Nanoparticle Induced Conformational Switch Between Alpha-Helix

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Nanoparticles Induced Conformational Switch Between Alpha Helix and Beta Sheet Attenuates Immunogenic Response of MPT63 Achinta Sannigrahi, Sayantani Chall, Junaid Jibran Jawed, Amrita Kundu, Subrata Majumdar, and Krishnananda Chattopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00354 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Nanoparticle Induced Conformational Switch Between Alpha-Helix and Beta-Sheet Attenuates Immunogenic Response of MPT63 Achinta Sannigrahi1#, Sayantani Chall1#, Junaid Jibran Jawed2, Amrita Kundu1, Subrata Majumdar2, 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 Molecular Medicine, Bose Institute, Kolkata, India

†

Corresponding author Email: [email protected]

#

Contributed equally

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Abstract: Although

significant

efforts

have

been

devoted

to

develop

nanoparticles-based

biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from beta-sheet to alpha-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a beta-sheet to alpha-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability towards NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Keywords: MPT63, Mycobacterium tuberculosis, Protein conformation, Mutation, Magnetite nanoparticles, alpha-helicity.

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Introduction: As biomolecules-based drug development (Biologics) are gaining momentum, improvement of their bioavailability and delivery procedures are becoming subjects of extensive research. One preferred delivery option is conjugation with a nanoparticle (NP), for which it is essential to investigate the effect of nano-surface binding on the activity of the biomolecule.1-3 Dynamic physicochemical interactions, kinetic and thermodynamic changes at NP surface may cause significant alteration in protein structure and function.4 The dependence of protein structurefunction on NPs size, surface characteristics and charge has been studied in detail. 5-7 The relevance of NP-biomolecules interaction becomes particularly important when the attached molecule is a protein, as different conformers of a protein can be separated by a very small energy barrier. Several research groups designed and developed conformational switches, which can modulate protein functions in various biological processes. This conformational switch may occur between a disordered and ordered state or, between a helix and a beta -sheet structure.8-10 We hypothesize that the binding of a protein onto nanoparticle (NP) surface can provide sufficient energy to trigger a conformational switch of a suitable protein molecule, the functional characteristics of which needs to be studied in detail. Although protein-NP binding has been studied extensively, the influence of the NP surface on protein conformational switch has not been well established yet. We verify here this hypothesis by studying the interaction between a suitably coated magnetiteNP and MPT63, a small major secreted protein from Mycobacterium tuberculosis. There are two reasons for choosing this protein as model. First, MPT63 is a target against TB, which has been shown to have immunogenic properties.11 Efforts are underway to develop NP -conjugated MPT63 as a therapeutic strategy against TB.12 There are two types of immune response, one is protective immunity also known as Th-1 response and another is suppressive immunity or Th-2 response. Th-1 response helps the host to restore immunity against any upcoming pathogenic infection or immune suppression, whereas increased Th-2 response is associated with a diseased condition and pathogenesis. MPT63 was found to be associated with increasing Th-1 cytokines profile which includes TNFα, IL-12 and the most potent anti-microbial reactive molecules NO.13 3


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These molecules are helpful in restoring host protective immune response, therefore MPT63 can be used as therapeutic candidates against infections. The applications of nano-conjugated immunogens against bacterial and auto-immune diseases has been well explored in literature.14-15 Second, although it contains predominantly beta-sheets16 (Figure 1), it has been shown to have propensity towards the formation of alpha-helices as monitored by the aid of biophysical techniques including steady state fluorescence, Fluorescence correlation spectroscopy and molecular dynamic simulation.17 Consequently, this protein is an ideal candidate to explore the possibility of developing a switch between a beta-strand to alpha-helix. We have studied the immunogenic response of MPT63 by monitoring the expression of tumor necrosis factor alpha (TNFα), Interleukin 12 (IL-12) and the production of nitric oxide (NO). TNFα and IL-12 are cytokines, which are small proteins present in host cells whose expression level changes in response to infection, inflammations etc. Cytokines are found to induce NO production in host cells, in a synergistic manner.13 For the present study, we used magnetic NPs because of their extensive therapeutic and delivery applications.18-19 Their use as contrast agents for magnetic resonance imaging has been well established.20 Cationic magnetic NPs has been shown to act as carriers for targeted delivery.21 Their applications in hyperthermic treatments has also been explored extensively.22 Suitably coated magnetic NP has been shown to clock the early events of aggregation of alpha-synuclein, a protein associated with the pathophysiology of Parkinson ’s disease.23 Several reports on the application of protein associated magnetic NPs is also available. For example, a recent study has shown effective antioxidant activity and efficient delivery to endothelial cells by incorporating an enzyme catalase into iron oxide NPs.24 Another report has shown that functionalization of iron oxide NPs with HSA protein is useful for thermal therapy.25 Recent research in our lab has shown that the interaction between magnetic NPs with a protein can depend on several factors including the concentrations ratio of protein and NPs and solution conditions.26,27 We show that the binding of arginine coated Fe3O4 NPs (Fe3O4@Arg NPs) to the wild type (WT) MPT63 triggers a local conformational switch from beta-sheet to alpha-helix, which is associated with partial unfolding. We find that the activity of MPT63 in the nano-conjugated form is reduced significantly, which could be restored by carefully using protein engineering to increase the flexibility of the sequence stretch between 19th and 30th amino acids. The present study 4


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emphasize that a careful optimization of protein sequence would be essential to develop a therapeutically active nano-formulation of MPT63.

Experimental Section: Materials: AR grade reagents including L-Lysine (C6H14N2O2), Polyethylene glycol (C2nH4n+2On+1) (MW ~20000) and tetrachloroauric acid (HAuCl4), L-arginine (C6H14N4O2), Ferrous Sulphate heptahydrate (FeSO4.7H2O) and Ferric Chloride tetrahydrate (FeCl3.4H2O) were purchased from Sigma–Aldrich (St. Louis, USA). Expression and purification of MPT63 and its mutants: The plasmid pQE30 containing the wild type MPT63 gene was provided by Dr. David Eisenberg (University of California, Los Angeles, U.S.). Plasmids were transformed in E. coli XL1-Blue cells. Site-directed mutagenesis (W26F, G20C, G25A) was carried out using a Quick Change Lighting site-directed mutagenesis kit (Agilent Technologies). The mutations were verified by DNA sequencing. pQE30 was transformed into XL1-Blue cells and there after these transformed strains were used to express and purify WT and mutant proteins. Single colony of transformed XL1- Blue cells were grown aerobically at 37 °C in a LB medium containing 100 µg/mL ampicillin and induced with 1mM IPTG for 5 h when the absorbance of the medium reached 0.5 at 600 nm. Then cells were harvested by centrifugation at 8000 rpm for 15 min at 4 °C and re-suspended in 40mL sonication buffer (50 mM Potassium phosphate, 300 mM KCl, 10mM imidazole, pH 7.8). 1mM phenylmethylsulfonyl fluoride (PMSF) was added to the sonication buffer prior to sonication. Cell lysates were then centrifuged at 12,500 rpm for 45 min at 4 °C and supernatants were mixed with 2 mL of Ni-NTA agarose resin (Qiagen), previously equilibrated in sonication buffer, and stirred at 4 °C for 1 h. After that, protein and resin slurry were poured into a column and washed with 10 volumes of sonication buffer followed by 10 volumes of wash buffer (50 mM potassium phosphate K3PO4, 300 mM KCl, 20 mM imidazole, pH 7.8).Same buffer was used for protein elution with a gradient of 20 to 500 mM imidazole. Fractions containing purified protein as 5


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assessed by SDS-PAGE (purity >90%), were pooled and dialyzed against 20 mM Sodium phosphate buffer pH 7.5 and aliquots were stored at -20 °C until required.17 Activity assay of wild type MPT63 and W26F mutant: RAW264.7 cells were maintained in DMEM media (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FCS (Gibco, USA). After overnight culture 100 nM of MPT63 and W26F were treated for 4 hr and 24 hr for mRNA isolation and for ELISA respectively. RNA was isolated as per the standard protocol using Trizol reagent and reverse transcribed with Revet-Aid TM M-MuLV reverse transcriptase (Fermentas, Ontario, Canada) followed by PCR using specific primers for IL-12, TNFα and GAPDH.13 Nitric oxide generation was measured using a calorimetric assay kit (Boehringer Mannheim Biochemicals). ELISA was performed using a specific antibody of IL-12 and TNFα (BD Biosciences, CA, USA) as per the manufacturer instruction. We treated the overnight grown macrophage culture with 100 nM of each of the protein separately, cells were isolated after 4 hr of incubation and kept in Trizol whereas the supernatant were collected separately after 48 hr. Supernatant thus obtained was used to perform ELISA for IL-12 and TNFα expression and the colorimetric assay was performed to observe the change in nitrite generation as these are the markers of inflammatory response. Synthesis of Arginine modified Fe3O4 NPs: The syntheses were performed following a similar protocol published earlier28 with small modifications. The first step was the generation of the magnetite NPs using co-precipitation method, in which FeSO4.7H2O and FeCl3.4H2O salts were used in water in the molar ratio of 1:2 under nitrogen atmosphere. At this stage, an aqueous solution of Arginine (5 mg/ml) was added to the suspension slowly and then allowed to stir for another 30min. After cooling to room temperature, the solvent was removed by magnetic decantation. The dispersion was made free of excess Arginine or any residual salts by multiple washing of the particles with water. The dispersion was then centrifuged (5000 rpm for 5 min) to remove bigger aggregates and the supernatant was stored for further use. The same procedure was followed for the preparation of uncoated NPs without adding Arginine. Here, after washing the uncoated NPs were made into aqueous dispersion via peptization with perchloric acid. The Arginine coated Fe3O4 NPs (Fe3O4@Arg) were characterized by various methods. The coated NPs were 6


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magnetic at room temperature both as solid and in water dispersed state (which was checked by holding a magnet by the side of the dispersion). Synthesis of polyethyleneglycol (PEG) and Lysine modified Fe3O4 NPs: Synthesis of PEG29 and lysine functionalized Fe3O4 NPs were carried out using previously reported protocol.23 Synthesis of Arginine modified Au NPs: For the synthesis of arginine stabilized gold nanoparticles (Au@Arg NPs), 5 ml of 5 mM arginine solution was added to the gold solution of final concentration 5×10-4 M under stirring at room temperature (pH 7.5). After few minutes of stirring, 25 µl from 0.01g ice cold NaBH4 stock was added drop wise. Solution color was rapidly changed indicating the formation of arginine stabilized gold NPs.30 Fluorescence assay for binding measurements: Protein-NPs binding was monitored by steady state fluorescence spectroscopy using the quenching of intrinsic tryptophan fluorescence of MPT63 and its mutants. Excitation wavelength of 295 nm was used to avoid any contribution of tyrosine residues. Emission spectra were recorded between 305 nm to 450 nm. Typical protein concentration of 5 µM was used for each binding experiments and protein solutions were titrated using 5 mg/ml stock concentration of four types of NPs suspensions (i.e., Fe3O4@Arg NPs, Fe3O4@PEG NPs, Fe3O4@Lys NPs, Au@Arg NPs). The data were fit using Hill equation as follows (Eqn 1):

ࡲ = ࡲ૙ +

(ࡲࢋ − ࡲ૙ )࢞࢔ … … … … (૚) ( ࢞࢔ + ࡷ࢔ )

Where, F and F0 refer to the fluorescence intensity of tryptophan in the presence and absence of NPs respectively. Fe denotes the minimum intensity in the presence of high concentration of NPs and K is the equilibrium dissociation constant. The parameter n is the Hill coefficient, which measures the cooperativity of binding. The parameter x is the concentration of NPs.31-32 Unfolding transition of WT-MPT63 and W26F mutant: Unfolding of wild type MPT63 and its mutant protein was performed using Guanidium hydrochloride (Gdmcl) by monitoring the quenching of tryptophan fluorescence of wild type and W26F mutant proteins. Typical protein concentration of 5 µM was used for unfolding experiments. Experimental data were fit using the two-state transition model as follows (Eqn 2): 7


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࢟ = ቄ(࢟࢔ + ࢓࢔ ∗ ࢞) + (࢟ࢊ + ࢓ࢊ ∗ ࢞) ∗ ࢋቂ−ቀ∆ࡳ / ቄ૚ + ࢋ

࢞ ቂ−ቀ∆ࡳ૙ +࢓∗ ቁቃ ૙.૞ૡ૛૟૜૙ ቅ

࢞ ૙.૞ૡ૛૟૜૙ቁቃ ቅ

૙ +࢓∗

(૛)

Where y denotes the observed fluorescence intensity, yn and yd refers to fluorescence signals at native and unfolded conditions respectively. ∆G0 stands for free energy of unfolding transition, m is the cooperativity and x denotes the concentration of Gdmcl. All data analysis and fitting were carried out using OriginPro 8.5. ANS Fluorescence Measurements: ANS stock solution was prepared in methanol. Final ANS and protein concentration used in the experiments were 100 µM and 1 µM respectively. Prior to the experiments ANS was added to the samples and the solutions were incubated for 10 minutes before the measurements of ANS fluorescence. Fluorescence spectra of ANS containing samples were recorded using an excitation wavelength of 370 nm and emission spectra were recorded between 450 nm and 650 nm at 25 °C.33 Molecular docking analysis and instrumentation part are discussed in the supporting information.

Results and Discussions: Characterization of nanoparticles: Fe3O4@Arg NPs were first characterized by X-ray diffraction analysis (Figure 2a). The presence of characteristic XRD peaks at 2θ = 30.2, 35.4, 43.1, 57.1 and 62.2, which corresponded to the (220), (311), (400), (511) and (440) planes (JCPDS card No. 19 -0629) substantiated the formation of Fe3O4. Further details of the size of Fe3O4@Arg NPs were obtained from HR-TEM analysis. The bright field images showed a composite like structure with one or more NPs surrounded by the organic amino acid coating (Figure 2b, S1a). Similar data were observed recently for PEG -coated magnetite NPs,29 where the thickness of the organic composite was found to be an obstacle to obtain a good high resolution (HR) picture (Figure S1a). However, analysis of the present high resolution images estimated that the average size of the core magnetite NPs were 5-7 nm, which was in good agreement with the XRD data. The EDX (Energy dispersive X-ray) performed with 8


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Fe3O4@Arg NPs showed the presence of Fe and O apart from Cu & C (from the grid) confirming the presence of iron oxide in coated NPs (Figure S1b). The SAED (selected area electron diffraction) analysis showed amorphous nature of arginine coated magnetite NPs (Figure S1c). FTIR spectra further supported the formation of arginine coated magnetite NPs. Figure 2c showed Fe˗O characteristic peak of magnetite (~582 cm-1) in the spectra. Also, the C=O and CO stretching bands from arginine were observed at ~1630 and ~1440 cm-1 respectively. While the peak at ~2920 cm-1 can be attributed to the C˗H stretching vibration of the amino acid residue, the N˗H stretching of the same appeared at ~3420 cm-1. This FTIR spectral behavior was significantly different from the FTIR spectra of bare magnetite NPs, which supported the formation of arginine functionalized magnetite NPs. The solution hydrodynamics of Fe3O4@Arg NPs was characterized by measuring the hydrodynamic diameter of the particles using dynamic light scattering (DLS). The data showed (Figure 2d) that the average hydrodynamic diameter of NPs were 178 nm. The suspension was found stable at least for one month (Figure 2e). After addition of WT MPT63 in the solution of the Fe3O4@Arg NPs, there occurred significant increase in the hydrodynamic diameter of the particles (Zavg ~290 nm) indicating the adsorption of proteins (Zavg ~11 nm) on NPs surface (Figure S2). The suspension of arginine capped NPs resulted in zeta potential value of 0.00042 at pH 7.5, which increased to 10.45 at pH 1.5. Thermal stability of the synthesized functionalized magnetite NPs was studied by TGA analysis (Figure 2f). Appearance of surface Plasmon resonance band at ~545 nm (Figure S3a) indicated synthesis of arginine stabilized gold NPs. DLS study revealed hydrodynamic size of gold NPs of around 120 nm (Figure S3b). Characterization of Fe3O4@PEG and Fe3O4@Lys NPs were done as reported earlier.23,29 Interactions of NPs with MPT63: The interaction between wild type MPT63 and Fe3O4@Arg NPs resulted in a large quenching of tryptophan fluorescence. Using the change in fluorescence, binding constant (Ka) was determined to be (6.6 ±0.1)×103 M-1 at pH 7.5 (Figure 3a, S4a, Table 1). There are various electronic factors, which may be responsible for the quenching of tryptophan fluorescence as the protein binds onto Fe3O4@Arg NPs surface. This observed behavior may have contributions from both static or dynamic quenching as evident by the nonlinear nature of the Stern-Volmer plot (Figure S4b). To obtain further insights into this, time 9


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resolved measurements were carried out in the presence of increasing concentration of Fe3O4@Arg NPs, which showed a linear dependence of the τ0/τ (the definition of τ0 and τ are given in supporting information section) with Fe3O4@Arg NP concentration (Figure S4c). The binding interaction between the NPs and wild type protein was found to be fast, completing within minutes (Figure S4d). Protein binding to the NPs was simultaneously monitored by measuring enhanced absorbance at 280 nm (Figure S4e). Ka values obtained from the fluorescence and absorbance measurements were found to be similar. The observed protein-NPs interactions seemed to have a strong electrostatic component, as the presence of NaCl (100 mM and 250 mM) resulted in a decrease in Ka (Figure S4f, Table 1). Far UV CD was subsequently used to determine whether Fe3O4@Arg NPs binding lead to any change in the secondary structures of the protein. Far UV CD spectrum of WT MPT63 in the absence of NPs was characterized by a prominent peak at 216 nm suggesting it to be a betasheet protein, a result confirmed by the crystal structure (Figure 1). The binding of Fe3O4@Arg NPs with WT MPT63 resulted in a decrease in the overall ellipticity indicating partial unfolding of the protein (Figure 3b). In addition, far UV CD showed a shift towards 222 nm (Figure 3c). The difference far UV CD spectrum calculated from the spectra obtained in the presence and absence of NPs suggested a prominent increase in alpha helical character of the protein (increase in the ellipticity at 222 nm, Figure 3d). FTIR spectroscopy further corroborated CD experiments. In the FT-IR analysis, the 1600-1700 cm-1 range denotes the amide-I region of a protein, which arises due to C=O stretching vibration. FTIR signature at 1633 cm−1 and 1638 cm−1 indicated the presence of beta-sheets, while 16451658 cm−1 corresponded to helical structure.34 The extent of helical content was found to increase substantially as a result of NPs binding, which was also accompanied by a decrease in the beta-sheet contents. This switch was clearly visible by the decrease in 1638 cm−1 band and a simultaneous increase in 1650 cm−1 (Figure 3e and f, Table 2). This observation was also found to be in good agreement with the change in the amide II region (N-H deformation). Analysis of amide–II region demonstrated NPs binding-induced conformational switch from beta-sheet (~1523 cm-1) to alpha -helix (~1545 cm-1) (Figure S5). To understand the functional aspects of this protein-nanoparticle conjugation, we studied immunogenic response of WT in the absence and presence of Fe3O4@Arg NPs (Figure 4). In the 10


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presence of Fe3O4@Arg NPs, a large decrease in immunogenic response was observed, which was measured by a large drop in the expression of TNFα, IL-12 along with a decrease in NO generation. The ELISA data were confirmed by mRNA expressions of these genes which showed similar effects (Figure 4). Figure 4 also contains immunogenic profile of W26F mutant which will be discussed later. CD and FTIR showed that NPs binding-induced partial unfolding and a conformational switch from beta-sheet to alpha-helix, resulting in the loss of activity of WT-Fe3O4@Arg nanoconjugates. The observed immunogenic response is unsatisfactory towards the development of a NPs conjugated therapeutic agent. Hence, it is necessary to understand the mechanism of NPs driven conformational changes of MPT63 so that the loss of function could be recovered presumably using a reverse change. In order to achieve that, we needed to determine the plausible binding site of the NPs at the protein. We used molecular docking studies for this purpose. The docking analyses suggested that the most stable conformation for WT MPT63-arginine binding would yield a binding energy of approximately -1.82 kcal (Figure 5a). These analyses further showed that binding site would be near the sequence stretch between 19th and 30th amino acids (Figure 5b). Further, sequence analysis of WT MPT63 using ChSeq Database35 revealed the existence of eight chameleon sequences. These were 19VGQVVL24, 27KVSDLK32, 28VSDLKS33, 34SDLKSST40, 56AIRGSV61, 52ATVNAI57, 51TATVNA56 and 69NARTAD74. These chameleon sequences are known to adopt both helical and strand conformations based on the nature of the environment and the neighbouring sequence of the protein. Computational analysis also showed the presence of two short overlapping stretches of chameleon sequences, namely KVSDLK (residue 27-32) and VSDLKS (residue 28-33) near N-terminal. Interestingly, these stretches overlapped with the possible binding sites (19th to 30th residues) of the protein. Detailed ab-initio calculations using Zhang Lab server36 (https://zhanglab.ccmb.med.umich.edu/I-TASSER-MR/) showed the presence of helical signature in the sequence stretch WKVSDLKSSTA (Figure 5c), although the crystal structure of the protein did not have any helix at that region. It has been shown recently that MPT63 can adopt helical structures during structure formation from unfolded to folded state.17 It is found that the helical structure is a locally stabilized state, which gets destabilized in the later stage of the folding as the long range contacts form.17 11


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Binding of MPT63 on NPs presumably at the above stretch induces conformational stress leading to partial unfolding, breaks crucial long distance interactions and as a result, the stabilization of the local helix could be achieved (scheme 1). Scheme 1

We used Scheme 1 to develop a strategy to minimize the unfolding, which would allow the protein to be active even in its Fe3O4@Arg NPs bound state. We decided to increase the flexibility of the 19th to 30th stretch using subtle amino acid replacement. A flexible stretch would allow the binding of Fe3O4@Arg NPs without inducing significant stress in other regions of the protein, minimizing unfolding. Three single site mutations are generated, namely G20C, G25A and W26F. Figure S6 showed the sequence distribution of hydrophobic scores for the three mutants (W26F, G25A, and G20C), along with wild type protein which was calculated using ProtScale (http://web.expasy.org/protscale/). We used the hydrophobicity scale as defined by Kyte and Doolittle.37 The point mutations resulted in small increase in the hydrophobicity at the 19th to 30th stretch. The change was maximum for W26F and minimum for G20C mutant. Indeed for the W26F mutant, a large increase in helical content was observed both by far UV CD and FTIR spectroscopy (Figure 6a-c, Table 2). In contrast, the behavior of G20C mutant was identical to the WT protein while G25A mutant showed behavior, which was intermediate between W26F and G20C/WT (Figure S7, Table 2). ANS fluorescence measurements showed experimentally that W26F mutant had higher extent of exposed hydrophobic surface compared to the WT protein (Figure S8a). We also found that the WT protein was slightly more stable (mid-point of transition 1.4 M) compared to the W26F mutant (mid-point of 1.2 M) (Figure S8b). We subsequently used ENCOM server (http://bcb.med.usherbrooke.ca/encom.php) to determine the effect of W26F mutation on protein conformational flexibility. The ENCOM calculates the vibrational entropy change of the W26F mutant with respect to the WT protein (∆Svib) for each position of the amino acid sequence.38 We found that ∆Svib values for W26F mutant were higher in approximately all positions throughout

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the sequence, while the positions near 26th, 66th and 130th were maximally affected (Figure 6d, e). It was observed that these point mutants of WT MPT63 differed slightly in their binding affinity towards Fe3O4@Arg NPs (Figure 2a, Table 1). According to docking results, the protein-NP complex was found to have slightly increased stabilization energy, as observed for W26F mutant (Figure 5d, -4.01 kcal). Although W26F mutant was characterized by the enhancement of the helical content, the decrease in the overall secondary structure (which was observed for the WT, G25A and G20C protein in the presence of Fe3O4@Arg NPs) was absent for this mutant protein (Figure 6a-c). For each case, far UV CD and FT-IR spectroscopy supported each other (Figure 6b, c, 7a-d). Since subtle change in the protein hydrophobicity played crucial role in the observed sheet to helix transition, we decided to decrease solution pH. This is because lowering of solution pH has been found to increase the effective hydrophobicity of protein chains.17 Indeed, decreasing pH to 1.5 resulted in the beta-sheet to alpha-helix switch for the WT and G20C proteins (Figure S9). For W26F, which achieved its maximum helical content at pH 7.5 itself, no further increase in the helical content was observed as solution pH was decreased (Figure S9, Figure 7b). Again, no observable changes in secondary organizations of WT as well as W26F were found due to binding with Fe3O4@Arg NPs at low solution pH ( pH 1.5) (Figure S9c, Figure 7b). At this point, we would revert back to Figure 4 which showed the immunogenic profiles of both WT and W26F mutant in the absence and presence of NPs (Figure 4). Interestingly, there was no effect of W26F mutant on the immunogenic profile, and the mutant was found to be equally active as the WT protein. Moreover, the binding of Fe3O4@Arg NPs with W26F did not result in any loss in the expression of TNFα, IL-12 or the generation of NO. As before, the data were cross confirmed with the mRNA expressions of these genes which showed similar effects (Figure 4). Our results showed that protein-NP conjugation of W26F preserved the activity of the protein, while for the WT the activity was compromised. As discussed above, local interactions are plausible stabilizing factors for the early helices. As the folding progresses and non-local interactions start contributing, the helices are replaced by beta-sheets (the native folded protein). The binding of NPs (and the lowering of pH or mutational stress) provide additional stability to locally formed helices. We found out that the binding is an 13


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important factor for the beta- sheet to helix switch. This is because, a PEG coated magnetite NPs, which did not show any binding to MPT63 (Figure S10), did not form the helices (Figure S11) whereas lysine functionalization showed changes almost alike with arginine functionalization. However, we failed to observe any direct correlation between the binding constants (determined from the mutants) and the extent of induced helical contents. Moreover, an arginine coated Au NPs showed binding ability 100 times stronger than Fe3O4@Arg NPs, although their helix forming ability was found to be identical (Figure S11, S12, Table 1). We hypothesize that initial binding is indeed needed for the partial unfolding and resulting sheet to helix switch, however, beyond a threshold, the binding strength is not necessarily correlated with the helix forming ability. To validate this hypothesis, we are in the process of developing materials using which we can fine tune the binding constants in the intermediate binding regime. This study outlines a dynamic interplay between different conformers of MPT63 (helix vs sheets vs partially unfolded), protein-NPs binding and the variation of active populations (in terms of protein immunogenic profiles). Different parameters, including a subtle change in protein sequence space, the involvement of strategic chameleon sequence, and the alteration in solution conditions would attenuate this interplay. Although a quantitative understanding of these parameters is elusive at this point, a quadrant analysis shown in Scheme 2 depicts a qualitative strategy towards the development of a NP conjugated therapeutic molecule based on MPT63. Scheme 2, which takes into account all results presented in this study, places varying populations of different species in different quadrants. The role of the chameleon sequences has been highlighted, whose change in color (through either a conformational switch or other factors) play crucial roles in defining the population variations of these four quadrants. We find that the quadrant 1 in Scheme 2 represented the active populations of the protein. Hence the beta-sheet to helix switch (which presumably represented a local event) is away for the active region of the protein, while the partial unfolding was a somewhat global event affecting the overall activity of the protein. The present study emphasized the importance of careful evaluation of both the local and global influence of an interacting nanoparticle in order to obtain a detailed insight into the activity and toxicity of a protein -based therapeutic material.

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Conclusions: Understanding of the interaction between NPs and proteins is important not only to determine the toxicity induced by the NPs introduced inside the physiological system, but also to develop NP conjugated bio-therapeutics. This is particularly true for the protein-based molecules, which are typically stabilized by multiple weak interactions and conformational switches between different forms are extensively investigated. WT MPT63, when attached to Fe3O4@Arg NPs, was found to lose its inflammatory profile, which made this protein unsuitable as a therapeutic agent against TB. Far UV CD and FT-IR studies clearly showed that the binding of Fe3O4@Arg NPs with WT MPT63 resulted in a conformational switch from beta-sheet to alpha-helix, which occurred presumably at the sequence stretch between 19th and 30th amino acids. The conformational switch was accompanied by partial unfolding of WT protein. Using different surface modifying agents and a multiple metal cores for the NPs, we showed that a binding was prerequisite for the conformational switch. Interestingly, no correlation between binding constant and extent of switch could be found once strong binding regime was achieved. A site directed mutant (W26F) was designed, which successfully reverted back the inflammation profile, which was lost for the WT protein. We discussed here a quadrant analysis to optimize the activity of MPT63 using the conformational switch, the extent of NP- induced unfolding and NPs-proteins binding as inputs.

Acknowledgement: Author A.S acknowledges University Grant Commission, Govt. of India for providing senior research fellowship. Author SC acknowledges DST (SERB) for providing NPDF (File No. PDF/2017/000453). The authors thank the central instrument facility of CSIR-IICB for the CD and FT-IR measurements. The authors thank Dr. Sanat Karmakar of Jadavpur University and Dr. Soumen Sarkar of Balurghat College, Kolkata for their critical helps in manuscript preparation. The authors thank the Director, CSIR-IICB for his help and encouragements.

Supporting Information: Molecular Docking analysis, Instrumentation and characterization of the nanoparticles were discussed in supporting information. Figure (S1-S12) were discussed in supporting information. 15


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Table 1: Binding constants and cooperativity of different experimental systems.

Systems

Binding constants (M-1)

Co-operative index (n)

WT-Fe3O4@Arg

(6.6±0.1)×103

2.1±0.1

W26F-Fe3O4@Arg

(7.2±0.2)×103

2.9±0.1

G20C-Fe3O4@Arg

(5.7±0.1)×103

2.1±0.1

G25A-Fe3O4@Arg

(8.8±0.2)×103

1.9±0.1

WT-Fe3O4@Lys

(7.3±0.3)×103

1.6±0.2

W26F- Fe3O4@Lys

(8.7±0.2)×103

1.5±0.1

G20C- Fe3O4@Lys

(5.3±0.3)×103

1.9±0.1

G25A- Fe3O4@Lys

(10.9±0.2)×103

2.1±0.2

WT-Au@Arg

(2.4±0.4)×105

1.9±0.3

W26F-Au@Arg

(2.6±0.2)×105

0.9±0.2

G20C-Au@Arg

(2.5±0.3)×105

1.7±0.3

G25A-Au@Arg

(2.5±0.2)×105

2.1±0.1

WT- Fe3O4@Arg+100mM NaCl

(4.8±0.4)×103

1.8±0.2

WT- Fe3O4@Arg+250mM NaCl

(2.7±0.3)×103

1.9±0.1

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Table 2: Extent of secondary structure (percentage) calculated from the deconvoluted FTIR data of different protein systems using 1638 cm-1 as β-sheet and 1650 cm-1 as α-helix signatures.

Systems

1638 cm-1(β-sheet)

1650 cm-1(α-helix)

WT

33.5

22.3

G20C

30.8

23.5

G25A

35.8

33.4

W26F

10.5

50.6

WT- Fe3O4@Arg

5.4

53.0

W26F- Fe3O4@Arg

4.5

53.5

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Figure 1: Pymol representation of the structure of WT MPT63 protein (PDB ID 1LMI). All three mutation sites are indicated as different colour spheres; red, magenta and green colours correspond to G20C, G25A and W26F mutations respectively.

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Figure 2: (a) XRD pattern of Fe3O4@Arg nanoparticles; (b) Transmission electron microscopy images of Fe3O4@Arg nanoparticles; Characterization of the arginine capped Fe3O4 nanoparticles employing (c) FTIR (d) DLS study; (e) Arginine capped magnetite nanoparticles exhibited significant stability with time; (d) TGA analysis further affirm formation of the NPs. Samples were prepared at pH 7.5.

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Figure 3: (a) Binding plot of WT MPT63 and its three mutants G20C, G25A, W26F with Fe3O4@Arg NPs at pH 7.5. Solid lines are obtained from the fitting using Hill equation. (b) CD titration spectra of WT MPT63 protein with increasing concentration of Fe3O4@Arg NPs at pHs 7.5. CD data indicate clearly beta-sheet to alpha-helix transition of WT at pH 7.5 along with partial unfolding of the protein (c) CD spectral shift of WT from beta to alpha-helix conformation upon binding with Fe3O4@Arg NPs. (d) Difference Far -UV-CD spectra of WT in absence and presence of Fe3O4@Arg NPs.Deconvoluted FTIR spectral signatures of amide-I region of WT in (e) absence and (f) presence of Fe3O4@Arg NPs. Here we observe significant drop of beta-sheet signature at 1638 cm-1 (pink) and rise in helicity at 1650 cm-1 (blue) due to NPs binding. This result clearly indicates that there occurs conformational transition from beta-sheet to alpha-helix (conformational switch) due to NPs binding with WT. All these experiments were carried out at physiological pH 7.5.

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Figure 4: Immunological activity of WT MPT63 and W26F mutant. ELISA and PCR were performed to analyze the expression of IL-12, TNFα and NO both (a) in absence and (b) in presence of Fe3O4@Arg NPs conjugation of MPT63 and W26F. Densitometry was performed using BioRad Quantity One software and normalized against GAPDH. ** considered to be significant (P value greater than 0.01) and *** is highly significant (P value greater than 0.001), n.s is non-significant; nMPT63 and nW26F represent nanoconjugated forms of the proteins. The symbols ‘plus’ and ‘minus’ mean the presence and absence of particular species respectively.

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Figure 5: (a) Pymol representation of most stable docked structure of MPT63 with Arginine where we can see (b) localization of arginine near 19-30 residual stretch. (c) Stretch of our interest near the mutation region WKVSDLKSSTA was modeled through Zhang lab I-TASSER server and it shows the helical content in this stretch though in crystal structure this helical stretch is absent. (d) Energetically most stable docked structure of W26F mutant with arginine.

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Figure 6: (a) Difference in far UV-CD spectrum calculated using far UV-CD spectra of W26F mutant (black) and G20C mutant (red) with respect to WT at pH 7.5. Presence of prominent peak at 222 nm for W26F indicates the presence of significant alpha helical content whereas G20C behaves like WT protein. Deconvoluted FTIR spectral signatures of amide-I region of W26F mutant in absence (b) and presence (c) of Fe3O4@Arg NPs. Here we observe no significant drop of beta-sheet signature at 1638 cm-1 (pink) and rise in helicity at 1650 cm-1 (blue) due to NPs binding. This result clearly indicates that there occurs no conformational transition from beta-sheet to alpha-helix (Conformational switch) due to NPs binding with W26F. All these experiments were carried out at physiological pH 7.5. (d) W26F mutation causes an increase in flexibility in the regions marked red obtained from ENCOM server. (e) Graphical output of the change in vibrational entropy due to single point mutation. We found that ∆Svib values for W26F mutant were higher in approximately all positions throughout the sequence, while the positions near 26th, 66th and 130th were maximally affected.

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Figure 7: Far UV CD titration spectra of (a) W26F mutant at pH 7.5 and (b) pH 1.5 with increasing concentration of Fe3O4@Arg NPs. These results clearly indicate insignificant structural perturbation of W26F mutant on NP’s binding at both pH 7.5 and 1.5. Far UVCD titration spectra of (c) G20C mutant at pH 7.5 and (d) G25A mutant at pH 7.5 with increasing concentration of Fe3O4@Arg NPs. Notable conformational switch (beta to alpha) was observed along with unfolding of proteins.

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Scheme2: Quadrant analysis of different proteins and NPs conjugated proteins with respect to their helical content and unfolding.

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Table of content:

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Nanoparticle Induced Conformational Switch Between Alpha-Helix

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