin conformations are allowed according to the Ramachandran plot [9]. The detailed molecular mechanism of gramicidin adopting various conformations in membranes is yet unknown. Gramicidin conformation in membranes, therefore, appears to be dependent on its ‘‘solvent history’’ [10]. The channel conformation of gramicidin in membranes has been characterized in molecular detail by solid-state NMR [11]. It was reported that the channel form of gramicidin is the most preferred and thermodynamically stable conformation in membranes and membrane-mimetic environments [12,13]. This form is characterized as single stranded b6.3 helical dimer which conducts cation, and is formed by the head-to-head (amino terminal to-amino terminal) single stranded b6.3 helical dimer [14]. In this conformation, the carboxy terminus remains exposed to the membrane–water interface, while the amino terminal is buried into the hydrophobic core. Tryptophan residues in this conformation remain clustered at the membrane– water interface around the entrance to the channel [14–16]. These residues are of prime importance in ion channel activity

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and conformation of gramicidin [17]. However, the conformation of gramicidin in interdigitated lipid bilayer is not the typical b6.3 helix as in the normal bilayer [18]. Ionic liquids (ILs), organic salts with a melting temperature below 100 °C, are a class of materials that have considerable potential to advance liquid formulation for protein based pharmaceuticals [19]. ILs have been developed as a successful alternatives for organic solvents in traditional chemical processes, and more recently they have been explored as replacements for aqueous media in enzyme-based processes [20]. ILs possesses mesmerizing features such as high flexibility, tunability and less volatility, in addition to chemical variety that widens the range of compounds. Due to this features, their dispersion to environment is checked to reduce the waste and pollution. Thus ILs gets an edge over other solvents and although being expensive compensates for the initial cost. [21]. In depth investigation, computational as well as experimental studies of the interaction between ILs having different alkyl chain length and varied head groups with phospholipid membranes have been reported and the results showed disruption of the phospholipid bilayer at higher concentration of ILs with long hydrocarbon side chain [22–24]. Some recent findings also reported that ILs has the ability to fuse and aggregate lipid vesicles depending upon the length of the alkyl chain at higher concentrations [25,26]. These studies involving interaction between ILs and membranes are very informative for determining their various aspects like, mode of action of such chemicals and their effect on the biological membranes. Insertion of peptides or proteins into the frame widens the scope further for better understanding and assessing of the role of ILs in case of biological molecules. So far, very little attention was paid in regard for their use as reaction media for membrane protein. Herein, the study serves the purpose well to evaluate the effect of interaction of IL with membrane bound peptide. In order to investigate the interaction of pyrrolidinium based IL, 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) on a gramicidin ion channel in lipid vesicles, we applied a combination of spectroscopic approaches, which includes circular dichroism (CD) spectroscopy, fluorescence quenching, red edge excitation shift (REES), fluorescence lifetime decay along with dynamic surface tension and field emission scanning electron microscopy (FESEM). BMOP behaves as a room temperature ionic liquid (RTIL), and it occurs in liquid form at room temperature [27]. To the best of our knowledge, this work represents the first report of interaction of IL with the channel form of gramicidin ion channel in lipid membranes.

typically consisted of 80–85% gramicidin A, 6–7% B, 5–14% C and <1% gramicidin D [29–31]. All chemicals used were of the highest purity available. Solvents used were of spectroscopic grade. Millipore water was used throughout the experiments. 2.2. Preparation of lipid vesicles All experiments were done using unilamellar vesicles prepared as stated earlier [12]. The molar ratio of gramicidin to lipid were kept 1:50 for all experiments. The sample was subjected to a high vacuum for overnight followed by hydrating the film with pH 7.2 buffer. Afterward, the samples were vortexed and subjected for sonication. The samples were then centrifuged and were incubated for a minimum of 8 h at 65 °C to induce the channel conformation [10,15]. Background samples were prepared in the same way except that gramicidin was omitted. All experiments were carried out at 303.15 K i.e. above the phase transition temperature of DMPC [32]. 2.3. Addition of BMOP The BMOP of varying concentrations ranging from 0.125 mM to 0.495 mM for spectroscopic and 0.263 mM to 200 mM for other techniques were utilized. The samples were left in the dark for a period of four hours before each experiment. 2.4. Steady state fluorescence measurements Steady-state fluorescence measurements were performed on a Cary Eclipse spectrofluorimeter (Varian, USA) equipped with a 150 W xenon lamp using 1 cm path length quartz cuvettes. Excitation and emission slits with a nominal band pass of 5 nm were used for all measurements. The background intensities of samples without gramicidin were subtracted from each sample spectrum. The spectral shifts obtained with different sets of samples were identical in most cases, or were within ±1 nm of the ones reported. The gramicidin/lipid ratio was kept low to avoid any instrumental errors. 2.5. Fluorescence quenching measurements

2. Materials and methods

Fluorescence quenching experiments of gramicidin in lipid vesicles were carried out by measurement of fluorescence intensity in separate samples containing increasing amounts of BMOP taken from a freshly prepared 50 mM stock solution. Samples were kept in the dark for at least 4 h before measuring fluorescence. Corrections for inner filter effect were made using the equation [33]

2.1. Materials

F ¼ F obs antilog½ðAex þ Aem Þ=2;

Gramicidin (from Bacillus brevis) and 1,2-dimyristoyl-sn-glycer o-3-phosphocholine (DMPC; diC14:0 PC) was purchased from Sigma Aldrich. IL, 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) used were synthesized in our lab previously [28]. Scheme 1 shows the schematic structure of BMOP. The gramicidin

Br

N

O

Scheme 1. Structure of 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP).

ð1Þ

where F is the corrected fluorescence intensity and Fobs is the background subtracted observed fluorescence intensity. The values Aex and Aem are the measured absorbances at the excitation and emission wavelengths. The data was analyzed by fitting of the Stern–Volmer equation [33]

F0 ¼ 1 þ K sv ½Q  ¼ 1 þ K q s0 ½Q  F

ð2Þ

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. Kq is the quenching rate constant of the fluorophore, Ksv is the Stern–Volmer quenching constant, [Q] is the molar concentration of BMOP (quencher) and s0 is the lifetime of the fluorophore without quencher. The Ksv value obtained from the slope of the Stern–Volmer plot of gramicidin-BMOP system.

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P 2 ai s hsi ¼ P i ai si

ð3Þ

where ai and si are the relative contribution and life time of different components to the total decay. A fit was considered acceptable when plots of the weighted residuals, and the autocorrelation function showed random deviation about zero with a minimum v2 values. 2.7. Circular dichroism measurements The CD measurements were carried out on a JASCO J-815 spectropolarimeter (Tokyo, Japan) and were calibrated with [+]-10-camphorsulfonic acid [35]. All measurements were carried out at 303.15 K with a thermostatically controlled cell holder attached to a Neslab Rte-110 water bath with an accuracy of ±0.1°K. The spectra were scanned in a quartz optical cell with a path length of 0.1 cm. All spectra were recorded in 1 nm wavelength increments with a 4 s response and a band width of 1 nm. For monitoring changes in secondary structure spectra were scanned from 200 to 250 nm at a scan rate of 50 nm/min. Each spectrum is the average of 4 scans with a full-scale sensitivity of 10 mdeg. All spectra were corrected for background by subtraction of appropriate blanks and were smoothed without any change in the overall shape of the spectrum. Data were represented as mean residue ellipticities [h] and were calculated using the formula:

½h ¼ hobs =ð10ClÞ

ð4Þ

where hobs is the observed ellipticity in mdeg, l is the path length in cm, and C is the concentration of peptide bonds in mol/L. 2.8. Dynamic surface tension measurements Surface tension measurements were performed using Du Nouy-Padday method on DeltaPi-4 (Kibron, Helsinki, Finland), equipped with four parallel microbalances and having a small diameter (0.51 mm) special alloy wire (Dyne Probes, cleaning by Blazer piezo micro torch). The signal were recorded as surface tension versus time, the adsorption of lipid vesicles and membrane-bound gramicidin vesicles in solvent were spread at the air–water interface to produce a final concentration of 150 lM [36]. Teflon multiwell plate with injection ports was used

2.9. Field emission scanning electron microscope characterization FESEM investigations were carried out with a FEI Nova NanoSEM 450 instrument at 5 kV energy, to visualize the surface morphology of transferred films and on silicon substrate. Samples of lipid vesicles having gramicidin were prepared on silicon substrate by the conventional drop cast method to avoid charge accumulation for better resolution through conductive substrate. 3. Results and discussion 3.1. Gramicidin conformation in membranes monitored by circular dichroism CD spectroscopy has been utilized here to monitor the effect of BMOP on the channel conformation of gramicidin in lipid vesicles. In channel conformation, the gramicidin exists as characteristic single-stranded b6.3 helical conformation having typical peaks of positive ellipticity around 218 and 235 nm, with a valley around 230 nm and negative ellipticity below 208 nm [12,37,38]. Fig. 1 shows the far UV-CD spectra of the channel form gramicidin in vesicles of DMPC in the absence and presence of BMOP. The addition of BMOP decreases the positive ellipticity around 218 nm without any spectral shift. This shows that BMOP interact with the channel form of gramicidin. However, the backbone CD spectra with different additions of BMOP showed the similar channel conformation of gramicidin in lipid vesicles. It is suggested that the interaction of BMOP associated with the change in orientation of the chromophores results in a slight conformational change of gramicidin. Hence, the CD spectrum of gramicidin is sensitive to the conformational changes with the addition of BMOP.

30 0 mM BMOP 0.125 mM BMOP 0.249 mM BMOP 0.372 mM BMOP 0.495 mM BMOP

25 20

(deg cm2 dmol-1)

Fluorescence lifetimes were calculated from time-resolved fluorescence intensity decays by the single-photon counting spectrometer equipped with pulsed nanosecond LED excitation heads at 280 nm (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK) at temperature 303.15 K. Lamp profiles were measured at the excitation wavelength using Ludox (colloidal silica) as a scatterer. The fluorescence lifetime data were measured to 10,000 counts in the peak, otherwise indicated. The excitation wavelength was set to 295 nm and emission was set at 340 nm. The instrumental response function was recorded sequentially using a scattering solution and a time calibration of 114 ps/channel. All experiments were performed using excitation and emission slits with a band pass of 8 nm. The goodness of fit was judged in terms of both a chi-squared (v2) value and weighted residuals. Data were analyzed using a sum of exponentials, employing a nonlinear least square reconvolution analysis from Horiba (Jobin Yvon, IBH Ltd). The impulse response functions (IBH DAS6 software) were used to analyze decay curves [34]. The mean fluorescence lifetimes hsi for tri-exponential iterative fittings and pre-exponential factors were calculated by using the following relation [33]:

for measurement of small subphase volumes. The sample was magnetically stirred for 5 min after injecting the subphase into the sample to provide complete mixing and the change in surface tension of vesicles upon interaction with IL dissolved in the subphase was monitored. The temperature control plate was attached with an external circulating water bath (Grant GD120 water thermostat) to control the subphase temperature at 303.15 K with an accuracy of ±0.1 K. The Delta Graph software was utilized to record the data on the computer.

MEAN RESIDUE ELLIPTICITY (x10-3)

2.6. Time-resolved fluorescence measurements

15 10 5 0 -5 -10 200

210

220

230

240

250

Wavelenghth (nm) Fig. 1. Far UV CD spectra of the channel form gramicidin in vesicles of DMPC in absence and presence of BMOP. Concentration of DMPC was 0.43 mM and the ratio of gramicidin to DMPC was 1:50 (mol/mol).

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3.2. Fluorescence characteristics and quenching of channel form of gramicidin by BMOP The fluorescence emission spectrum of channel conformation of gramicidin exhibits an emission maximum of 333 nm, when excited at 280 nm as shown in Fig. 2. The emission maximum of channel formation was in agreement with the previously reported literature [15,37]. From the results it was observed that the emission maximum wavelength of channel form of gramicidin b6.3-helical conformation remains almost constant, i.e. around 333 nm. Further, the maximum fluorescence intensity and the centre of mass of fluorescence emission were found to have only minor discrepancies, i.e. both of these corresponded to the same wavelength. The decrease in fluorescence intensity of a fluorophore refers to fluorescence quenching for any process. Usually the fluorescence quenching is classified as dynamic and static quenching. Dynamic quenching results from the collision between the fluorophore and quencher, while static quenching results from the formation of a ground-state complex between the fluorophore and quencher [39]. Fluorescence quenching experiments were carried out with the addition of BMOP to the channel conformation of gramicidin in vesicle. Earlier, quenching experiments along with the gramicidin in the membrane-bound form as well as in aqueous solution has been performed with different quenchers [15,40–42]. These studies are useful to investigate the relative orientation, the degree of exposure and accessibility of tryptophans residues of proteins in membranes [43]. The quenching effect of BMOP on different proteins was studied elsewhere [28,44]. As shown in Fig. 2. The fluorescence intensity of channel form gramicidin decreases regularly with increasing concentration of BMOP. In the case of membrane-bound peptides having a heterogeneous distribution of tryptophans as in non-channel conformation of gramicidin, a blue shift was observed if acrylamide preferentially quenches it [27,44]. In our case, no blue shift was observed in fluorescence emission maximum upon quenching by BMOP. In channel form the tryptophan residues are clustered at the membrane–water interface and Trp-9 and Trp-15 are involved in stacking interaction. The tryptophans in non-channel form is placed shallower compared to that of the channel form [16,45]. In multi-tryptophan

120

3.3. Red edge excitation shift of channel form of gramicidin in membrane Red-edge excitation shift, i.e. REES is the shift of fluorescence emission maximum toward higher wavelength as a result of a shift in excitation wavelength toward the red edge of the absorption band. This effect is observed with polar fluorophores in motionally restricted region. REES is a quite useful tool to study the behaviour of membrane-bound molecules in motionally restricted environment, and also the membrane organization and dynamics of peptides bound to membranes [38,48,49]. The tryptophans in gramicidin channel conformation are located in motionally restricted region of the membrane. It has been previously reported that tryptophan in non-channel form is more dynamic, i.e. less ordered compared to that of channel form [15]. The interfacial region of membrane is characterized by unique motional and dielectric characteristics. This region is quite distinct from the isotropic deeper regions of the membrane and the bulk aqueous phase [50–52]. Quite distinguishable the region shows slow rates for solvent relaxation and also participates in intermolecular charge interactions [53] as well as hydrogen bonding through the polar head group [54]. These structural features count for the slow rate of solvent reorientation was accounted for microenvironments giving rise to significant REES effects. It is, therefore, the membrane interface which is most likely to display red-edge effects [48]. The progressive shift of excitation wavelength from 280 to 305 nm shifted the emission wavelength of gramicidin in vesicles from 333 nm to that of 340 nm in absence of BMOP, but in the presence a shift from 333 nm to 338 nm was seen as shown in Fig. 4. The REES in the presence and absence of BMOP to the gramicidin in vesicles were 5 nm and 7 nm respectively. The difference in REES of 2 nm after BMOP addition was observed in channel form

1.14

100

1.11

Fo/ F

Fluroscence Intensity (a.u.)

0 mM BMOP 0.125 mM BMOP 0.249 mM BMOP 0.372 mM BMOP 0.495 mM BMOP

peptides like gramicidin, the contribution of individual tryptophans is complex due to the environmental sensitivity as each tryptophan having heterogeneity in fluorescence parameters [46]. From the Stern–Volmer plot (Fig. 3), the Stern–Volmer quenching constant, Ksv, was calculated, which was found to be 0.24  103 L M1. Also plot shows an upward curvature, which hints that the quenching process is to be static in nature [47]. The bimolecular quenching constant (Kq), was also calculated from Eq. (2) and was found to be 6.92  1010 L M1 s1. The value of Kq also suggested that the process follows the static quenching mechanism. These results were further supported by the time resolved measurements.

80

1.08

1.05

1.02

60 300

320

340

360

Wavelength (nm)

0.1

0.2

0.3

0.4

0.5

[IL] (mM) Fig. 2. Fluorescence emission spectra of channel form gramicidin in lipid vesicle with addition of BMOP of different concentrations. The excitation wavelength was at 280 nm.

Fig. 3. The Stern–Volmer plot of the quenching of channel form gramicidin in lipid vesicle by BMOP.

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Table 1 Fluorescence lifetime measurements of the channel conformation of gramicidin in lipid vesicle with addition of different concentration of BMOP.

Emission maximum (nm)

340

338

336

Conc. (mM)

s1

s2

s3

(ns)

(ns)

(ns)

0.000 0.125 0.249 0.372 0.495

0.64 0.67 0.68 0.60 0.69

1.82 2.05 1.76 1.89 1.78

5.07 4.65 4.90 5.03 5.00

a1

a2

a3

v2

s⁄m

s0/s

56.74 54.91 55.42 57.95 55.16

28.03 23.94 27.47 27.14 28.39

15.23 19.12 17.11 14.91 16.45

0.94 1.02 0.97 0.97 1.20

3.09 3.09 3.07 3.07 3.09

1.00 0.99 1.00 1.00 0.99

The excitation wavelength was 295 nm; emission was monitored at 340 nm. See Section 2 for other details. ⁄ Calculated using Eq. (3) [33].

334

280

285

290

295

300

305

Excitation wavelength (nm) Fig. 4. Effect of changing excitation wavelength on the wavelength of maximum emission for the channel form gramicidin (j) with addition of BMOP (h). The ratio of gramicidin to DMPC was 1:50 (mol/mol).

gramicidin in vesicle suggests possible interactions of BMOP with membrane-bound gramicidin. These findings are consistent with the results obtained from steady state spectra. 3.4. Fluorescence lifetimes of channel form of gramicidin in membrane The fluorescence lifetime is a faithful indicator of the microenvironment and polarity of the fluorophore in which it is located [55]. The fluorescence lifetime of tryptophan is quite sensitive to solvent temperature and excited state interactions [56,57]. Fig. 5 shows the decay profile of the gramicidin in lipid vesicle with different concentrations of BMOP. The decay parameter fitted well tri-exponential function, which shows the decay of channel form gramicidin in lipid vesicles. Table 1 shows the decay profile of the tryptophan lifetimes of gramicidin in lipid vesicles along with different statistical parameters used to check the goodness of fit.

Eq. (3) was used to calculate the mean fluorescence lifetimes (s⁄m). The tryptophan lifetimes are known to be reduced when exposed to the polar environments [58]. The lower mean fluorescence lifetime of the tryptophan in channel conformation compared to non channel is mainly attributed to the polarity of the interfacial region where tryptophan residues are located. With the addition of different concentration of BMOP to the membrane-bound gramicidin, the mean fluorescence lifetime showed minor variations and remained almost constant. These minor variations in a mean fluorescence lifetime possibly may arise due to different mixtures of conformations and different average tryptophan positions. In addition, the s0/s ratio was also constant nearly equal to 1 as shown in Table 1. Therefore, it was again suggested that the quenching process operating here was static [33].

3.5. Dynamic surface tension This experiment was utilized to gain valuable insight into the effect of BMOP on gramicidin in lipid vesicles. Fig. 6 shows the surface tension isotherms of gramicidin in lipid vesicles and those corresponding to their mixture with BMOP at the air–water interface. The concentration of BMOP in the trough was fixed at 0.263 mM, 50 mM, 100 mM and 200 mM, while the concentration of gramicidin vesicles was kept constant. The critical micelle concentration (CMC) of BMOP (100 mM) has been reported earlier [27]. As with increase in time the dynamic surface tension curve of membrane bound gramicidin showed greater decrease with the increasing

Vesicles+gA Vesicles+gA + 0.263 mM BMOP Vesicles+gA + 50 mM BMOP Vesicles+gA + 100 mM BMOP Vesicles+gA + 200 mM BMOP

Suraface Tension (mN/m)

70

60

50

40

30

0

2000

4000

6000

8000

10000

Time (s) Fig. 5. Time-resolved fluorescence decay of the channel conformation gramicidin in lipid vesicle. Excitation wavelength was at 295 nm and the emission wavelength was monitored at 340 nm. The relatively broad peak on the right is the decay profile, fitted to a tri-exponential function. The lower plot shows the weighted residuals. The ratio of gramicidin to DMPC was 1:50 (mol/mol).

Fig. 6. Dynamic surface tension curve of pure vesicles having gramicidin and with the following BMOP concentrations (0.263 mM, 50 mM, 100 mM and 200 mM) with time. The ratio of gramicidin to DMPC was kept 1:50 (mol/mol). The experiments were performed with a final subphase phospholipid concentration of 150 lM at 303.15 K. The ratio of gramicidin to DMPC was 1:50 (mol/mol).

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concentration of BMOP. In order to check the behaviour of surface tension isotherms and hence the adsorption at air–water interfaces, BMOP at high concentration (ie. above CMC) was utilized. Rosen [59] suggested that dynamic surface tension curve have four regions: (1) the induction region, (2) the rapid fall region, (3) the meso-equilibrium region, and (4) the equilibrium region. As can be seen from Fig. 6 the surface tension of vesicles having gramicidin decreased and needs greater time to reach equilibrium upon adsorption at the air–water interface. From Fig. 6 it is very perceptible that no induction region exists here because of the more hydrophobic nature of the membrane. Further, the addition of BMOP to these gramicidin vesicles reduced the surface tension and equilibrium time considerably due to strong hydrophobic– hydrophobic interactions, as a result faster adsorption of BMOP with gramicidin vesicle occurs at air–water interface. The dynamic surface tension curves of pure vesicle and vesicle having gramicidin in the presence of BMOP differ remarkably as shown in Fig. 7. It is quite notable that the pure vesicle compared to vesicle having gramicidin, shows a slow reduction in surface tension. The vesicle having gramicidin shows faster adsorption with BMOP at

Surface Tension (mN/m)

70

Vesicle+ 50 mM BMOP Vesicle+gA+50 mM BMOP

60

50

40

30

20 0

2000

4000

6000

8000

10000

Time (s) Fig. 7. Dynamic surface tension of pure vesicle and vesicle having gramicidin with the BMOP concentration at 50 mM with time. The ratio of gramicidin to DMPC was kept 1:50 (mol/mol). The experiments were performed with a final subphase phospholipid concentration of 150 lM at 303.15 K. The ratio of gramicidin to DMPC was 1:50 (mol/mol).

air–water interface. Thus, the difference in the equilibration time clearly suggests binding of BMOP with gramicidin. The remarkable change in the surface tension hints toward the interaction between gramicidin in lipid vesicle with BMOP that might result in the formation of complexes with improved surface properties. At higher concentration of BMOP i.e. near CMC, surface tension isotherm shows sharp decrease (shown in Fig. 6), and equilibration time was very less at higher concentrations of BMOP. As the concentration was increased, more BMOP was adsorbed to the interface and hence the surface tension decreases [60]. One reason for such decrease may be due to the increase in the surface activity and complex formation as a result of interaction between the gramicidin in lipid vesicle and BMOP. The result confirms the interaction between BMOP and gramicidin in lipid vesicles as stated by previous discussed techniques. 3.6. FESEM characterization The Surface morphology of drop casted film was visualized through FESEM imaging having pure gramicidin containing lipid vesicular solution and mixed with BMOP. In Fig. 8 (Panel A) the surface morphology of the lipid vesicles having gramicidin in the absence of BMOP has been represented. Panel B and C shows the vesicles having gramicidin in the presence of BMOP at 50 mM and 100 mM concentration respectively. The high concentration of BMOP was utilized to visualize the interacting behaviour with vesicles. The Insets shown in Fig. 8 Panel B and C are the magnified image of the single vesicle having gramicidin with BMOP. Surface of the vesicle without BMOP is found smooth, whereas, BMOP makes the surface of the vesicles having gramicidin become rough (Panel C). The BMOP appears to bind with lipid vesicle and presumably to the gramicidin inserted in those vesicles (as discussed in the results shown above) which is not evident here due to the high concentration of BMOP used for the experiment. The image shows that the addition of BMOP interacts with the vesicle having gramicidin and the size of the vesicle increase after interaction. It has been known recently that IL possess ionic conductivity and lack electric conductivity which makes them quite suitable for SEM observation giving contrast images without noise, and keeps the sample in wet condition shown in Fig. 8, Panel B and C [61,62]. At CMC (100 mM) the size of the vesicles increases largely and the BMOP forms a film around the gramicidin vesicle, this type of behaviour of the pyrrolidinium based IL with vesicles has been reported earlier [23].

Fig. 8. SEM images of the vesicles at pH 7.2 and the inset represents the magnified image of a single vesicle. Panel A represents the lipid vesicles having gramicidin at pH 7.2 and. Panel-B and C represents the SEM images of lipid vesicles having gramicidin with addition of BMOP of 50 and 100 mM respectively at pH 7.2 drop-casted on the silicon substrate. The inset in Panel B and C represents the magnified image at same condition. The ratio of gramicidin to DMPC was 1:50 (mol/mol).

U.K. Singh et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 1–8

The above results signify that the membrane bound gramicidin in its channel form have strong interactions with BMOP and well supported by the above discussed results. The spectroscopic studies the steady state and time-resolved fluorescence provided the static quenching mechanism operative during the interaction. The CD spectra confirm that gramicidin in its channel form and after interaction with BMOP gramicidin remains in its channel form. The dynamic surface tension results supported the faster adsorption of BMOP with membrane bound gramicidin at the air–water interface which further supported the interactions between them. Finally, the FESEM images provided a valuable insight to validate the interaction of channel form gramicidin with BMOP. 4. Conclusion In summary, for the first time the effect of IL (BMOP) on the channel form gramicidin in lipid vesicle has been reported. The results obtained from REES and CD spectra reveals that the addition of BMOP shows interaction with the channel form of gramicidin vesicle without any considerable effect on its conformation. The results obtained from intrinsic fluorescence spectra indicated that BMOP effectively quenched the fluorophore (Trp) of membrane-bound gramicidin through static quenching mechanism. More specifically, time resolved spectra support the static quenching mechanism. In addition, the dynamic surface tension results strongly suggested that the presence of BMOP increases the surface activity of the membrane bound gramicidin ion channel due to hydrophobic interactions at air–water interface. The FESEM images showed that BMOP forms a layer over gramicidin vesicle at higher concentration. The comprehensive studies of IL with membrane have emerged as a topic of prime importance for designing of biocompatible ionic liquids utilizing their unlimited tunable property which would have potentially reduced toxic effect on membranes and biological molecules. Further, to expand the horizon beyond the membrane inserted peptides it would be important to extrapolate the biological effect of ILs to the membrane proteins to decipher the effect of ILs and comprehend their mechanism of interaction as well. We strongly believe that the work serves as the basis in designing of green ILs and useful for the future use of membrane inserted peptides in structure–activity relationship studies. Thus, it seemed to be quite promising for the application of ILs in advancement in protein pharmaceuticals, biochemical and biotechnological industries. Acknowledgments Dr. Rajan Patel thanks to Science and Engineering Research Board, New Delhi for providing research grant with Sanction Order No. (SR/S1/PC-19/2011) and one of the author (Upendra Kumar Singh) is thankful to UGC, New Delhi for JRF fellowship. References [1] W. Veatch, L. Stryer, The dimeric nature of the gramicidin A transmembrane channel: conductance and fluorescence energy transfer studies of hybrid channels, J. Mol. Biol. 113 (1977) 89–102. [2] O.S. Andersen, R.E. Koeppe, Molecular determinants of channel function, Physiol. Rev. 72 (1992) S89–S158. [3] J.A. Killian, Gramicidin and gramicidin–lipid interactions, Biochim. Biophys. Acta 1113 (1992) 391–425. [4] B.A. Wallace, Common structural features in gramicidin and other ion channels, BioEssays 22 (2000) 227–234. [5] D.A. Kelkar, A. Chattopadhyay, The gramicidin ion channel: a model membrane protein, Biochim. Biophys. Acta 1768 (2007) 2011–2025. [6] O.S. Andersen, R.E. Koeppe 2nd, Bilayer thickness and membrane protein function: an energetic perspective, Annu. Rev. Biophys. Biomol. Struct. 36 (2007) 107–130.

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