Binding of the Iminium and Alkanolamine Forms of Sanguinarine to

Oct 29, 2014 - Amandeep Kaur , Imran Ahmd Khan , Parampaul Kaur Banipal , Tarlok Singh Banipal. Spectrochimica Acta Part A: Molecular and Biomolecular...
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Binding of the Iminium and Alkanolamine Forms of Sanguinarine to Lysozyme: Spectroscopic Analysis, Thermodynamics and Molecular Modeling Studies Chandrima Jash, Pavan V Payghan, Nanda Ghoshal, and Gopinatha Suresh Kumar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp5068704 • Publication Date (Web): 29 Oct 2014 Downloaded from http://pubs.acs.org on November 6, 2014

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Binding

of

the

Sanguinarine

to

Iminium

and

Lysozyme:

Alkanolamine Spectroscopic

Forms

Analysis,

Thermodynamics and Molecular Modeling Studies



Chandrima Jash, Pavan V. Payghan,‡ Nanda Ghoshal,‡ and Gopinatha Suresh Kumar* †



Biophysical Chemistry Laboratory, Chemistry Division

Structural Biology and Bioinformatics Division

CSIR-Indian Institute of Chemical Biology Kolkata 700 032, INDIA

Address for Correspondence Dr. G. Suresh Kumar Biophysical Chemistry Laboratory, Chemistry Division CSIR-Indian Institute of Chemical Biology Kolkata 700 032, INDIA Phone: +91 33 2499 5723, Fax: +91 33 2473 0284 e-mail: [email protected]

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ABSTRACT Sanguinarine (SGR) exists in the charged iminium (SGRI) and neutral alkanolamine (SGRA) forms. The binding of these two forms to the protein lysozyme (Lyz) was investigated by fluorescence, UV/vis absorbance and circular dichroism spectroscopy, and by in silico molecular docking approaches. Thermodynamics of the binding was studied by microcalorimetry. Both forms of sanguinarine quenched the intrinsic fluorescence of Lyz but the quenching efficiencies due to binding derived after correction of the inner-filter effect varied. The equilibrium binding constants at 25±1.0 oC for the iminium and alkanolamine forms were 1.17 × 105 and 3.32 × 105 M-1, respectively, with approximately one binding site for both forms on the protein. Conformational changes in the protein in the presence of the SGR were confirmed by absorbance, circular dichroism, 3D fluorescence and synchronous fluorescence spectroscopy. Microcalorimetry data revealed the binding of the SGRI form to be endothermic and predominantly involving electrostatic and hydrophobic interactions, while for SGRA form it was exothermic and dominated by hydrogen bonding interactions. The molecular distances (r) of 3.27 and 3.04 nm, respectively, between the donor (Lyz) and the acceptors (SGRI and SGRA forms) were calculated according to Förster’s theory. This data suggested both forms to be bound near Trp-62/63 residues of Lyz. Stronger binding of the SGRA over the SGRI form was apparent from both structural and thermodynamic results. Molecular docking studies revealed that the putative binding site for SGR analogs reside at the catalytic site. Docking results are in accord with spectroscopic and thermodynamic data which further validate the stronger binding of the SGRA over the SGRI form. The binding site was situated near the deep crevice on the protein surface and was close to several crucial amino acid residues, such as Asp-52, Glu-35, Trp-62 and Trp-63. This study advances new knowledge on the structural nature and thermodynamics aspects of the binding of the putative anticancer alkaloid sanguinarine on lysozyme. 2

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The Journal of Physical Chemistry

Key words: Protetin, Alkaloid, Interaction; Fluorescence; Docking

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INTRODUCTION In pharmacology-guided approach to drug design and development, identification of new drug targets or deriving information on small molecules binding to potential cellular targets was the primary discovery engine. In this context natural products of plant origin have gained increasing importance as potential lead compounds1 because of their high abundance in nature, remarkable potency and relatively low toxicity.2 Quaternary benzo[c]phenanthridine alkaloids are one such group known for their remarkable therapeutic utility.3-7 Sanguinarine (Figure 1), the putative anticancer agent and prominent member of this group, is one of the most widely distributed alkaloids in many botanical species.8-10

Figure 1. Chemical structures of (A) SGRI form and (B) SGRA form, and (C) 3D representation of lysozyme with six tryptophan residues.

Sanguinarine (Figure 1) is a lead compound in cancer drug research exhibiting multiple pharmacological properties and inducing apoptosis in many cancer cell lines through a variety of mechanisms.11-17 It has strong affinity to various nucleic acid structures like duplex, triplex and quadruplexes in vitro, and also exhibits excellent phototoxicity that may be harnessed for phototodynamic therapy.18 Sanguinarine is also a strong DNA topoisomerase inhibitor7, the enzyme considered to be the primary cellular target of many anticancer drugs. 4

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Its interaction with chromatin has also been reported.19 In aqueous solutions the alkaloid display an interesting pH-dependent structural equilibrium between the iminium (cationic) (SGRI, Figure 1A) and alkanolamine (neutral) forms (SGRA, Figure 1B) with a pKa of around 8.06.20 At pH 6.4, the cationic form persists while at pH 9.2, only the neutral form is present. The charged iminium form only binds to nucleic acids.21 Binding of the two forms of SGR, studied recently with serum proteins and hemoglobin, has revealed contrasting results.22,23 The binding affinity to Human serum albumin was observed to be higher for the SGRA form, while the SGRI form showed higher affinity to hemoglobin. As the efficacy and effectiveness of SGR in therapeutic applications may depend on its ability to associate with enzymes and proteins such information may also be crucial for an understanding of its distribution to required sites and toxicity to cells. Lysozyme (Lyz; muramidase, Figure 1C) is an antimicrobial enzyme that is found in various tissues and protective secretions like saliva, tears, milk and mucus. It can damage the protective bacterial cell walls by cleaving the beta-glycosidic linkage between the N-acetylmuramic acid and N-acetyl-glucosamine of the peptidoglycan thereby protecting against bacterial infections. The enzyme is also useful in food preservation and as an antimicrobial agent.24-26 Anti-inflammatory, antiviral, antiseptic, antihistamine and antineoplastic activities of Lyz are also known. Lysozyme has been the choice, as a model protein for studies on understanding folding and dynamics, structure-function relationship, and ligand-protein interactions27,28, the attraction owing to its small size, high stability, natural abundance and ability to bind to drugs.29 Lysozyme is a monomeric globular protein having 129 amino acids. It has six tryptophan (Trp) and three tyrosine (Tyr) residues, and four cross-linked disulfide bonds with a number of alpha helices, beta sheets, turns and loops in its secondary structure.30,31 The active site of the protein is reported to have a deep crevice that separates the protein between two domains connected by an alpha helix. One domain (amino acids 405

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85) consists mostly of beta sheet conformations, while the second domain (amino acids 89 to 99) is more alpha helical in nature.32 From the crystal structure of the protein, it has been observed that three Trp residues (Trp-62, 63 and 108) are placed near to the substrate binding site.33 Lysozyme is capable of reversibly binding to a number of endogenous and exogenous compounds.34 An understanding of the binding characteristics of therapeutically important alkaloids with Lyz may facilitate developing possible delivery modes for availability at required sites leading to therapeutic usage. The results of such studies may also facilitate the development of new small molecules as effective inhibitors for amyloid-related diseases as Lyz under appropriate conditions is known to easily form typical amyloid fibrils.35 Recently, the interaction studies of many natural products to Hen egg white Lyz and that of protoberberine alkaloids coralyne, palmatine and berberine to chicken egg white Lyz have been reported.36,37 For this investigation, we studied the interaction of the two forms of SGR with lysozyme (chicken egg white) with the help of spectroscopy, calorimetry and molecular docking techniques to understand in details the binding aspects and thereby provide a molecular basis for Lyz-SGR interaction. The protein is positively charged at pH 6.4 and neutral at pH 9.2, the two buffer conditions in our study. The structure of the protein at these pH values is more or less similar. The association constants of SGRI and SGRA forms binding to Lyz were determined by isothermal titration calorimetry (ITC) experiments in combination with a number of spectral techniques and the thermodynamic profiles associated with the complexation were characterized in details. Alterations in the protein structure on complexation with the two structural conformers of SGR were studied by synchronous fluorescence, circular dichroism (CD), and three-dimensional (3D) fluorescence spectroscopy techniques. Finally, using molecular modeling studies we probed the binding location of the 6

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alkaloid on the protein and also verified the forces involved in the binding interaction. The results reveal salient features of the unique protein binding properties of SGR molecule that may be useful for drug development and delivery. MATERIALS AND METHODS Materials. All chemicals used in this study were of analytical quality. Deionised and distilled water was used for the preparation of the experimental buffer solutions. The buffers were clarified through membrane filters (pore size 0.22 µm) before use. Chicken egg white lysozyme (purity ≥ 98%, M = 14.3 kDa) and sanguinarine chloride hydrate (purity ≥ 98%, M = 367.78 Da) were purchased from Sigma-Aldrich Co. (USA). The commercial sample of Lyz was purified by passing through a CM-cellulose column as reported.38 The sample was then desalted on a Sephadex G-50 column, dialyzed in the cold (SpectraPor MWCO, 3500 membrane), and freeze-dried. SGR sample was used as received. All the samples were dissolved in either citrate-phosphate (CP) buffer, pH 6.4 or carbonatebicarbonate (CB) buffer, pH 9.2. In both buffers the [Na+] was 10 mM. In these buffers SGRI and SGRA forms persisted in 100% abundance. The concentration of the samples were estimated by using molar absorption coefficient values of 37,750 M-1 cm-1 (280 nm) for Lyz,37 and 30,700 M-1 cm-1 and 21,600 M-1 cm1 (327 nm) for SGRI and SGRA forms, respectively.21 Equipments and Spectral Measurements. Electronic absorption spectroscopy studies were done at 25 ± 1.0 oC on Jasco spectrophotometer (Model V660, Jasco, Hachioji, Japan) using matched 1.0 cm light path length quartz cuvettes (Starna Cells, Inc., USA). Fluorescence spectra were measured on a Shimadzu model RF-5301 PC (Shimadzu Kyoto, Japan) or a Hitachi F-4010 (Hitachi Ltd., Tokyo, Japan) spectrofluorimeter in quartz cuvettes of 1.0 cm path length. The excitation and emission bandpass filters for all measurements were set up at

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5 and 10 nm, respectively. The temperature of the sample holder was kept at 25±0.5 oC using a circulating bath (Eyela UniCool, Tokyo Rikakikai Co. Ltd., Japan). Fluorescence quenching study was performed by exciting the protein sample at 295 nm as this exclusively excites the intrinsic Trp residues. SGRI and SGRA forms were excited at their wavelength maxima via 469 and 328 nm, respectively. Fluorescence spectral studies at different temperatures were done on the Hitachi F-4010 unit interfaced with a Lab Companion circulator (model RW-2015G). Synchronous fluorescence spectra were measured in the region 220–375 nm with ∆λ set at 15 and 60 nm, respectively. Three-dimensional (3D) fluorescence spectra were obtained at 25±1.0 oC on the PerkinElmer LS55 luminescence spectrometer. The protein fluorescence emission spectrum was measured in the 270-500 nm range. The excitation wavelength was initially kept at 200 nm and 15 scans were performed up to 340 nm at an increment of 10 nm. Here, the concentration of the protein used was 1 µM and the protein-alkaloid ratio was 1:5. Fluorescence polarization anisotropy measurements were performed following the protocol of Larsson et al.39 on the Hitachi unit as reported.40 The excitation and emission wavelengths were set at 327 and 420 nm, respectively. After each addition of an aliquot of the protein sample to the alkaloid solution, the solution was mixed and stable complex formation was ensured before noting the readings. Each reading was an average of six successive measurements. Anisotropy values were calculated using the equation38 A = (Ivv - IvhG)/(Ivv + 2IvhG)

(1)

where I is the intensity; the subscripts refer, respectively, to the vertical or horizontal positions of excitation and emission polarizers and G is the ratio Ihv/Ihh, the instrumental correction factor for correcting the polarizing effects in the emission monochromator and detector.41 Anisotropy data were plotted against increasing protein concentration and the association constant was evaluated by fitting of the data to the Hill equation. 8

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Secondary and tertiary structural changes in Lyz on binding with SGR were determined using CD spectra measured on a spectropolarimeter (model J815) from Jasco Company. The CD instrument was routinely calibrated with the standard, D-10-camphorsulfonic acid. The cuvette temperature was maintained at 25 oC using the Peltier cell holder and temperature controller. The CD spectra were acquired with a sensitivity of 10 milli degree, scan speed of 50 nm per min. and step size of 0.5 nm, time constant was 1 s. and band width of 0.2 nm. Signal-to-noise ratio was improved by acquiring five scans for each sample. The curves were smoothed after base line correction for each spectrum. The CD spectra were plotted as wavelength versus molar ellipticity (θ) and expressed as mean residue molar ellipticity (MRE), in units of deg cm2 /dmol. Isothermal titration calorimetry experiments were conducted in a VP-ITC unit (MicroCal, LLC., Northampton, MA, USA). Samples were extensively degassed prior to titration to prevent the formation of air bubbles. The experiments were done as follows. The syringe of the calorimeter was loaded with a solution of the protein (1 mM for for titration with SGRI form and 0.5 mM for SGRA form). Successive injections of 10 µL aliquots of Lyz solution into the solutions of SGRI and SGRA forms present in the calorimeter cell were programmed. Stirring was carried out by the rotating syringe (351 rpm). Blank experiments, where protein/alkaloid were injected into the buffers, were done under identical conditions to get the heat change due to dilution and these heats of dilution were appropriately subtracted from the heat of the reaction. The resulting corrected injection heats in terms of kcal/mole were plotted as a function of mole ratio of alkaloid/protein, fitted with a model of “one set of binding sites” with minimum χ2 value, and analyzed using inbuilt software to give the equilibrium binding affinity (Kb), the binding stoichiometry (N), and the standard molar enthalpy of complex formation (∆Ho). The standard molar Gibbs energy (∆Go) values for

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binding and the entropy contribution (T∆So) were calculated using the standard thermodynamic relations viz. ∆G O = − RT ln K b

(2)

and

T ∆SO = ∆H O − ∆GO

(3)

where T (in Kelvin) is the absolute temperature at which the experiment was conducted and R is the ideal gas constant (1.9872041 cal. /mol/ K). Periodic electrical calibration of the calorimeter unit was performed and heat exchange verified using standard experiments prescribed in the MicroCal manual so that the mean energy per injection was below 1.30 µcal and standard deviation was below 0.015 µcal. pH values of the buffers were observed to remain unchanged in the temperature range 15-35 oC reported here. The structural basis of molecular recognition was investigated using Induced Fit Docking Protocol in Schrodinger Maestro-v96012 9.6 package.42 3D coordinates for Lyz with a resolution of 2.0 Å were obtained from RCSB Protein Data Bank (PDB ID 2LYZ) .43 Ligand structure for the SGRI form (CID 5154) was retrieved from PubChem compound database. The SGRA form structure was built on SGRI skeleton using Maestro suite. Pre-processing tools like protein preparation wizard and LigPrep of Schrödinger software were used for preparing protein and ligand structures, respectively, before submitting for docking. Lyz coordinates were assigned with correct bond orders while adding hydrogens. The protein structure was generated at two different pH conditions of 6.4 and 9.2. Restrained minimization was performed using the OPLS-2005 force field. RMSD convergence criterion of 0.30 Å was set for all heavy atoms, allowing hydrogens to move freely while keeping backbone intact. The ionization states for ligands were generated using the ionizer module of Maestro suite; the pH for SGRI form was kept at 6.4 while that for SGRA was 9.2. Binding 10

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site identification was done by using different bioinformatic tools, viz. CASTp44 MetaPocket 2.045,46 and PatchDock.47,48 CASTp algorithm works by identifying surface topography for locating the functional regions of the protein. MetaPocket uses 8 different methods forming a consensus method that predicts binding sites. PatchDock algorithm works on shape complementarity principles, while carrying out simultaneous minimization of steric clashes. For PatchDock docking the redundant solutions were discarded with 1.5 Å clustering RMSD. Top ranked twenty poses were considered for final analysis. Poses were screened based on number of interactions and consistency in binding pattern. Finally, the putative binding site was confirmed by combining the results from CASTp MetaPocket 2.0 and PatchDock, guided by previous experimental reports44-48 on the important amino acids contributing to the known binding site. The ligands were docked on the protein using Induced Fit Docking Tool of Schrödinger package. Glide grid set up was specified with box size of 24 Å using the centroid of the residues defining the binding sites such as Arg-45, Asn-46, Trp-62, Trp-63, Trp-108, Val-109 and Ala-110. A soft potential docking with van der Waals radii scaling of 0.5 was used for initial Glide49 docking with SP mode to generate 20 ligand poses. Protein flexibility was taken care of by Prime50 refinement using OPLS parameter sets. Residues within 24 Å were subjected to conformational search and minimization, keeping the rest fixed. Prime output of twenty protein conformations was further subjected to Glide XP (Extra Precision) redocking using default hard potential function at the van der Waals radii scaling of 1.0. The top ranked poses were used for the final analysis. Discovery Studio 4.051, Pymol52, and Swiss PDB viewer53 were used for visualization. RESULTS AND DISCUSSION Quenching of Lysozyme Fluorescence by Sanguinarine. Fluorescence studies are commonly employed to evaluate the binding interaction between small molecules and proteins54,55 as the photophysical characteristics of the fluorophores of the protein is sensitive 11

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to the polarity of the surroundings. Therefore, a change in the intrinsic fluorescence property of Lyz in the presence of the alkaloid may provide information on the mode and nature of the interaction. For this experiment, excitation was performed at 295 nm, the region where only the Trp moieties of the protein are excited, giving fluorescence emission spectrum with maximum around 340 nm. Lyz has six Trp residues at positions 28, 62, 63, 108, 111 and 123 (see Figure 1).37 The residues at 28, 108, 111, and 123 are located in the alpha domain and those at positions 62, 63 and 108 are present in the substrate-binding cleft. Trp-62 and 63 are close to the hinge region between the alpha and beta domains. It is known from previous studies that Trp-62 and 63 are the residues most exposed to the solvent and hence highly susceptible to chemical modifications.56 When Lyz is unfolded partially, Trp-62 and 63 are more exposed to light and the fluorescence of the protein enhances. Detailed studies using chemical modification and fluorescent lifetime measurements have been reported in the literature to understand the individual contribution of Trp residues in Lyz to the overall fluorescence emission intensity of the protein.57,58 Such studies have clearly revealed that the Trp residues at positions 62 and 108 contributed largely to the fluorescence emission of Lyz, while those residues at 28, 63, 108, 111 and 123 make only small contributions. The Trp residue at position 63, lying close to the active site hinge region is not buried in the hydrophobic core. Trp-62, on the other hand, is exposed most fully to the solvent, while Trp-108 is away from the hydrophilic environment.59 Furthermore, a sequential inter tryptophanyl energy transfer between the Trp residues 108, 63 and 62 has also been suggested to occur during emission.57 Therefore, the exposed Trp-62 and Trp-63 moieties could be the key residues affected and quenched by ligand binding, and the fluorescence data may provide information on their local environment. Previously we had observed quenching of the protein fluorescence on binding of protoberberine alkaloids.37 In that study it was suggested that the alkaloids were bound close to the Trp-62 residue.37 The 12

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cyclic oligosaccharide β-cyclodextrin was reported to bind near Trp-62 and 63 residues leading to increase in the fluorescence intensity of the protein.60

Figure 2. Fluorescence emission spectra of Lyz (1 µM) in the presence of SGRI (A) and SGRA (B) forms. Curves (1− 6) in panel A correspond to 0, 1, 2, 3, 4 and 5 µM of SGRI form, and curves (1−11) in panel B denote 0, 0.38, 0.76, 1.14, 1.52, 1.90, 2.28, 2.66, 3.04, 3.42 and 3.80 µM of SGRA form, respectively. The excitation wavelength was 295 nm.

Fluorescence spectral changes of Lyz in the presence of SGRI and SGRA forms are illustrated in Figure 2. Increasing the concentration of the alkaloid, in both cases, leads to quenching of the fluorescence of Lyz, the effects reaching a saturation point in both cases, but at a higher concentration of SGRI compared to SGRA form. The quenching mechanism may be due to inner-filter effect, collisional quenching (dynamic quenching), or bindingrelated quenching (static quenching),61 the latter two effects being differentiated conventionally by their differential response toward temperature. Since SGR has strong absorption at the excitation wavelength (Figure 3), the center of the cuvette will receive less light and therefore, the fluorescence intensity of Lyz would be diminished. The absorbance at the emission wavelength of the fluorophore would also reduce the light reaching the detector, which would lead to a decrease in the protein fluorescence intensity. Therefore, a correction for the inner-filter effect of SGR on Lyz was applied. This was done by measuring the absorbance values (Aex and Aem) at the λex and λem for each concentration of sanguinarine and 13

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then multiplying the observed fluorescence intensity value with a correction factor of ~10(Aex+Aem)/2 using the equation described earlier.23

Figure 3. Absorption spectra of sanguinarine (SGRI, curve 1) (SGRA, curve 2) at 5 µM concentration. The wavelength maxima of the peaks in each case is denoted.

The strong alkaloid induced quenching was accompanied by a small red shift of the wavelength maxima for the SGRI and SGRA forms by 3 and 2 nm, respectively. Since the fluorescence intensity decreased after correction for inner filter effect, and this is caused by either collisional or binding induced quenching, further analysis was performed to distinguish between the static and dynamic quenching effects. It is known that diffusion coefficients are larger at higher temperatures and the dynamic quenching constants increase with increasing temperature. On the other hand, temperature increase may lead to decreased stability of the complexes in static quenching mechanism. Thus, to differentiate between the static and dynamic quenching mechanisms in the interaction of the SGRI and SGRA forms with Lyz, temperature dependent fluorescence spectral experiments were conducted at three temperatures viz. 15, 25 and 35 oC and the data analyzed with the classical Stern-Volmer equation60 14

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F0 = 1 + kq τ0 [Q] = 1 + ΚSV [Q] F

(4)

where F0 and F denote the fluorescence intensities at the wavelength maxima of Lyz alone and in the presence of sanguinarine, kq is the apparent bimolecular quenching rate constant,

Ksv is the Stern-Volmer quenching constant, τo is the average fluorescence life time of Lyz 8

without the SGR which is usually taken to be 10 s-1, and [Q] is the quencher concentraion.61 Figure S1 (Supporting Information) shows the linear dependence of F0/F on the molar

Table 1. Fluorescence Spetral Data for SGR Binding to Lyz at Three Temperaturesa T/oC

(KSV) M-1

(Kq) M-1 s-1

(KA) M-1

N

(KDR)M-1

15

1.64 × 105

1.64 ×1013

1.73 × 105

0.96

1.77 × 105

25

1.12 × 105

1.12 × 1013

1.14 × 105

0.98

1.15 × 105

35

0.97 × 105

0.97 × 1013

0.97 × 105

1.01

0.94 × 105

alkanolamine

15

4.28 ×105

4.28 × 1013

4.28 × 105

0.99

4.54 × 105

form

25

3.22 × 105

3.22 × 1013

3.37 × 105

0.97

3.46 × 105

35

2.26 × 105

2.26 × 1013

2.27 × 105

1.02

2.15×105

SGR

iminium form

a

The data reported are the mean of four experiments. KSV is the Stern–Volmer quenching constant,

KA is the binding constant, and KDR is the static quenching constant from double reciprocal plot analysis.

concentration of SGR at the three temperatures. The calculated KSV and kq values are summarized in Table 1. It can be seen that the values of KSV and kq decreased on raising the experimental temperature, and the magnitude of the kq values were > 2.0×1010 M-1 s-1, suggesting that in both the cases a static quenching mechanism occurred.57 In other words, the the protein fluorescence quenched in the presence of both forms of SGR as a result of specific complex formation between them at the ground state and not due to dynamic collision effects.

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Both SGRI and SGRA forms are excellent fluorophores; the latter presents an emission spectrum with stronger intensity than the former.23 The emission spectral maxima are at 565 nm and 420 nm, respectively, for SGRI and SGRA forms (Figure 4) under the present buffer conditions and these are away from the fluorescence maximum of the protein.

Figure 4. Fluorescence spectra SGRI (curve 1) and SGRA (curve 2) forms. The concentration of SGR was of 2 µM and the λex were 470 and 327 nm, respectively, for SGRI and SGRA forms.

So the effect of Lyz on the fluorescence spectrum of SGR was also studied. The results are presented in Figure 5.

Figure 5. Fluorescence spectra of SGRI (A) and SGRA (B) forms (2 µM) treated with Lyz. Curves (1−7) in panel A denote 0, 0.5, 1, 2, 3, 4 and 5 µM of Lyz, and curves (1−9) in panel B denote 0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 µM of Lyz. The λex were 470 and 327 nm, respectively, for SGRI and SGRA forms. 16

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A large quenching of the SGRA fluorescence was effected by Lyz (Figure 5B) compared to the relatively low changes in the case of SGRI (Figure 5A). No changes were seen in the wavelength maximun in either case. The stronger fluorescence quenching of the SGRA form over the SGRI form suggested a stronger interaction of the former with Lyz.

Fluorescence Resonance Energy Transfer from Tryptophan to the Bound Alkaloid. The formation of SGR-Lyz complexes may lead to transfer of excited energy from the donor (Lyz) to the acceptor (SGR). Apart from revealing more details on the binding, measurement of energy transfer efficiency can also divulge information on the distance between the bound ligand and the site of interaction on the protein, and this is useful for understanding structural and conformational aspects of donor-acceptor complexes.37 In an environment of the protein, the proximity of the ligand molecule to the Trp moiety of the protein is understood through the fluorescence resonance energy transfer (FRET) study. If the emission spectrum of donor significantly overlaps with the absorption spectrum of acceptor, these donor-acceptor pairs may be considered in Förster distance and the possibility of energy transfer as described above could be envisaged. The Förster theory discloses that the efficiency of energy transfer (EFRET) may depend on the orientation of the transition dipoles of the donor, and the distance separating the donor and acceptor which should be in the range 2-8 nm.62,37 The EFRET depends on the inverse sixth power of the distance between donor and acceptor (r), and of the critical energy transfer distance or Förster radius (Ro). A situation of 1:1 donor to acceptor concentration prevails when the efficiency of transfer is 50%, and EFRET is given by the equation

 F RO 6 EFRET = 1 −  = 6 6  F0  RO + r

(5)

RO being obtained by the relation R 6O = 8.8 × 10 −25 κ 2 n −4 φJ

(6) 17

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∫ F(λ )ε(λ )λ dλ 4

J=

where,

0 ∞

∫ F(λ)dλ 0

Here, κ2 is the spacial factor of orientation, n is the refractive index of the medium, and φ is the quantum yield of the donor. F(λ) and ε(λ) denote the fluorescence intensity of the donor and the molar absorption coefficient of the acceptor, respectively, at the wavelength λ. Using the values of κ2 = 2/3, n = 1.336 and φ = 0.14 for Lyz,37 the values of E, J, Ro and r were calculated to be 0.28, 2.97×10 1.88×10

-14

3

-14

3

-1

cm. L. mol , 2.79 nm and 3.27 nm for SGRI-Lyz, and 0.27,

-1

cm. L. mol , 2.59 nm and 3.04 nm for SGRA-Lyz interaction (Figure S2,

Supporting Information). The ‘r’ between SGR and the Trp residue in Lyz is much lower than the 8 nm upper limit, supporting the high probability of energy transfer from Lyz to the bound SGR.37 It may be noted that a similar scenario was also observed previously in the case of coralyne, palmatine and berberine.37 It may be observed that the distance of SGRI and SGRA forms from the protein is somewhat close to each other in magnitude indicating them to be bound at nearby sites on the protein. Furthermore, in both cases the relationship 0.5RO < r < 1.5RO is obeyed suggesting high possibility of energy transfer from the protein to the alkaloid, which can also explain the efficient quenching of Lyz fluorescence upon binding of SGR. Thus, the results obtained demonstrate that in both systems the conditions of energy transfer theory via. (a) Lyz can produce fluorescence light (Figure 2), (b) sufficient overlap is observed between the fluorescence spectrum of Lyz and the UV-vis spectra of SGRI and SGRA forms (Figure S2, Supporting Information), and (c) the distance separating the protein and SGR in both systems is less than 8 nm, the upper limit are obeyed. Therefore, the results unequivocally support that the binding of SGR to Lyz leads to energy transfer, resulting from the formation of strong ground-state complexes. Hence, determination the binding constants 18

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and number of binding sites of SGR-Lyz complexation was attempted from the fluorescence spectral data.

Evaluation of the Binding Constants and Number of Binding Sites of Sanguinarine-Lyz Complexation. The results presented in the previous sections clearly proposed that the quenching of the fluorescence of SGR involved strong interaction and subsequent complex formation with Lyz. In such cases involving small molecules binding independently to a set of identical sites on a macromolecule, the equilibrium binding constant (KA) and the number of binding sites (n) may be estimated from the following equation37 log

(F0 - F) = logKA + n log[Q] F

(7)

The binding affinity values of SGRI and SGRA forms to Lyz at the three temperatures studied were determined from the plots of log(F0-F/F) against log[Q] (not shown). The values evaluated are depicted in Table 1. Examination of the data reveals moderate affinity for both forms of the alkaloid to Lyz, and that the SGRA form has binding affinity (3.32×105 M-1) higher than the SGRI form (1.18×105 M-1). Furthermore, it may be observed from the magnitude of the ‘n’ values that there is only one kind of binding site for both forms of the alkaloid on the protein. It is likely that both forms of the alkaloid are bound close to a Trp residue in the protein. It may be mentioned here that we confirmed the 1:1 binding of SGRA and SGRI forms to Lyz by Jobs plot using the fluorescence spectral data. The plots of difference in fluorescence at 339 nm versus mole fraction of alkaloids (Figure S3, Supporting Information) crossed at χ = 0.47 and 0.49, respectively, in the case of SGRI and SGRA forms, proving the number of molecules of SGRI and SGRA binding to Lyz to be close to unity. Quantitative analysis of the fluorescence quenching data was also performed by the double reciprocal plot methodology using the equation given below37

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1 1 1 = + (F0 - F) F0 KDR F0 [Q]

(8)

Here, the quenching constants (KDR) were derived from the ratio of the intercept to slope of the double reciprocal plots that describes the efficiency of the ground state quenching. In Figure S4 (Supporting Information) the double reciprocal plots of Lyz-SGR interaction at the temperatures 15, 25 and 35 oC studied are depicted. Data presented in Table 1 reveals that the SGRA form has a higher affinity to Lyz than the SGRI form. The decreasing magnitude of the KDR values at higher temperatures corroborates the temperature dependence of the KSV values (Table 1) and is again consistent with the static quenching mechanism proposed.

Measurement of Steady-State Fluorescence Anisotropy. This technique is used as a probe to assess the extent of flexibility and or tumbling motion of small molecules upon binding to a macromolecule. It is based on the concept that larger molecules tumble slowly compared to smaller molecules that can tumble faster. Anisotropy results can also guide us on the probable location of the small molecule in the heterogeneous environment of the protein. Support for the binding of SGR to Lyz was obtained from an increase in the fluorescence polarization anisotropy values. Binding of the alkaloid resulted in a reduction of its mobility and motion resulting in an increase of anisotropy. The anisotropy value of the SGRA form increased from 0.01 to 0.04 on binding to Lyz (Figure S5, Supporting Information). The change in the case of SGRI form was only marginal. This data also provides support for the stronger interaction of the SGRA to the protein compared to the SGRI form. The change in fluorescence anisotropy (r) value of SGRA form upon binding to Lyz allows the determination of the binding constant (K) independently, using the following equations described by Ingersoll and Strollo63 viz. 1 1 1 = 1+ fB K [L]

(9)

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and fB =

r − rF R(rB − r) + r − rF

(10)

Here fF and fB represent the fractional fluorescence intensities of the free and bound forms of SGRA and rF and rB are the corresponding anisotropy values. A correction factor R = FB ⁄ FF,, which is the ratio of the intensities under the same conditions is introduced as SGRA form fluorescence intensity decreased on binding to the protein. Following the above relation double reciprocal plots of 1⁄ FB versus 1 ⁄[L] were drawn and found to give linear fits yielding the binding constant to be 3.02×105 M-1 from the slope. Consistency of the estimated value from anisotropy results with those evaluated from the fluorescence quenching data (vide supra in Table 1) establishes the validity of this technique.

Studies on Conformational Changes of the Protein from Synchronous Fluorescence Spectroscopy. Conformational changes in Lyz consequent to the binding of the two forms of the alkaloid were assessed using synchronous fluorescence spectroscopy.64 This technique essentially explores the microenvironment of the amino acid moieties by measuring the emission data. The difference between excitation and emission wavelengths (∆λ) controls the shape and intensity of synchronous fluorescence spectra. The synchronous fluorescence spectra of Lyz will provide information on the environment around Tyr residues when ∆λ is 15 nm and that near the Trp residues when it is 60 nm. The effect of SGR on the synchronous fluorescence of Lyz (∆λ = 60 nm) led to systematic quenching with a large bathochromic shift in the emission maximum (9 nm) for the SGRI form and almost no shift for the SGRA form (Figure 6A,B). Thus, with the SGRI form a shift to a more hydrophilic environment for the Trp residues of Lyz leading to more exposure to the solvent appears to occur. On the

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other

hand,

there

is

almost

no

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in

the

maximum

Figure 6. Syn-chronous fluorescence (∆λ = 60 nm) spectra of Lyz (1 µM) with SGRI (A) and SGRA (B) forms. Curves (1–8) in panel A and B denote 0, 0.5, 2, 4, 6, 8, 10 and 11 µM of SGRI form and 3.8, 1.14, 1.9, 2.66, 3.42, 4.18 and 5.32 µM of SGRA form, respectively.

emission wavelength (not shown) with ∆λ=15 nm for both forms of SGR revealing that no change happens in the microenvironment around the Tyr residues. Therefore, binding of SGR leads to alteration in the polarity around the Trp residues, i.e. that around Trp-62 and Trp-63, while that around Tyr residues remained unaltered. This result is consistent with those derived from quenching and energy transfer experiments confirming the potential role of these Trp residue(s) of Lyz in the interaction phenomena.

Hydrophobic Probe Displacement Study. To determine the potential binding site of SGR on Lyz, we performed an ANS (8-aninilo-1-napthalenesulfonic acid) displacement study. ANS is known to be a sensitive probe useful to understand microenvironmental changes in proteins and more often used to derive information on the hydrophobic binding region.23 In this assay binding studies were performed in the presence of ANS under identical conditions, and the the variation of the relative fluorescence (F/F0) against SGR concentration was plotted (ESI Fig. S6). We find that, SGR at a concentration of 9 µM had a better quenching effect on fluorescence of Lyz than ANS, i.e. SGRA form could quench about ~63%, SGRI 22

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56%, and ANS approximately 33% and 34%, respectively, at the two pH conditions. When SGR is added to the ANS–Lyz complex, the fluorescence intensity decreased by about 41% and 52% respectively, for SGRI and SGRA forms. The reduction of fluorescence intensity of ANS–Lyz complex indicated that SGR can compete against ANS moderately for its binding site.

Absorption Spectral Studies. The structural changes that occur in Lyz on interaction with the two alkaloid forms can also be inferred from absorption spectral studies. Figure 7 depicts the UV/vis- absorption spectral changes of Lyz in the presence of SGRI and SGRA forms. Two absorption peaks, a strong one around 201 nm followed by a weak one around 280 nm characterizes the protein spectrum. This is in agreement to the literature report.37,65 The 201 nm absorbption peak is due to the CO-NH bonds and the 280 nm peak results from that of the aromatic amino acid residues, particularly the Trp residues. A fall in the absorbance of the peaks, with a bathochromic shift of the 280 nm peak with SGRI form and hypsochromic shift of the same peak with SGRA form occurred on titration. Similar types of changes have been reported to occur in the Lyz spectrum on binding with many small molecule ligands. 37,65 The change in the absorption spectra of the protein induced by SGR observed here may indicate an unfolding of the backbone conformation of the protein resulting from change in the environment around the Trp residues by an increase in the hydrophobicity in the case of SGRA form and the hydrophilicity in the presence of SGRI form. This inference is also in agreement with the fluorescence data. Since dynamic quenching influences the excitation state of the molecule and has no effect on the absorption spectrum, the spectral change observed here may also lend support to the static quenching effect of the protein fluorescence by both forms of the alkaloid.

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Figure 7. Absorption spectral changes of Lyz (1.07 µM) on titration with SGRI (A) and SGRA (B) forms. In panel (A) and (B) curves (1–6) denote 0, 1, 2, 3, 4 and 5 µM of SGRI form and 0, 1, 2, 3, 4 and 5 µM of SGRA form, respectively. Inset shows the amplified view of the 250-350 nm regions of the spectra.

Conformational Studies from Circular Dichroism Spectroscopy. The conformational effects in the protein on the binding of the two forms of SGR were studied using CD spectral analysis in the far and near-UV regions. The far UV region (< 250 nm) spectral changes reflect on the protein secondary structure, whereas those in the near-UV region (>250 nm) arise from the tertiary structural variations. Lysozyme has a large alpha-domain. It consists of four alpha-helices and a 310-helix, and a smaller beta-domain with a triple-stranded antiparallel beta-sheet, an irregular loop with two disulfide bridges and a 310-helix. The protein divides itself into two domains, by the deep active cleft, one of them having mostly betasheet structure and the other containing N and C-terminal segments that have alpha helical

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structure. The ligand induced conformational changes in Lyz can be effectively monitored by circular dichroism experiments.66 The measured far-UV CD spectrum of Lyz is characterized by a positive band around 194 nm and two minima centred at 208 and 222 nms, respectively (Figure 8) as reported in the literature.37 This is characteristic of a predominantly alpha-helical protein. The 208 nm peak emanated due to π–π* transition of the alpha-helix and the 222 nm band is ascribed to the n– π* transition, both for alpha-helix and unordered structures. Titration of the protein with increasing concentrations of SGRI and SGRA forms led to a notable reduction in ellipticity of the peaks in the far-UV CD spectrum without any shifts in the wavelengths (Figure 8 A,B). This is indicative of a reduction in the alpha-helical content suggesting that the binding of both forms of the alkaloid induced changes in the secondary structure of Lyz. Both forms of SGR are optically inactive and hence are devoid of CD in the entire UV/vis region. The induced alterations in the secondary structure were quantified from the observed ellipticity values in terms of MRE (deg. cm2. dmole-1) values (at 222 nm) calculated applying the reported37 equation as follows MRE =

θobs (m deg) 10 × n × C × l

(11)

where θobs is the observed ellipticity in milli degrees, C is the concentration (in Moles), n is the number of amino acid residues and l (cm) is the length of the light path. The helical contents of the native protein and alkaloid bound protein structures were calculated from the MRE values at 222 nm37 %alpha − helix =

MRE 222nm − 3000 × 100 −36000 − 3000

(12)

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Lyz was found to contain ~ 29% alpha-helix at pH 6.4 and ~31% alpha-helix at pH 9.2. These values are similar to those reported previously at pH 7.0.37

Fig. 8. Far-UV CD spectrum of Lyz (5 µM) on titration with SGRI (A) and SGRA (B) forms, and near UV CD spectrum of Lyz (25 µM) on titration with SGRI (C) and SGRA (D) forms. In panel (A) curves (1–7) denote 0, 1, 2, 4, 6, 8 and 10 µM of SGRI, and in (B) curves (1–7) denote 0, 0.76, 1.52, 3.04, 4.56, 6.08 and 7.6 µM of SGRA forms. In panels (C) and (D) curves (1–3) denote 0, 20 and 50 µM of SGRI and SGRA forms.

In the presence of saturating amounts of SGRI (10 µM) and SGRA (7.6 µM) forms, the alpha-helical composition of Lyz was reduced to 21 and 19% from the initial 29 and 31%.. Thus, the binding of the alkaloid induced unfolding, leading to significant loss of the helical stability, resulting in strong secondary structural changes in Lyz. It is likely that binding of SGR leads to protein unfolding concomitantly exposing the hydrophobic cavities and the aromatic residues. Near-UV CD studies were performed to understand the changes that might have occurred in the tertiary structure of Lyz on binding of the two structural analogues of the alkaloid. The circular dichroism signal in this region (250-300 nm) arises due to the presence of disulfide 26

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bonds and the aromatic amino acid residues.37 The transitions of the Trp residues result in CD signals at 283, 290 and 295 nm.37 In the presence of SGRI and SGRA forms there were small variations in the CD spectra (Figure 8 C,D). This showed that some changes in the environment of the side chains of the aromatic amino acids occurred due to binding with the indole rings of the Trp residues (62, 63 or 108, the former two residues lying on the molecular surface and the latter one towards the end of the cleft) leading to an unfolding of the tertiary structure of the protein. This change, when considered in the light of the far UV CD spectral alterations and other spectral results, may point to the strong interaction of the alkaloid with the protein. It is interesting to note that neither the SGRI nor the SGRA form acquired induced CD in the proteneous environment.

Three-Dimensional Fluorescence Spectroscopic Studies. Protein conformational changes occurring as a result of binding of small molecules can be evaluated from 3D fluorescence studies which is a relatively new analytical technique.67,68 Comparative data can give information on the conformational aspects from microenvironmental changes in the protein. A shift in the excitation or emission wavelength near the fluorescence peak, emergence of a new peaks or disappearance of existing peaks may divulge an important clue on the nature of the conformational changes in the protein. When Lyz is excited at 280 nm the intrinsic fluorescence of Trp and Tyr residues are revealed and there is negligible contribution from the third fluorescent amino acid namely the phenylalanine (Phe) residues. In the figure of three dimensional spectra (Figure 9) two representative fluorescence peaks (peaks 1 and 2) are clearly seen. Peak 1 essentially reveals the spectral behavior of the Trp residues. On comparing with the absorption spectrum of Lyz, the peak at 280 nm can be inferred to be caused due to the π-π* transitions of the aromatic amino acids. The Trp, Tyr and Phe residues in the binding cavity of protein have conjugated π-electrons and can easily involve

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themselves in charge transfer interactions with other electron deficient species or π-electron systems. The 3D spectra and contour maps of Lyz-SGRI and Lyz-SGRA form complexes are presented in Figure 9 and the spectral parameters derived from these spectra are presented in Table 2. In Figure 9A and C, peaks ‘a’ and ‘b’ represent 1st order Rayleigh scattering peak (λex = λem) and 2nd order Rayleigh scattering peak (λem = 2λex), respectively. Peak 1 (λex = 280 nm and λem = 350 nm) is the characteristic intrinsic fluorescence spectral behaviour of Trp and Tyr residues.37

Figure 9. 3D-fluorescence spectra and contour maps of Lyz−SGRI form complex (A,B) and Lyz−SGRA form (C,D) complex.

The binding of both forms of SGR to Lyz leads to a decrease in the fluorescence intensity and increase of the Stokes shift (∆λ). This suggests a change in the protein conformation, increase in the polarity surrounding the Trp residues, and concomitant decrease of the hydrophobicity; these are consistent with the synchronous fluorescence data. The Stokes shift value of Lyz-

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SGRI form complex was lower than that of Lyz-SGRA complex indicating that the energy transfer from Trp to bound alkaloid is higher in the latter case.

Table 2. 3-D Fluorescence Data for Lyz and Lyz-SGR Complexes peak 1

peak 2

system position

Stokes shift

position

Stokes shift

(λex/λem/intensity)

∆λ (nm)

(λex/λem/intensity)

∆λ (nm)

(nm/nm/F)

(nm/nm/F)

Lyz (pH 6.4)

280/354/148.12

74

230/352/202.55

122

Lyz-SGRI form

280/358/71.55

78

230/358/116.73

128

Lyz (pH 9.2)

280/354/105.74

74

230/354/147.20

124

Lyz-SGRA form

280/358/73.29

78

230/358/100.90

128

Peak 2 (λex = 220 nm and λem = 350 nm) in Lyz results from the characteristic n→π* transition of the polypeptide backbone. The fluorescence intensity of both peaks diminished in the complex but to different extents leading to the inference that unfolding of polypeptide chain occurs leading to conformational change resulting in more exposure of some hydrophobic regions.

Insights into the Thermodynamics of the Binding. The binding of a small molecule like SGR to Lyz may involve the formation of weak noncovalent forces like electrostatic, hydrophobic, vanderWaals, and hydrogen bonds. Knowledge of the thermodynamic parameters may provide clue to the extent of involvement of these forces in the complexation process.69 The binding associated thermodynamics was evaluated from isothermal titration 29

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calorimetry (ITC) measurements. The thermodynamic parameters viz. standard molar Gibbs energy change (∆G°), standard molar enthalpy of binding (∆H°) and the entropy contribution (T∆S°) along with the binding affinity (Kb), and stoichiometry (N) were derived from ITC experiments. The ITC profiles for the binding of SGRI and SGRA forms of the alkaloid with Lyz

are

presented

in

Figure

10.

Figure 10. Calorimetric titration profiles of SGR-Lyz complexation. The top panels depict raw heat data for the sequential injection of 1 mM and 0.5 mM of Lyz into solutions of (A) SGRI (50 µM), and (B) SGRA (50 µM) form at 298.15 K, and control titration heats of Lyz into respective buffer (not in scale). In the bottom panels the integrated heat data (points) fitted to “one set of binding sites” model (best fit lines) against the mole ratio of Lyz/SGR is shown.

It can be observed that the binding of the SGRI form is an endothermic process and that of the SGRA form is exothermic in nature. Hydrophobic interactions are small and endothermic, while electrostatic interactions are exothermic and higher in magnitude than the hydrophobic interactions.70 The results of ITC studies are summarized in Table 3. At pH of 6.4, the protein is essentially positive in net charge and SGRI form is a cation. So hydrophobic interactions will be predominant and binding may occur via hydrophobic 30

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interactions between the alkaloid and the amino acid residues of the protein. Here the ∆H° value is positive. A positive value of ∆H° may support the involvement of hydrophobic interactions. On the other hand, at pH 9.2 Lyz is neutral71 and SGR is also in its neutral form; the interaction may therefore take place predominantly via hydrogen bonding. The negative ∆H° and positive T∆S° values advocate the involvement of hydrogen bond formation and hydrophobic interactions in the protein-SGRA form complex. The exothermic nature of the reaction at pH 9.2 may also support the role of hydrogen bonding interactions playing a major role here. Notably, the ITC profiles in both the cases have only one binding event. The protein binding affinity of the SGRI form at 298.15 K was evaluated to be (1.17 ± 0.08) × 105 M−1 and the same for the SGRA form was (3.32 ± 0.08) × 105 M−1. The higher binding affinity of the SGRA form over the SGRI form was thus evident from the calorimetric data also and this corroborates with the results from spectroscopic studies described earlier. In this context it is worthwhile to recall that SGRA form also binds to serum albumins more strongly than SGRI form but in both the cases the calorimetric profile was exothermic.22 Both forms of the alkaloid bind to Lyz in 1:1 ratio as revealed from the N values depicted in Table 3. The binding stoichiometry for SGR was around 1.0 and this received support from Job plot data also (Figure S3, Supporting Information). It can be observed from the data presented in Table 3 that the standard molar Gibbs energy change at 298.15 K for the binding of SGRA form was slightly higher (by about 0.6 kcal/mol) than that of the SGRI form. In both the cases the reaction was entropy driven, but with a small favorable enthalpy change in the case of SGRA form and a small unfavorable enthalpy contribution in the case of SGRI.

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Table 3. ITC data for Lyz-SGR complexation at Different Temperaturesa binding constant alkaloid

temp (K)

SGRA form

(T∆S0)

(∆G0

(∆Cp)

kcal/mole

kcal/mole

kcal/mole

(cal/mol.K)

N (Kb) (×105 M-1)

SGRI form

(∆H0)

288.15

1.77 ± 0.27

0.85

1.20 ± 0.13

8.13

-6.92 ± 0.13

298.15

1.17 ± 0.08

0.84

1.13 ± 0.98

8.05

-6.92 ± 0.98

308.15

0.95 ± 0.05

1.02

1.07 ± 1.06

8.04

-6.97 ± 1.06

288.15

4.46 ± 0 .33

0.86

-0.97 ± 0.09

6.48

-7.45 ± 0.09

298.15

3.32 ± 0.08

1.12

-1.38 ± 0.06

6.14

-7.52 ± 0.06

308.15

2.06 ± 0.18

1.26

-1.54 ± 0.09

5.96

-7.49 ± 0.09

a

The data in this table are averages of four independent determinations.

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Temperature dependent thermodynamic study was performed to gain more knowledge on the nature of the driving forces involved in the binding interaction. The same experimental procedures were used in the range 288.15-308.15 K. The data at the three temperatures studied are depicted in Table 3. It can be seen that with increasing temperature, the binding became weaker. The ∆H° values for the SGRI form decreased while it became more negative in the case of SGRA. The negative enthalpy of binding in the case of the SGRA form at all the temperatures divulged favorable exothermic binding. Here with increase of the temperature, the entropy contributions decreased but enthalpy value became more negative, so ∆G° is conserved. This suggests that SGRA form Lyz complex formation is controlled by dominant enthalpy contributions. For SGRI form binding, the entropy contributions decreased only marginally with increasing temperature but remained a favorable factor to the Gibbs energy as positive ∆Ho decreased with the temperature, i.e. the reaction is endothermic at higher temperatures. In both cases there is an enthalpy-entropy compensation behavior due to the conservation of ∆G° in the temperature range studied. Change in heat capacity (∆Cpo) for small ligands binding to proteins can be evaluated from the slope of the plot of variation of ∆Ho values with temperature. Heat capacity values can advance significant insights into the nature and magnitude of the binding forces involved in the binding interaction and also are indicative of structural alterations that occur on binding. These are obtained from the first derivative of T versus ∆Ho plots (Figure S6, Supporting Information). The ∆Cpo values obtained for the binding of SGRI and SGRA forms to Lyz are -6.5 and -28.5 cal/mol·K, respectively. The non-zero but negative values of ∆Cp o in both cases indicate the binding to be specific and involving the burial of non-polar surface area.72 A linear change of the standard molar enthalpy values with temperature was observed (Figure S7, Supporting Information). This behavior suggests the absence of any measurable shift of the pre-existing equilibrium between the conformational states of the protein. 33

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It is known that large ∆Cpo values generally are indicative of changes in hydrophobic or polar group hydration. Consequently, such values are often thought to point to strong hydrophobic effects in the binding interaction. The ∆Cpo value for Lyz-SGRI form interaction is relatively small in magnitude compared to that of Lyz-SGRA interaction. The strikingly low magnitude of ∆Cpo in the case of SGRI form may also be corroborated with the lower structural changes that occurred in Lyz on SGRI binding compared SGRA binding as revealed from the CD results. Furthermore, the relatively high heat capacity value of the Lyz-SGRA form system clearly suggests the involvement of a stronger hydrophobic desolvation effect in the protein consequent to ligand binding; hydrophobic interactions between SGRA form and the active site of Lyz may play a major role at the higher pH as suggested above. Thus, the difference in the ∆Cpo values may point to the overall differences in the extent of hydrophobic interaction and the conformational changes in these systems.

Molecular Docking Studies. Molecular recognition pattern for SGR to Lyz protein provides evidence to the binding region. Ample literature is available about the catalytic site of Lyz and binding of different ligands and antigens at this site.73-75 A combined analysis from different binding site prediction methods coupled with the known literature depicts putative binding site for SGR forms lie near the catalytic site (Figure 11 A). At this orthosteric site we studied the binding modes with Lyz guided by IFD docking, exclusively at pH 6.4 for SGRI form and pH 9.2 for the SGRA form.

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Figure 11. Surface representation of Lyz bound with SGRA (A) direct interacting residues in brown, remaining binding site in light yellow; (B) hydrophobic contribution for the binding of SGRA; dotted pink line represents Pi-hydrophobic contact between SGRA and Trp-108 , light pink for Pi-alkyl contact with Ala-107; hydrophobic scale (-3 to 3) : from hydrophilic blue to hydrophobic gray.

The SGR binding pocket is lined up with the residues Glu-35, Asn-44, Asn-46, Thr-47, Asp52, Gln-57, Trp-62, Trp-63, Ala-107 and Val-109. Each of these residues contributes in the formation of binding site which carries complementarity to the SGR moiety needed for its molecular recognition. Both SGRI and SGRA forms lie close to Trp-63 at distances of 2.093 and 1.881 Å, respectively. Location of Trp-63 is at the substrate-binding hinge region (the door of the binding site) of the alpha and beta-domains. It is accessible for binding as it is not buried in the hydrophobic core. The interaction of fluoroquinolones with Lyz has been reported to involve the Trp-63 residue.54 The higher binding of SGRA over SGRI form appears to be due to the presence of crucial H-bond interaction at Glu-35 residue, the cleavage point of the active site, which was absent with SGRI form. Hydrophobic interactions with Trp-108 and Ala-107 residues also contribute for the higher affinity of the SGRA form (Figure 11 B). Interaction-wise differences between the two forms of the alkaloid are shown in Figure 12.

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Figure 12. Hydrogen bonding network and positioning of cleavage point residues (Glu-35-Asp52) with docked SGRA (A) and SGRI (B) forms. Black dotted lines are used to show H-bonding and important residues are shown in purple colour.

Hydroxyl group of SGRA form interacts with catalytically important Glu-35. On the same side hydroxyl group also interacts with Val-109 residue, coming from the loop. This interaction further strengthens the binding of the SGRA form. The SGRA form positions itself towards the catalytic site while the SGRI form positions horizontally in between Asn-44 and Trp-63, bit away from the catalytic site towards Asp-52 which is counterpart of Glu-35 at the catalytic cleavage point. It shows electrostatic interactions with both forms based on Discovery Studio 4.0 interaction calculations. We also observed the structural changes induced in Lyz upon binding of both forms of the alkaloid. Backbone RMSD values (comparing Lyz structure before and after the binding) was found to be 0.75 Å for the SGRA and 0.23 Å for the SGRI form. RMSD calculations when restricted to only binding site, values were 0.72 Å and 0.19 Å for SGRA and SGRI form, respectively. Based on our data we conclude that SGRA binding induces stronger structural changes over SGRI binding which is in accordance with our near and far UV CD data. It provides us with the clue related to probable origin of induced changes that lie near Val-109, situated at helix-loop junction. This supports the higher conformational changes induced by alkanolamine binding over the iminium binding. 36

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An analysis based on distance comparison between docking and FRET results have also been carried out. Distances were calculated between O1 of the ligand and NE1 of Trp residues of the protein. The values from Trp-28, 62, 63, 108, 111, 123 are 13.25, 9.27, 4.82, 3.27, 11.93 and 17.06 Å, respectively, for iminium and 13.08, 8.88, 4.80, 3.13, 11.30 and 16.62 Å, respectively, for the SGRA form. Comparative analysis based on Docking and FRET suggests that the distance trend goes hand in hand, however, exact distance matching is limited due to the core differences governing two methods. As docking gives zoomed view with atomistic details, all the Trp to the ligand distances are well below the FRET observations. Low distance range for SGRA over the SGRI form from both the methods validates higher binding to the former. FRET studies consider protein and ligand as a single entity and provide the distance information between the donor (protein) and the acceptor (ligand). For a protein with multiple Trp residues overall trend of distance range at molecular level associated with their interaction carries hint for binding affinity as well. Therefore, this comparative analysis helps us to further consolidate our findings.

Table 4. Binding Parameters of Sanguinarine-Lyz Complexation from Molecular Docking Studies alkaloid

GlideScore

IfdScore

H-bonded

electrostatic

hydrophobic

interactions

interactions

interactions

Trp-63,

Trp108 (Pi-Pi T Asn-46,

SGRA form

-5.649

-278.986

Val-109,

shaped), Ala-107 Asp-52

Glu-35

SGRI form

-3.522

(Pi-alkyl)

Trp-63,

Asp-52,

Asn-44

Glu-35

-259.365

----------

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Overall summary of parameters which were used for comparing SGRA with SGRI form has been presented in Table 4. It is evident from GlideScore and Ifdscore that SGRA form molecular recognition results in stronger binding as compared to that of the SGRI form.

CONCLUSIONS Spectroscopic evidence suggests that both forms of sanguinarine bind Lyz through ground state complex formation. The binding affinity of the neutral SGRA was stronger than that of the charged SGRI form. The interaction involves close contact with Trp-62 and 63 at the cleft region of Lyz as inferred from fluorescence spectral data. Energetics of the interaction revealed endothermic binding for the SGRI form and exothermic binding for the SGRA form as electrostatic interaction plays a major role in the binding of the former and hydrophobic interactions in the case of the latter. Circular dichroism, synchronous fluorescence and 3D fluorescence results point to the onset of stronger conformational changes in Lyz upon binding of the SGRA form compared to the SGRI form. Molecular recognition of sanguinarine occurs at the catalytically important cleavage point with residues, Glu-35 and Asp-52. The SGRI form does not interact via H-bonding with any of these residues. On the other hand, the SGRA form interacts directly with Glu-35 by H-bonding and with Trp-108 and Ala-107 through hydrophobic interactions. Docking results support pH-wise binding differences accurately, viz. the higher binding for the alkanolamine and lower binding for the iminium form. We believe SGR can be a very prospective small molecule ligand due to its binding at the catalytically important location of Lyz protein.

ACKNOWLEDGMENTS The Council of Scientific and Industrial Research (CSIR), Government of India network project GenCODE (BSC-0123) supported this work. C. Jash is a Department of Science and Technology, Government of India INSPIRE Senior Research Fellow. N.G. thanks CSIR, New Delhi for providing financial support. P.V.P. thanks CSIR for a Project Fellowship. The 38

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authors express their sincere thanks to Dr. Basudeb Achari, Ex. Deputy Director of CSIRIICB for his valuable inputs. We appreciate the constructive comments of the editor and the reviewers that helped us to significantly improve this manuscript.

ASSOCIATED CONTENT Supporting information Figures S1-S7 depicting Stern–Volmer plots for the quenching of Lyz fluorescence, Overlap of Lyz fluorescence spectrum and absorption spectra of SGR, Job plots for the binding of SGR to Lyz, double reciprocal plots Lyz-SGR complexation, fluorescence anisotropy data, ANS displacement data and plots of variation of standard molar enthalpy change with temperature for the binding of SGR to Lyz are available free of charge via the Internet://httppubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel: :+91 33 2499 5723. Fax: +91 33 2473 5197 E-mail: [email protected]

Notes The authors declare no competing financial interest.

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