Interaction of KMP-11 with Phospholipid Membranes and Its

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The Interaction of KMP-11 With Phospholipid Membranes and Its Implications in Leishmaniasis: The Effects of Single Tryptophan Mutations and Cholesterol Achinta Sannigrahi, Pabitra Maity, Sanat Karmakar, and Krishnananda Chattopadhyay J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11948 • Publication Date (Web): 09 Feb 2017 Downloaded from http://pubs.acs.org on February 11, 2017

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The Journal of Physical Chemistry

The Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: The Effects of Single Tryptophan Mutations and Cholesterol

Achinta Sannigrahi1, Pabitra Maity2, Sanat Karmakar2, Krishnananda Chattopadhyay1† 1

Structural Biology & Bio-Informatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.

C. Mallick Road, Kolkata 700032, India 2

Department of Physics, Jadavpur University, 188, Raja S. C. Mallick Road, Kolkata – 700032.

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Abstract: KMP-11 is a small protein, which is believed to control overall bilayer pressure of Leishmania parasite. Recent results suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 play an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this paper, we investigated the interaction between KMP-11 and phospholipid membranes, using an unsatuarated

(PC 18:1) 1,2-dioleoyl-sn-glycero-3-

phosphocholine (DOPC) and a saturated (PC 12:0) 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) lipid, as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady state as well as time resolved fluorescence spectroscopy, isothermal titration calorimetry and zeta potential measurements were used for the determination of the binding constants for the wild-type and single site tryptophan mutants. Single site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane binding affinities of the partially exposed and buried tryptophan mutants (Y5W,Y48W respectively) were found to be greater than the wild type protein. In the presence of cholesterol, the binding constants of the wild type and Y48W mutant were found to decrease with the increase in cholesterol concentration. This was in contrast to the Y5W and F77W mutants, in which the binding constants increased by adding cholesterol. The present study highlights the interplay between the conformational architechture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of Leishmania parasite inside host macrophages.

†

Corresponding author: email: [email protected]

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Introduction: KMP-11 is a small (11 kD) protein, which is highly conserved in all stages of Leishmanial life cycle. This protein is being developed as a potential vaccine candidate against visceral Leishmaiasis1. It acts as a potential B and T cell immunogen during Leishmania infection. KMP11 is a major constituent of cell surface of kinetoplastid, and is expressed in both promastigotes (sand fly phase) and amastigotes (human phase). However, the surface expression of KMP-11 is considerably higher in amastigotes than in promastigotes, indicating the role of molecular architechture of this protein in the parasite relationship with the mammalian host2. In both phases, the protein was found to associate with membrane structures in cell surface, localizing itself around flagellar pocket, intracellular vesicles and flagellum3. It has been shown that cholesterol depletion from macrophage plasma membranes using MβCD results in significant reductions of the extent of Leishmania infection. It has also been suggested that membrane cholesterol is specifically required for the efficient attachment and internalization of the parasite on to macrophage cells leading to productive infection4. These results emphasize the importance of studying the effect of membrane binding on KMP-11 and the role of cholesterol in order to obtain insights into

Leishmaniasis. Interestingly, there is no systematic study available in

literature on KMP-11-lipid interactions and/or to investigate the role of cholesterol on this interaction. KMP-11 is a predominantly α-helical protein (Figure 1), containing significant sequence homology with human apolipoprotein A-1. Similar to ApoA1, KMP-11 contains amphiphilic character5. Tryptophans are abundant in membrane proteins. These residues reside preferentially near the lipid-water interface, where it is thought to play a significant anchoring role6. Interestingly, KMP-11 does not have any tryptophan residue in its WT sequence. In this paper, we have studied in detail the interaction between KMP-11 and phospholipid membranes. In doing so, we have quantitatively determined the binding constant of the protein for its binding with phospholipid membranes. Subsequently, we have inserted tryptophan residues in different portions to obtain a number of mutant proteins with varying solvent



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exposures. The positions of the inserted tryptophan residues in KMP-11 are shown in Figure 1. In one of the mutant proteins (Y48W), tryptophan residue stays buried deep inside the protein core, while in F77W, the tryptophan residue is solvent exposed. In the third mutant (Y5W), the tryptophan residue has been found to be partially exposed. This way, we are able to investigate how solvent exposure of a tryptophan residue could modulate the membrane binding property of a protein. Finally, we have studied the effect of cholesterol on the membrane binding of WT and tryptophan mutants. The implications of these results on the survival of Leishmania parasite inside host macrophage have been discussed.

Experimental Materials: Dioleoylphosphotidyl choline (DOPC), Dilauroylphosphotidyl choline(DLPC) and cholesterol were purchased from Avanti polar lipids Inc.(Alabaster,AL). DiI C-18 dye was obtained from Invitrogen (Eugene,Oregon,USA) and acrylamide from Sigma-Aldrich (St. Louis,USA). All other necessary chemicals were obtained from Aldrich (St.Louis,USA) and

Merck

(Mumbai,India).

Preparation of small and large unilamellar vesicles: An appropriate amount of lipid in chloroform (concentration of stock solution is 25 mg/ml) was transferred to a 10 ml glass bottle. Organic solvent was removed by gently passing dry nitrogen gas. The sample was then placed in a desiccator, connected to a vacuum pump, for a couple of hours to remove the traces of the leftover solvent. Required volume of 20 mM sodium phosphate buffer at pH 7.4 was added to the dried lipid film so that the final desired concentration (10 mM) was obtained.The lipid film with the buffer was kept overnight at 4 °C to ensure efficient hydration of phospholipid heads. Vortexing of hydrated lipid film for about 30 min produces multilamellar vesicles (MLV). Long vortexing was occationally required to make uniform lipid mixtures. LUV and SUVs were prepared by extruding the MLV with LiposoFast using AVESTIN extruder (Otawa,Canada). MLV suspensions were extruded through a polycarbonate membrane having pore diameters of 80 and 120 nm7. This results in the formation of well 4 ACS Paragon Plus Environment

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defined size of SUV (average diameter ~78 nm) and LUV (average diameter≈120 nm), as measured by dynamic light scattering (DLS). Vesicle solutions were degassed prior to all measurements, as air bubbles introduced in the sample during the extrusion may lead to artifacts. For preparing SUV containing cholesterol of different mole percentages, we have added required amount of cholesterol with the lipid in a chloroform solution.

Structure modelling of KMP-11 Structure modelling of KMP-11 has been discussed earlier1. Sequence analysis of KMP-11 using NCBI protein-protein BLAST8 does not show any close homolog with available solved structures. Profile based search performed using PSI BLAST9, however, indicates the presence of few remote homologs whose crystal structures are available. Since the sequence identities of the remote homologs are low, we have used a composite approach using ITASSER (iterative threading assembly refinement)10 of Zhanglab server, which combines various techniques such as threading, ab-initio modeling and atomic-level structure refinement methods. From the I-TASSER analyses, we have chosen a model structure which provides the best confidence score11.

The purification of the WT and mutants of KMP-11 Recombinant KMP-11 constructs (both wild type and mutants) were expressed and purified using

Ni-NTA

affinity

chromatography. For this purpose, Qiagen

supplied

protocol

(Qiaexpressionisttm, Qiagen, Germany) was used after slight modifications. The modifications included the use of 20mM imidazole in both lysis and wash buffers during cell lysis. The purified protein fractions were checked using 15% SDS -PAGE gel electrophoresis.The collected fractions containing the protein were dialyzed using 20 mM sodium phosphate buffer at pH 7.4 to remove excess imidazole. The concentration of the protein was determined using BCA Protein Assay Kit (Pierce, Thermo Scientific, USA)1.

Tryptophan quenching experiments: Steady state fluorescence spectroscopy and acrylamide quenching measurements in free and in membrane bound conditions were carried out using a PTI fluorimeter (Photon Technology International,USA). A cuvette with 1cm path length was used for the fluorescence 5 ACS Paragon Plus Environment


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measurements. For the tryptophan fluorescence quenching experiments, an wavelength

of 295 nm

was

used

to

eliminate the contributions

from

excitation the tyrosine

fluorescence. Fluorescence data were recorded using a step size of 1 nm and an integration time of 1sec. Excitation and emission slits were kept at 5nm in each case. Emission spectra between 305 nm and 450 nm were recorded in triplicate for each experiment. Typical protein concentration of 10 µM was used for each quenching experiment and 1:200 protein-lipid molar ratio was maintained. The protein solutions were incubated at room temperature for 1 hour and then titrated using a stock of 10M acrylamide. Necessary background corrections were made for each experiment. Acrylamide Quenching Data Analysis: Assuming I & Io are the tryptophan fluorescence intensity of the proteins in the presence and absence of acrylamide concentration [Q], the Stern-Volmer Equation12 can be represented as follows:

= 1 + [Q]

( 1)

is the Stern-Volmer constant, which can be determined from the slope of the linear plot of I0/I vs. acrylamide concentrations [Q]. It is important to point out here that the values obtained from the steady state fluorescence experiments may have complications arising from the components of static quenching. This is because, steady state fluorescence intensity may contain both static and dynamic components. Fluorescence lifetime of tryptophan residue does not change by static quenching; and hence time resolved fluorescence experiments can un-ambiguously determine the dynamic

quenching

constant.

We

have

carried

out

time

resolved

fluorescence

measurements with tryptophan mutants (Y5W, Y48W and F77W). Dynamic quenching constants measured by the lifetime data show identical trend (Figure S2 and Table S1). Fluorescence

lifetimes

were

measured

using

a

time-correlated-single-photon-counting

(TCSPC) equipment (Fluorocube Fluorescence Lifetime System by Horiba JovinYvon, Japan) using a picosecond pulsed diode laser with excitation at λex= 295 nm. The total intensity decay curves, I(t), were fitted to a bi-exponential function: 6 ACS Paragon Plus Environment

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=

Ʈ

+

Ʈ

(2)

where Ai, Ʈi denote the amplitude and lifetime of the i-th species. The fitting was done by an iterative procedure using the DAS 6.2 data analysis software supplied by IBH. Reduced and weighted residuals served as parameters for ensuring goodness of fit. The average lifetime of the tryptophan residue has been calculated using the following equation: = A Ʈ + A Ʈ /A Ʈ + A Ʈ

(3)

The average lifetimes in the absence and presence of acrylamide quencher have been denoted as T0avg and Tavg respectively12.

Binding assay using fluorescence spectroscopy We have used a fluorommetric assay for studying the binding of WT KMP-11 and its tryptophan mutants with SUV or LUV composed of DOPC, DLPC and DOPC-cholesterol mixtures. All samples were prepared in 20mM sodium phosphate buffer at pH 7.4. A set of samples were prepared using 1mM concentration of uniformly synthesized lipid vesicles. In each sample vial, 0.5 wt% of membrane specific DiI C-18 dye13 was added and the samples were kept at 37°C for overnight incubation. Subsequently, required amount of protein was added into the vials by maintaining lipid/protein molar ratio between 1:0 and 50:1. Samples were then incubated at room temperature (250C) for two hours.The steady state fluorescence emission spectra of the dye was taken at an excitation wavelength of 600 nm. The peak intensity values at 670 nm for DOPC and at 685nm for DLPC were plotted with protein concentration. The data have been fit using the Hill equation, as follows (Equation 4): = +

! " #$ #$ % &$

where F and

"

(4)

refer to the fluorescence intensity of DiI-C18 in the presence and absence of

protein, respectively.

! denotes

the minimum intensity in the presence of higher concentration

of protein and K being the equilibrium dissociation co-efficient of lipid-protein complex . The n is the Hill coefficient, which measures the cooperativity of binding and x being the concentration of protein.



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Isothermal Titration Calorimetry (ITC): The heat flow obtained from the binding of KMP-11 with phospholipid vesicles was measured using high sensitivity ITC (MicroCal iTC 200, UK). All experiments were performed at 30 0C. Solutions were degassed under vacuum prior to filling the sample and reference cells up. Solutions of protein sample (10µM) and DOPC SUV (2 mM) were loaded in the calorimetric cell of volume of 350 µl and in the syringe of volume of 40 µl, respectively.In an experiment, a series of 20 injections each with 2µl from the syringe was injected into the ITC cell containing KMP11 at 240 seconds intervals. Each injection produces a characteristic heat signal, arising from the released or absorbed heat from the lipid-protein interaction, leading to exothermic and endothermic signals. Heat of dilution was determined by injecting the buffer into the ITC cell containing KMP-11. ITC thermogram was obtained from the integration of heat signal and subtracting the heat of dilution arising due to each injection. ITC thermogram was then fit to a model (one site binding) provided by Microcal Origin to determine the binding constant (K) and the molar enthalpy of interaction (∆H). The Gibbs free energy G was obtained from the relation '( = −*ln 55.5 and hence entropic contribution can be eatimated via '( = ∆0 − ∆1. Here, the concentration of water 55.5 M was used to correct the unit of K to molar fraction.

Dynamic Light Scattering and zeta potential measurement: Zeta potential and size distribution were measured at room temperature (~25 °C) with the Zetasizer Nano ZS (Malvern Instruments, UK).The Zetasizer Nano uses 4 mW He–Ne Laser of wavelength 632.8 nm.The detector is positioned at the scattering angle of 173°. The detected scattered light is sent to signal processing correlator. Extruded vesicles, prepared with varying concentrations of protein, were loaded in a folded capillary cell for both zeta and size measurements. Each zeta potential measurementconsisting of 100 runs and size measurement 10–100 runs has been performed. Zeta potential was measured from the electrophoretic mobility (µ ) using a model described by the Smoluchowski and Hückel equation14. 345

2 = 678

(5)


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Where, η and 9 are the coefficient of viscosity and the permittivity of the aqueous medium, respectively. The Henry function, :;< , depends on the inverse Debye length κand the radius a of the vesicle. The hydrodynamic radius of the vesicles was measured from the dynamic light scattering (DLS) experiments. In DLS, the intensity fluctuations of scattered light were measured and the intensity auto correlation function was fit to exponential decay function to obtain the values of diffusion constant. Einstein-Stokes relation of < =

=> ?@4A

was used to calculate the hydrodynamic radius of

the vesicle. D is the diffusion constant and kT is the thermal energy. For both size and zeta potential measurements,100 µM concentration of DOPC SUV was used.

Circular Dichroism spectroscopy: Far UV-CD spectra of wild type KMP-11 and tryptophan mutants were recorded using a JASCO J720 spectropolarimeter (Japan Spectroscopic Ltd.). Far-UV CD measurements (between 200nm and 250 nm) were performed using a cuvette of 1mm path length. Protein concentration of 10µM was used for the CD measurements. Scan speed was 50nm/min; with response time being 2 sec. Bandwidth was set at 1nm. Three to five CD spectra were recorded in continuous mode and averaged.

Results and Discussion: Characterization of the mutant proteins of KMP-11 using far UV CD, steady state fluorescence and time resolved fluorescence quenching studies. Figure S1 shows the primary structure of KMP-11. These three mutants are selected by virtually inserting tryptophan residues at different positions of KMP-11, which span almost the entire sequence of KMP-11(Figure S1). Surface accessibility calculations suggested that tryptophan residue in Y5W, F19W, F30W, F51W, F62W, F73W and Y89W would be partially exposed. In contrast, that in Y48W was predicted to be buried, while the same calculation indicated the residue in F77W would be fully exposed1. We prepared Y5W, Y48W and F77W using site directed mutagenesis to be representative of three tryptophan classes.



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Far UV CD spectroscopy is a common method to study the secondary structure of a protein. Far UV CD spectroscopy shows the presence of double minima at 209nm and at 222nm (Figure 2a). The observed CD profile suggest that KMP-11 is alpha helical, supporting the predicted structure10 (Figure 1). Far UV CD spectra of the mutant proteins were found identical, indicating that the tryptophan mutations did not result in any significant change in the secondary structure. Steady state tryptophan fluorescence experiments were carried out to monitor the tryptophan environments of the mutant proteins and the results are shown in Figure 2b. In the case of Y48W, the tryptophan emission maximum was found to be at 326 nm, which is typically observed in a globular protein when the tryptophan residue is buried inside the hydrophobic core. For F77W, in contrast, the emission maximum was observed to be at 350 nm. For F77W, this emission maximum at 350 nm was accompanied by a decrease in the emission intensity when compared to that observed for Y48W.The tryptophan emission maximum at 350nm typically suggests a solvent exposed tryptophan residue. The emission maximum (at 350nm) matches with the emission maximum of free tryptophan in aqueous buffer. This observation suggests that the tryptophan residue in this mutant (F77W) is solvent exposed. In the case of Y5W, the emission maximum was observed at 340 nm, while the intensity value was found to be between Y48W and F77W. Fluorescence experiments suggest that the tryptophan residue present in Y5W is possibly partially exposed. While the steady state tryptophan fluorescence experiments provide preliminary idea about the varying exposure of the tryptophan residues in the mutant proteins, these results have been further supported by acrylamide quenching experiments (Figure 2c). The values of the Stern Volmer constant have been determined using a straight line fit as shown in Figure 2c.The measured Stern Volmer constant has been found to be the maximum in the case of F77W (5.8M-1), while the mutant Y48W has the minimum value (2.2 M-1). The value for the Y5W (3.8M-1) has been found to be between those observed for Y48W and F77W. The quenching results complement

the steady state fluorescence data, which is described in the previous

paragraph. Since the acrylamide quenching results obtained using steady state fluorescence methods may have complications arising from the static components of fluorescence quenching, quenching experiments are repeated using time resolved fluorescence spectroscopy (Figure S2, Supporting Information). The

observed Stern-Volmer quenching constants (Kd,M-1) and 10 ACS Paragon Plus Environment

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bimolecular quenching constants (Kq, M-1sec-1 ) from the time resolved fluorescence data of the inserted tryptophan residue (Y5W,Y48W, and F77W) follow identical trend (Figure S2, Table S1).

Interaction of WT KMP-11 with lipid vesicles: We have systematically investigated the binding interaction of WT and three different mutants with phospholipids membranes. We have chosen phospholipids, such as DOPC (18:1 unsaturated, Transition temperature ~-18°C15) and DLPC (12:0 saturated, Transition temperature ~-10C), as they exhibit fluid phase at room temperature, which is biologically more relevant. Their vesicle preparation is comparatively straightforward16. A. J. Schroit et al have determined the composition of different phospholipids in macrophage phagolysosome membrane17. Their results show that unsaturated phospholipids, such as 18:1 PC (DOPC), is a major constituent of the membrane18. It is well-known that unsaturated fatty acid component (PC 18:1) remains in higher amount in macrophage membrane19 due to its role in the enhancement of macrophage phagocytic activity. In contrast, the extent of saturated fatty acids (PC 12:0) is comparatively less in macrophage membrane17. For these reasons, we have chosen DOPC and DLPC for the present study. We understand that the use of different chain lengths for the unsaturated and saturated lipids is not ideal, and same chain length should be used for better comparison. However, it is difficult to prepare unilamellar vesicles using DSPC (18:0) or DPPC (16:0) at room temperature as the chain melting temperature (Tm) of them is much higher (550C for DSPC and 420C for DPPC). Since these lipids exhibit gel phase at room temperature, they may not be suitable as biomimicking model membrane. In addition, we have not used the mixture of two lipids to avoid the complexity of the system. Consequently, in this study we cannot rule out the possibility of the effect of saturation and chain length on the lipid-protein interaction. The interaction between KMP-11 and lipid vesicles was studied using a membrane specific fluorescence probe DiI C-18. This dye itself does not have significant fluorescence in aqueous buffer when excited at 600 nm, while the addition of phospholipid vesicles significantly enhances its fluorescence intensity (Figure S3a). In the presence of DOPC lipid vesicles, DiI C18 shows maximum fluorescence intensity at 670 nm whereas for the case of DLPC it shows maximum at 685 nm. Subsequent addition of KMP-11 decreases the fluorescence intensity 11 ACS Paragon Plus Environment


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(Figure 3a). The fluorescence emission intensity of DiI C-18 in the presence of DOPC SUV,DOPC LUV and DLPC SUV were found to decrease with increasing protein concentration (Figure S3a,b; Figure 3a). A slight blue shift was observed for DOPC SUV, but no significant change of wavelength maxima was noticed for DOPC LUV(Figure 3b) and DLPC SUV. The lipophilic carbocyanines, such as DiI C18 is known to emit fluorescence when incorporated into lipid bilayers. In contrast, they are weakly fluorescent in aqueous solution20. The high environment sensitivity of this dye, hence, makes it an excellent candidate to monitor proteinlipid binding.

There may be several processes that can occur in the excited state of the

fluorophore leading to a decrease in the intensity (quenching) due to protein binding. We measured time-resolved fluorescence to obtain further insights into these processes. We have found that the presence of protein does not affect the fluorescence property of the fluorophore, suggesting that the dye does not have a strong binding affinity towards the protein(Figure S3a). This result rules out the possibility of complex formation with the protein in the ground state of DiI leading to any static quenching. We have found out that the average lifetime of the dye (in the presence of the membrane) decreases as we add the protein, suggesting a strong contribution of dynamic quenching presumably through the solvent environments(Table S2). This quenching may occur due to the expulsion of the dye molecules as a result of protein binding. Alternatively, the hydration dynamic at the membrane-solvent interface may get altered due to lipid-protein interaction modulating the relaxation behavior of DiI. It is also known that DiI diffuses laterally within the membrane and therefore the change in the translational diffusion due to protein adsorption may lead to dynamic quenching. There are several literatures where DiI C18 is used to monitor the lipid rafts21 and also large-scale inhomogeneity in living cell surface22 as well as in giant vesicles containing protein23 . The association constant (Ka) as well as cooperativity index (n) were determined from the fitting of the fluorescence intensity data using equation (4). It was clearly evident that Ka for DOPC SUV (2.70 x 105M-1) is higher compared to that of DOPC LUV(0.917X105 M-1), but less than DLPC SUV (9.52 ×105 M-1). This result indicates that the binding of wild type KMP-11 follows the order: DLPC SUV>DOPC SUV> DOPC LUV (Figure 3a). All subsequent experiments (described below) were carried out using SUVs of DOPC and DLPC. It is evident from these results that with increasing membrane curvature binding affinity of wild type KMP-11 increases 12 ACS Paragon Plus Environment

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because the increase in bilayer curvature from LUV(~ 120 nm hydrodynamic radius) to SUV(~80 nm) leads to considerable change in packing of lipid molecules in bilayer which may facilitates the penetration by wild type KMP-1124.Again it can be concluded that this proteinmembrane binding depends on the lipid composition of bilayer. Subsequently, we used isothermal titration calorimetry (ITC) to monitor the binding between wild type KMP-11 and DOPC SUVs. The idea of these experiments was to complement the fluorescence experiments (described above) using a different method. The binding heat was found to be exothermic (Figure 3c), with molar enthalpy change of ∆H=-660.9± 64.20 cal/mol. The apparent binding constant Kapp was estimated as 1.65 × 105 M-1.The values of ∆G and ∆S were found to be -9.5 kcal/mol and 0.021 cal/mol/deg. The binding constant obtained from ITC (1.65 × 105 M-1) was found to be in good agreement with the association constant obtained from the fluorescence experiments (2.7 × 105M-1). In order to determine the change in the surface potential of the SUVs upon binding with the protein, we measured the values of zeta potential. Zwitterionic DOPC SUV showed small negative zeta potential (~ - 3 mV) at pH 7.4. After addition of the protein, the zeta potential of DOPC SUVs decreased remarkably with increasing concentration of wild type and mutant proteins (Figure S4a).This is expected because KMP-11 posseses negative charge at physiological pH. KMP-11 has a net negative charge of -1.7 as determined by the Protein calculator v3.4

server25(http://protcalc.sourceforge.net/) (Figure S5).

The increase

in

hydrodynamic diameter of DOPC SUV (~78nm to 100 nm ), as measured from DLS, with increasing the concentration of protein further suggested the binding of protein on the membrane (Figure S4b). KMP-11-lipid interactions: the effect of tryptophan mutants: In the previous section, we have discussed the binding between KMP-11 and SUV, which we monitored using fluorescence spectroscopy, isothermal titration calorimmetry, and zeta potential measurements. Subsequently, we monitored the binding between SUV and three tryptophan mutants (Y5W, Y48W and F77W) using steady state fluorescence spectroscopy. Zeta potential measurements were also carried out to complement the fluorescence results (Figure S4a). Figure 4a summarizes all steady state fluorescence data, while the values of Ka and n are shown in 13 ACS Paragon Plus Environment


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Table 1 and Table 2.The association constant was found to follow the order: Y5W > Y48W > wild type ≥ F77W for both DOPC and DLPC system. There could be two possible reasons for the above binding trend. First is the steric factor. The protein-lipid interaction has been shown to depend on the hydrophobicity of the interacting regions26. Since the lipid remained the same (DOPC&DLPC SUV), we can assume that the hydrophobicity exerted by the lipids remained constant, while the hydrophobicity of the mutants would play important roles. Figure 4b shows sequence distribution of hydrophobic scores for the three mutants (Y5W, Y48W and F77W), which was calculated using ProtScale (http://web.expasy.org/protscale/). We used the hydrophobicity scale as defined by Kyte and Doolittle27. Figure 4b shows clearly that for F77W, a large decrease in the hydrophobicity score takes place at the region around the mutation site, while other mutants do not show any significant change. The observed low value of the association constant for F77W, hence, is not unexpected. Using the HeliQuestCompuParam28 (http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParamsV2.py) analysis, the extent of hydrophobic patches inside the protein was estimated. From the helical wheel model analyses of the WT sequence (figure S6), two hydrophobic patches were identified. One of them (patch 1) were found present around residues 1-18, while the second patch consisted of the residues 40-57. The mutation sites at the 5 th and 48 th positions resided in these two patches. Interestingly, no hydrophobic face was observed around the sequence 70-87 (region of the 77th. Mutation site). When the tyrosine residues present in the patch 1 or 2 were mutated by a more hydrophobic tryptophan residue (Y5W and Y48W), the effective hydrophobicity increased significantly, resulting in more efficient membrane binding for these two mutants. Since the regions around 77 does not have a hydrophobic patch, this effect is expected to be less. In addition to the sterics, the electrostatic interaction may also play important role. The binding between a protein and lipid has been shown to have a strong electrostatic component. In this model, protein-lipid binding depends on the surface exposure of tryptophan side chain. This is because; tryptophan can effectively bind to lipid bilayer through strong hydrogen bonding using the carbonyl and phosphate moieties. Further, cation-π interactions to lipid choline moieties facilitate the binding leading to the localization of the tryptophan near membrane glycerol backbone29.Among the two mutants (Y48W and Y5W), the former has significantly less solvent exposure, which can be evident from the values of Ksv (Figure 2c). It may be noted that F77W may provide the strongest electrostatic component (the Ksv value for F77W is the highest), but 14 ACS Paragon Plus Environment

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for this case the strong steric factor seems to play the predominanting role. The third consideration may arise from the orientations of proteins in membrane (OPM) calculations, which was carried out using the OPM server30(http://opm.phar.umich.edu/server.php). It was observed that the binding of KMP-11 occured through N-terminal targeting. Figure 4c shows the results of OPM calculation, which suggest that the hydrophobic face near 1-18 residue is inserted into the bilayer. The Y5W mutation would hence increase the hydrophobic face of that region. Favorable roles of the above three considerations may be responsible for the observed maximum lipid binding affinity of the Y5W mutant. KMP-11-lipid interactions: the effect of cholesterol Abundance of cholesterol in macrophage membrane is essential for efficient attachment and internalization of Leishmanial parasite. The depletion of cholesterol from macrophage membrane has been shown to cause significant reduction in the extent of Leishmania infection4. It is clearly evident from Figure 5a that association constants for the wild type and Y48W mutant decrease with increasing cholesterol concentration, whereas, these values increase for the partially exposed Y5W and fully exposed F77W mutants (Table. 1,Table. 2). Similar trend was found for both DOPC and DLPC systems (Figure 5a,b).We have also found that co-operativity of binding for wild type (Figure. S7a) decreases (change of n~2 to n~1) with increasing cholesterol concentration in DOPC bilayer. Interestingly, F77W mutant showed completely different behaviors (Table. 1, Figure. S7b), while no significant change in co-operativity was observed for Y48W and Y5W mutants (Figure. S7c,d, Table 1). In contrast for DLPC system, no significant change in co-operativity was observed for wild type, F77W and Y5W whereas slight decrease of co-operativity was observed for Y48W mutant(Figure. S8a,b,c; Table 2). Subsequently, we carried out acrylamide quenching measurements of the tryptophan mutants (Y48W, F77W and Y5W) in their membrane (DOPC SUV) bound states in the absence and presence of cholesterol (Figure. 5c; Table 3)31. We observed that Ksv values of the mutants in free solvent were higher than those obtained in membrane bound condition. Interestingly, the Ksv values for the Y48W mutant were found to increase in the presence of cholesterol (25 mol%), while the opposite trends were observed for the F77W and Y5W mutants (Figure. 5c;Table 3). We believe that unfavorable lipid binding of Y48W in the presence of cholesterol 15 ACS Paragon Plus Environment


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facilitates positioning of the protein at the surface of the lipid bilayer, at which condition higher value of Ksv is expected. This is because; fluorescence emission from the tryptophan residue positioned at the surface is easier to quench than when the residue is inside the bilayer. The situation is opposite for F77W and Y5W, at which the increase in the cholesterol concentration increases lipid binding and decreases Ksv. For these mutants, the favorable lipid-protein interaction in the presence of cholesterol facilitates the positioning of the protein deep inside lipid bilayer, a condition in which Ksv is expected to decrease. Cholesterol triggers the survival of the parasite inside host macrophage: Implications in Leishmaniasis: It has been observed that cholesterol depletion from macrophage membrane decreases the infection propensity of Leishmania4. We find here a decrease in the binding affinity of the WT and Y48W mutant towards the phospholipid membrane with increasing cholesterol concentration. Figure 6 shows a schematic representation of the role of KMP-11 in the survival of parasite. Once the promastigote is transformed into the infectious promastigote, there occurs a post-translational modification in KMP-11, which is expressed in high copy number on the surface of the parasite to produce monomethylated arginine residue of KMP-1132. After endocytosis, the promastigote is converted into the amastigote inside the phagolysosome. In phagolysosomal environment, the protein is detached from the parasite surface resulting in an increase in its solution concentration. It has been reported that macrophage phagocytic vesicle contains high amount of cholesterol18. The presence of membrane cholesterol inhibits its binding to the phagolysosomal membrane, and as a result, the protein undergoes degradation. In subsequent steps, the synthesis of nitric oxide free radical takes place via the formation of methylated arginine residues33. Nitric oxide synthesis is catalyzed by nitric oxide synthase34, and free methylated arginine residues act as a competitive inhibitor of nitric oxide synthase enzyme affecting the nitric oxide synthesis pathway35. As a matter of fact, nitric oxide free radicals help in the destruction process of the harmful parasites inside macrophage. We speculate that cholesterol induced degradation of the protein in effect inhibits nitric oxide radical generation helping the parasite to survive. In case of the tryptophan mutants (Y5W & F77W), binding affinities towards membrane increase with increasing membrane cholesterol, which is not expected to be favorable for the Leishmanial parasite. Although work is in progress in our laboratory to validate this hypothesis, it may be noted that the evolutionary KMP-11 does not inherently contain tryptophan residues to presumably generate productive infection. 16 ACS Paragon Plus Environment

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Macrophage membrane is high in unsaturated phospholipids (like DOPC) and low in saturated phospholipids (like DLPC)17. Figure S9 clearly shows that the cholesterol induced reduction in the binding of wild type KMP-11 with DOPC SUV is higher than that with DLPC SUV membrane. This result implicates that DOPC lipid component presumably helps in the degradation of the protein promoting the survival of the parasite. Investigations are currently underway in our laboratory to validate this hypothesis.

Conclusions The role of KMP-11 in the immunological activity of cell is poorly understood and overlooked in the literature, though it is believed that KMP-11 plays crucial roles in the survival of Leishmania parasite inside macrophage by generating methylated arginine. It is known that the cholesterol depletion from membrane decreases the Leishmanial infection. Therefore, the observed decrease in the binding constant of the wild type protein with increasing membrane cholesterol may have important consequences on Leishmanial infection. From our result, it is evident that KMP-11-lipid interaction is dependent on the lipid types and membrane cholesterol helps in the reduction of binding affinity of KMP-11 towards membrane. Being detached from parasite surface, the protein could not effectively get adsorbed on macrophage surface. Subsequently, the protein can undergo degradation readily to produce nitric oxide synthase inhibitory methylated arginine. Mutation of the wild type protein affects the binding pattern substantially, suggesting a vital role of tryptophan in the lipid-protein interation. Tryptophan mutants apperently binds to the phospholipid membrane with stronger affinity than the wild type protein. Binding constant of wild type obtained from fluorescence assay complements the results estimated from isothermal titration calorimetry. Interestingly, the binding affinity of F77W and Y5W mutants increases with increasing cholesterol concentration, while it decreases with increasing cholesterol concentration for wild type and for Y48W. Our findings highlight the crucial secondary functions of lipid constituent and cholesterol component towards successful infection through parasite survival.



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Supporting Information: Two additional tables (Table S1 and S2) and

Acknowledgement: S. Karmakar acknowledges the financial support from the UGC major research project F. No.42−769/2013 (SR) and DBT funded research project (BT/PR8475/BRB/10/1248/2013). K. Chattopadhyay acknowledges the financial support from the network project grant, HOPE. P. Maity and A. Sannigrahi are supported by a Research Fellowship from the University Grant Commission, Govt. of India.

References 1. Sharma, S.; Sarkar, S.; Paul, S. S.; Roy, S.; Chattopadhyay, K., A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties. Scientific reports 2013, 3. 2. de Mendonça, S. C. F.; Cysne-Finkelstein, L.; de Souza Matos, D. C., Kinetoplastid membrane protein-11 as a vaccine candidate and a virulence factor in Leishmania. Frontiers in immunology 2015, 6. 3. Sahoo, G. C.; Rani, M.; Dikhit, M. R.; Ansari, W. A.; Das, P., Structural modeling, evolution and ligand interaction of KMP11 protein of different leishmania strains. Journal of Computer Science & Systems Biology 2011, 2009. 4. Pucadyil, T. J.; Tewary, P.; Madhubala, R.; Chattopadhyay, A., Cholesterol is required for Leishmania donovani infection: implications in leishmaniasis. Molecular and biochemical parasitology 2004, 133 (2), 145-152. 5. Fukushima, D.; Yokoyama, S.; Kezdy, F.; Kaiser, E., Binding of amphiphilic peptides to phospholipid/cholesterol unilamellar vesicles: a model for protein--cholesterol interaction. Proceedings of the National Academy of Sciences 1981, 78 (5), 2732-2736. 6. de Jesus, A. J.; Allen, T. W., The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch. Biochimica et Biophysica Acta (BBA)-Biomembranes 2013, 1828 (2), 864876. 7. Maity, P.; Saha, B.; Kumar, G. S.; Karmakar, S., Binding of monovalent alkali metal ions with negatively charged phospholipid membranes. Biochimica et Biophysica Acta (BBA)-Biomembranes 2016, 1858 (4), 706-714. 8. Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J., Basic local alignment search tool. Journal of molecular biology 1990, 215 (3), 403-410. 9. Altschul, S. F.; Madden, T. L.; Schäffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research 1997, 25 (17), 3389-3402. 18 ACS Paragon Plus Environment

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10. Roy, A.; Kucukural, A.; Zhang, Y., I-TASSER: a unified platform for automated protein structure and function prediction. Nature protocols 2010, 5 (4), 725-738. 11. Zhang, Y., I-TASSER server for protein 3D structure prediction. BMC bioinformatics 2008, 9 (1), 1. 12. Lakowicz, J., Principles of fluorescence microscopy. Kluwer Academic, New York: 1999. 13. Ramadurai, S.; Holt, A.; Krasnikov, V.; van den Bogaart, G.; Killian, J. A.; Poolman, B., Lateral diffusion of membrane proteins. Journal of the American Chemical Society 2009, 131 (35), 12650-12656. 14. Hunter, R., Zeta potential in colloid science. 1981. 15. Leonenko, Z.; Finot, E.; Ma, H.; Dahms, T.; Cramb, D., Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy. Biophysical journal 2004, 86 (6), 3783-3793. 16. Lewis, B. A.; Engelman, D. M., Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. Journal of molecular biology 1983, 166 (2), 211-217. 17. Schroit, A.; Gallily, R., Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity. Immunology 1979, 36 (2), 199. 18. Mason, R. J.; Stossel, T. P.; Vaughan, M., Lipids of alveolar macrophages, polymorphonuclear leukocytes, and their phagocytic vesicles. Journal of Clinical Investigation 1972, 51 (9), 2399. 19. Spector, A. A.; Yorek, M. A., Membrane lipid composition and cellular function. Journal of lipid research 1985, 26 (9), 1015-1035. 20. Texier, I.; Goutayer, M.; Da Silva, A.; Guyon, L.; Djaker, N.; Josserand, V.; Neumann, E.; Bibette, J.; Vinet, F., Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging. Journal of biomedical optics 2009, 14 (5), 054005-054005-11. 21. Klymchenko, A. S.; Kreder, R., Fluorescent probes for lipid rafts: from model membranes to living cells. Chemistry & biology 2014, 21 (1), 97-113. 22. Atala, A.; Soker, S., Enhancement of angiogenesis to grafts using cells engineered to produce growth factors. Google Patents: 2015. 23. Kahya, N., Protein–protein and protein–lipid interactions in domain-assembly: lessons from giant unilamellar vesicles. Biochimica et Biophysica Acta (BBA)-Biomembranes 2010, 1798 (7), 13921398. 24. Wetterau, J. R.; Jonas, A., Effect of dipalmitoylphosphatidylcholine vesicle curvature on the reaction with human apolipoprotein AI. Journal of Biological Chemistry 1982, 257 (18), 10961-10966. 25. Lomonosova, A. V.; Ovchinnikova, E. V.; Kazakov, A. S.; Denesyuk, A. I.; Sofin, A. D.; Mikhailov, R. V.; Ulitin, A. B.; Mirzabekov, T. A.; Permyakov, E. A.; Permyakov, S. E., Extremophilic 50S ribosomal RNAbinding protein L35Ae as a basis for engineering of an alternative protein scaffold. PloS one 2015, 10 (8), e0134906. 26. de Planque, M. R.; Killian*, J. A., Protein–lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring (review). Molecular membrane biology 2003, 20 (4), 271-284. 27. Kyte, J.; Doolittle, R. F., A simple method for displaying the hydropathic character of a protein. Journal of molecular biology 1982, 157 (1), 105-132. 28. Gautier, R.; Douguet, D.; Antonny, B.; Drin, G., HELIQUEST: a web server to screen sequences with specific α-helical properties. Bioinformatics 2008, 24 (18), 2101-2102. 29. Allen, A. J. d. J. a. T. W., BBA Biomembrane 2013, 1828, 864-876. 30. Lomize, M. A.; Pogozheva, I. D.; Joo, H.; Mosberg, H. I.; Lomize, A. L., OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic acids research 2012, 40 (D1), D370-D376. 31. Morillas, M.; Swietnicki, W.; Gambetti, P.; Surewicz, W. K., Membrane environment alters the conformational structure of the recombinant human prion protein. Journal of Biological Chemistry 1999, 274 (52), 36859-36865. 19 ACS Paragon Plus Environment


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32. Jardim, A.; Funk, V.; Caprioli, R.; Olafson, R., Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein. Biochemical Journal 1995, 305 (1), 307-313. 33. Fang, F. C.; Vazquez-Torres, A., Nitric oxide production by human macrophages: there's NO doubt about it. American Journal of Physiology-Lung Cellular and Molecular Physiology 2002, 282 (5), L941-L943. 34. Olekhnovitch, R.; Bousso, P., Induction, propagation, and activity of host nitric oxide: Lessons from Leishmania infection. Trends in parasitology 2015, 31 (12), 653-664. 35. Jadeski, L. C.; Lala, P. K., Nitric oxide synthase inhibition by N G-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors. The American journal of pathology 1999, 155 (4), 1381-1390.


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Table 1 shows the values of the binding constants and cooperativity indices for the binding of wild type and mutant proteins with DOPC SUVs in the presence of different concentrations of cholesterol. Figure 5a plots these values as a function of cholesterol concentrations. Mole % of cholesterol

Binding constant(Ka, M-1) and Hill co-efficient (n) values in different cholesterol percentage Wild type 5

F77W

Y48W

5

5

Y5W 5

in DOPC

Ka (× 10 )

0

2.7 ± 0.135

2.018± 0.161

2.63±0.156

1.413±0.067

3.57± 0.161

1.98 ±0.158

7.69±0.384

2.003 ±0.15

5

1.58±0.079

2.185± 0.174

6.25±0.312

0.69 ±0.320

2.85± 0.185

1.90± 0.120

18.18±0.909

2.065 ±0.165

10

1.92±0.096

1.55± 0.124

10.2±0.510

1.75± 0.240

3.03±0.142

1.93±0.109

16.66±0.833

2.021 ±0.161

15

1.04±0.052

1.405 ±0.112

12.5±0.625

1.84± 0.147

2.87±0.143

2.12 ±0.121

17.54±0.877

2.047± 0.163

20

1.02±0.051

1.119± 0.089

14.28±0.714

1.81±0.144

2.56±0.166

2.086 ±0.086

17.24±0.862

2.03 ±0.162

25

0.78±0.039

1.003± 0.197

14.7±0.735

2.27±0.181

2.77±0.138

1.96 ±0.086

16.39±0.819

1.985 ±0.158

n

Ka (× 10 )

n

Ka (× 10 )


n

Ka( × 10 )

n


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Table 2 shows the values of the binding constants and cooperativity indices for the binding of wild type and mutant proteins with DLPC SUVs in the presence of different concentrations of cholesterol. Figure 5b plots these values as a function of cholesterol concentrations. Mole % of cholesterol

Binding constant(Ka, M-1) and Hill co-efficient (n) values in different cholesterol percentage Wild type

F77W

Y48W

Ka (× 105 )

0

9.52 ± 0.476

2.57±0.176

10.18 ± 0.509

1.686±0.221

15.6±0.527

1.909±0.121

17.37± 0.868

1.735±0.298

5

9.09± 0.454

2.38±0.231

40.01± 2.0005

0.918±0.197

15.85±0.213

1.133±0.076

20.04± 1.002

2.205±0.097

10

5.37 ± 0.268

1.45±0.116

40.17± 2.0085

1.118±0.239

10±0.318

0.86±0.231

20.12± 1.006

2.102±0.105

25

6.06± 0.303

2.13±0.097

45.66± 2.283

1.739±0.212

6.66±0.414

0.755±0.312

21.23± 1.061

2.483±0.217

n

Ka (× 105)

n

Ka (× 105)

Y5W

in DLPC


n

Ka( × 105)

n

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Table 3 Acrylamide quenching of tryptophan fluorescence of three different mutants in DOPC membrane bound condition containing different cholesterol concentration. Proteins

Ksv values(M-1) at different cholesterol percentage in DOPC SUV 0%

Y48W Y5W F77W

1.732±0.057 2.989±0.074 2.959±0.120

25% 2.120±0.068 2.402±0.087 1.993±0.048

Figures:



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Figure 1: The modeled structure of the wild type KMP-11. The positions of the tryptophan residues in the mutant proteins are shown by red color.


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Figure 2 (a) Far UV-CD spectra of the wild-type (pink), Y5W(red), F77W(black)and Y48W(blue)proteins. (b) Tryptophan fluorescence spectra and (c) acrylamide quenching plots (Fo/F vs. acrylamide concentrations) of F77W (black), Y5W(red) and Y48W(blue) proteins. Figures 2(b) and 2(c) do not have the data for the wild type protein because the wild-type KMP-11 does not have any tryptophan residue. All the experiments have been carried out using 20 mM sodium phosphate buffer at pH 7.4. Neccesary background corrections were performed for each experiment. 25 ACS Paragon Plus Environment


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Figure 3(a) Fluorescence intensity originated from DiI C-18 in the presence of DOPC SUV (black ), DLPC SUV (red) and DOPC LUV(blue) were plotted against the concentration of wild type KMP-11. The lines through the data are the fits using the Hill equation (b)Wavelength shift of DiI C-18 dye in DOPC SUV(red)and DOPC LUV(black) plotted against the increasing protein concentration.(c) Binding Isotherm of wild type KMP-11 with DOPC SUV. Solid line is obtained from the fit using one site binding model provided by microcal origin. 26 ACS Paragon Plus Environment

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Figure 4 (a) Quenching of fluorescence intensity of DiI C-18 with increasing concentration of wild type and mutant proteins. The solid lines are obtained from the fit with Hill equation. Error bars are the standard deviations. (b) Plot of the Hydrophobicity score against amino acid positions of the wild type and other three mutants. These plots show similar profiles for the wild type , Y5W and Y48W mutants. However, significant difference was observed for F77W mutant. (c) Orientation of the wild type protein on phospholipid vesicle surface (obtained using the OPM server). This shows the insertion of N-terminal region of KMP-11 inside the bilayer.



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Figure 5 (a)The binding constants of wild type, Y48W , Y5W and F77W as function of cholesterol concentration. Inset shows the change of binding constants of wild type(black solid line) and Y48W mutant(red solid line) with concentration of cholesterol in DOPC membrane.(b)The binding constants of wild type, Y5W and F77W as function of cholesterol concentration in DLPC membrane (c) Acrylamide quenching of tryptophan residue in three different mutant Y48W,Y5W and F77W in membrane bound condition in presence and absence of cholesterol where Y48W mutant shows reverse quenching than the other mutants in presence of cholesterol.


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Figure 6 Schematic representation of the role of KMP-11 in survival of parasite in macrophage. Cholesterol reduces the affinity of wild type KMP-11 towards membrane and enhances the probability of degradation to produce mono-methylated arginine which inhibits nitric oxide synthase enzyme from production of Nitric oxide radicals and helps the survival of parasite inside macrophage.



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Interaction of KMP-11 with Phospholipid Membranes and Its

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