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Explication of human γD-crystallin interactions with its aggregation inhibitor Schiff base at molecular level
Journal Pre-proof Explication of human γD-crystallin interactions with its aggregation inhibitor schiff base at molecular level Shiwani Rana, Kalyan Sundar Ghosh PII:
S0022-2860(19)31665-5
DOI:
https://doi.org/10.1016/j.molstruc.2019.127556
Reference:
MOLSTR 127556
To appear in:
Journal of Molecular Structure
Received Date: 22 July 2019 Revised Date:
22 November 2019
Accepted Date: 7 December 2019
Please cite this article as: S. Rana, K.S. Ghosh, Explication of human γD-crystallin interactions with its aggregation inhibitor schiff base at molecular level, Journal of Molecular Structure (2020), doi: https:// doi.org/10.1016/j.molstruc.2019.127556. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
hγD-Crystallin
+
Schiff base aggregation inhibitor
OH
N HO
Explication of human γD-crystallin interactions with its aggregation inhibitor Schiff base at molecular level Shiwani Rana and Kalyan Sundar Ghosh* Department of Chemistry, National Institute of Technology Hamirpur, Himachal Pradesh
*Corresponding Author Tel: +91-1972-254104; Fax: +91-1972-223834; e-mail: [email protected]
1
Abstract Cu(II)-induced aggregation of human γD-crystallin (HGD) was reported to be inhibited by a Schiff base (SB). In this work, interactions between HGD and its aggregation inhibitor were probed by using different fluorescence based techniques, circular dichroism and molecular docking. Ground-state complexation between HGD and SB caused fluorescence quenching of tryptophan residues of HGD. Thermodynamic parameters suggest the involvement of hydrogen bonding and/or van der Waals interactions between the complexing species. From FRET, donor (tryptophan of HGD) to acceptor (SB) distance was calculated as 3.56 nm. Complexation of SB with HGD did not result any conformational alteration as confirmed by the synchronous fluorescence and CD spectroscopy. Molecular docking envisaged binding of SB in the Cterminal domain of HGD, which is presumably associated with the process of aggregation. Therefore, in addition to the reduction of free Cu(II) concentration in lens by virtue of the chelation ability of the Schiff base, the binding of the inhibitor with HGD can also inhibit its aggregation.
Key words: Human γD-crystallin; Schiff base; molecular interactions; fluorescence quenching.
2
1 Introduction Opacification of eye lens due to cataract is the most prevalent cause of blindness. ~90% of the lens protein content is composed of crystallins. These proteins are reasonably stable with high solubility and responsible for the transparency of the lens. But with ageing, ocular β- and γcrystallins tend to aggregate due to their partial unfolding under stressful conditions. In this situation, α-crystallin acts as chaperone [1-3] through complexation with partially unfolded β/γcrystallins [4] and prevents the aggregation leading to cataract. Post-translational modifications such as oxidative damages in crystallin proteins increase the chance of their aggregation and hence the lens opacity gets enhanced [5, 6]. Generally, Cu(II) and Zn(II) ions play imperative roles in oxidative protein aggregation in Parkinson’s and Alzheimer’s diseases [7-9]. These metal ions were also found to induce amorphous aggregation of HGD [10]. In normal eye lens, tight binding between Cu(II) and α-crystallin prevents Cu(II)-mediated generation of reactive oxygen species [11] and assists α-crystallin to exhibit its cytoprotective effect [12]. Additionally, binding of Cu(II) and Zn(II) with α-crystallin enhances its chaperone activity [13, 14]. But in aged lens, Cu(II) binding capacity of α-crystallin is weakened due to complexation between αcrystallin and β/γ-crystallins [15]. So the free α-crystallin content as well as its chaperone activity is reduced in the aged lens. This will cause an increment in the free copper ion concentration in aged and cataractous lens [16, 17]. Under such circumstances, effective chelators of Cu(II) can open a new therapeutic window for the prevention of cataract. Antioxidant activity along with strong metal chelating ability of the Schiff bases was exploited for the inhibition of metal-mediated protein aggregation [18]. Inhibition of Cu(II)-mediated aggregation of γ-crystallins by mono and diimine Schiff bases was reported earlier by our group [19, 20]. The best Schiff base inhibitor (SB) of the monoamine series can also improve the
3
chaperone function of human αA-crystallin in a cooperative manner possibly through its interactions with the chaperone site of αA-crystallin [21]. Mechanistically, strong Cu(II)complexation ability of the above mentioned inhibitor (Fig. 1) was accounted for its aggregation inhibition activity. The presence of two phenolic –OH groups in this inhibitor molecule seems to be crucial for demonstrating aggregation inhibition. Recently, we have also reported inhibition of oxidative aggregation and tryptophan (Trp) damages of HGD by two ployphenolic compounds rutin and hesperetin respectively [22, 23]. OH
A N
B HO
Fig. 1: Structure of Schiff base (SB)
In the present work, molecular interactions between HGD and SB have been studied using fluorescence quenching, synchronous fluorescence, circular dichroism and docking techniques to gain a deeper mechanistic insight about the mechanism of aggregation inhibition by SB. The quenching of fluorescence of a molecule can occur due to ground-state complextion, energy transfer, collision quenching, and excited-state reactions. Steady state fluorescence quenching of the Trp residues of a protein on addition of a small molecule has been exploited frequently for studying their interactions. The quenching studies provide information about quenching and binding constants, mode of binding, number of binding sites and quenching mechanism. Synchronous fluorescence spectroscopy offers information about the conformational changes of the Trp and tyrosine (Tyr) residues in the protein due to binding of small molecule. Förster
4
resonance energy transfer (FRET) between a protein and small molecule is used generally to determine the interaction efficiency and the distance between the donor and the acceptor [24]. Circular dichroism (CD) spectroscopy is commonly used to probe the changes in the secondary structure of proteins in the presence of another molecule. Molecular docking predicts the preferred binding region of a molecule in a macromolecule like protein or nucleic acid. Binding affinity between them and the strength of association can also be predicted using docking studies.
2 Materials and methods The Schiff base (SB) used in this study was synthesized as reported by Chauhan et al. [19]. Experiments were performed in 10 mM phosphate buffer (pH 7.0) unless mentioned specifically. All consumables were procured from Himedia and SRL (India).
2.1 Overexpression and purification of HGD The protein was overexpressed and purified following the method described earlier by Chauhan et al. [19].
2.2 Fluorescence studies Emission spectra (300-450 nm) of HGD (4 µM) were recorded in Shimadzu RF 5301PC before and after successive addition of SB (0-26 µM) through excitation at 295 nm. Inner filter effect correction was made on recorded fluorescence intensity. Binding parameters for HGD-SB complexation were determined at four distinct temperatures by using double logarithmic plot as used elsewhere [25]. Quenching constant and fractional accessibility of fluorophores was
5
determined using modified Stern-Volmer equation [26]. Considering enthalpy change as constant in this range of temperature, the change in enthalpy (∆H), entropy (∆S) and Gibbs free energy (∆G) were estimated using van’t Hoff plot.
Furthermore, synchronous fluorescence spectra of HGD (4 µM) were recorded on successive addition of SB (0-50 µM). The wavelength difference (∆λ) between emission and excitation monochromators was fixed at 60 nm.
2.3 Circular dichroism Far-UV CD spectra of HGD (15 µM) and its complex with SB (15 µM) were recorded from 190 to 250 nm in 5 mM phosphate buffer (pH 7.0) in JASCO J-1500 spectrophotometer.
2.4 Docking HGD crystal structure was extracted from protein data bank (Id: 1HK0). SB conformation with minimum energy was generated by applying AMMOS force field using FROG web-server [27]. This conformation of SB was used further for docking with HGD using Autodock 4.2. Docked conformations were visualized using PyMol.
3 Results and discussion HGD had shown intense emission at 328 nm due to the presence of multiple tryptophan residues in the protein. But, HGD intrinsic fluorescence at 328 nm was decreased on consequent addition of SB (Supplementary materials Fig. S1). Quenching of Trp emission of HGD pointed towards the possibility of binding interactions between HGD and SB. The emission λmax of HGD was
6
unaltered, which suggests that Trp microenvironment remain unaffected during protein-ligand interactions.
Trp quenching of HGD by SB was studied at four different temperatures (294K, 299K, 304K and 310K). Using the quenching data, binding constant (K) and the number of binding sites (n) were determined by applying double logarithmic plots (Fig 2A). The quenching constant (Ksv) and fractional accessibility of fluorophore (fa) were also calculated using modified Stern-Volmer plots (Fig. 2B). The binding and quenching parameters for HGD-SB complexation were mentioned in Table 1. With increase in temperature, HGD-SB binding constant was found to be decreased suggesting the involvement of hydrogen bonding and/or van der Waals interactions between the protein and small molecule. The number of binding sites in HGD had been decreased at higher temperatures, which also indicates weaker binding of SB with HGD at higher temperatures.
0.0
log(F0-F/F)
294K 299K 304K 310K
9
(B)
294K 299K 304K 310K
F0/F0-F
-0.5
(A)
6
-1.0
3 -1.5 -6.0
-5.5 -5.0 log [Q]
-4.5
0.00
0.15 1/[Q] µM-1
0.30
Fig. 2: (A) Double logarithmic plots and (B) modified Stern-Volmer plots of HGD-SB system at 294, 299, 304 and 310 K. [HGD]: 4 µM; [SB]: 0 to 26 µM; λex: 295 nm.
7
On the other hand, increase in quenching constant with rise in temperature, suggests the probability of collisional quenching by SB. This is in contrary to the formation of HGD-SB complex at ground state. To realize this abnormality, the bimolecular quenching constant (kq) was calculated at different temperatures (mentioned in Table 1) using the equation Ksv = kq x τ0. The excited state lifetime (τ0) of moderately fluorescent Trp residues of HGD (Trp 42 and Trp 131) in absence of the small molecule is 3 ns [28]. The calculated kq values ~were 1013 M-1s-1 at all experimental temperatures. Therefore, the chance of dynamic quenching was discarded as the maximum kq value is 1010 M-1s-1 in case of dynamic quenching [24]. To understand this anomalous observation, Arrhenius theory was also used. In static quenching, the quenching efficiency lowers down with increase in temperature due to the lower stability of fluorophorequencher ground state complex. But according to Arrhenius theory, efficiency of quenching increases with temperature and this effect predominated over the effect of temperature for decreasing KSV. kq is related to the energy of activation (Ea) as follows: = −
(1)
where, A is a pre-exponential factor and T is the temperature.
Ea was calculated as 19.85 kJ mol-1 from the Arrhenius plot (Fig. 3) and found higher as compare to its commonly observed values for protein-small molecule binding [29-31]. This observation suggests that the effect of temperature is significant on the quenching process and firmly discard the chances of dynamic quenching. A similar type phenomenon was also noticed earlier for the binding of chloramphenicol with β-lactoglobulin [32]. Finally, it may be concluded that quenching mechanism of Trp fluorescence of HGD by SB is static in nature.
8
31.4
R2=0.933
ln kq
31.2 31.0 30.8 0.00321
0.00330 -1 0.00339 1/T (K )
Fig. 3: Arrhenius plot for the quenching of HGD fluorescence by SB.
Table 1: Parameters related to the binding and quenching of HGD by SB Temp (K) 294 299 304 310
Double logarithmic K (103 M-1) n 6.02±0.25 0.87±0.08 2.82±0.12 0.78±0.04 1.23±0.05 0.71±0.02 0.32±0.02 0.58±0.02
Modified Stern-Volmer Ksv (10 M-1) fa 7.70±0.21 0.54±0.04 8.60±0.18 0.56±0.05 10.80±0.31 0.48±0.02 11.36±0.22 0.51±0.03 4
kq (1013 M-1s-1) 2.57±0.09 2.87±0.12 3.60±0.11 3.79±0.13
Different types of intermolecular forces basically control the binding of small molecule with protein and they can be predicted on the basis of sign and magnitude of the changes in enthalpy and entropy [33]. These intermolecular forces include hydrogen bonding, van der Waals, electrostatic and hydrophobic interactions. Thermodynamic parameters were calculated using van’t Hoff plot (Fig. 4) for HGD-SB complexation and are given in Table 2. Negative change in free energy implies spontaneous binding. Decrease in the enthalpy and entropy was also noticed due to the binding of SB with HGD. During HGD-SB complexation, the change in enthalpy basically drove the process and contributed significantly to the free energy change (∆G).
9
Negative ∆H and ∆S values infer the involvement of hydrogen bonding and/or van der Waals forces between HGD and SB.
9
ln K
8 7 6 0.00328 0.00336 1/T (K-1) Fig. 4: van’t Hoff plot for the HGD-SB complexation. Table 2: Thermodynamic parameters for binding of SB with HGD Temp. (K)
-1
∆G (KJ mol )
294
-21.6±0.7
299
-19.6±0.5
304
-17.6±0.5
310
-15.2±0.6
-1
∆H (KJ mol )
-138.0±4.5
-1
-1
∆S (J mol K )
-396.2±14.7
Quenching of Trp fluorescence of HGD by SB indicates that the fluorophore of HGD and SB lies close to each other. Therefore, transfer of energy from HGD to SB is expected to occur. Fig. 6 demonstrates the overlap between the fluorescence spectrum of HGD (4 µM) and the absorption spectrum of SB (4 µM). This suggests the chances of efficient energy transfer between them. The Förster resonance energy transfer (FRET) parameters like overlap integral (J), Förster distance and (R0) and the distance between donor and acceptor (r) were calculated [24]. The overlap integral (J) was computed as 1.63×10-14 M-1cm3 by integrating the overlap area of two spectra in 10
Fig. 5. Then R0 was determined by using above calculated J value and the quantum yield of
400
0.03
Absorbance
Fluorescence intensity
HGD as 0.058 [34]. Furthermore, r was calculated as 3.56 nm.
200
0
400 Wavelength (nm)
0.00 500
Fig. 5: Overlap between HGD fluorescence (solid) and SB absorption spectra (dotted). [HGD] and [SB]: 4 µM each; λex: 295 nm.
Synchronous fluorescence spectroscopy can also provide indications regarding the changes in Trp and Tyr microenvironment within a protein due to ligand binding [26]. Information regarding Trp residues may be extracted from the synchronous spectra, if the wavelength difference (∆λ) between the excitation and emission monochromators is maintained at 60 nm. Changes in the synchronous fluorescence spectra of HGD at ∆λ=60 nm in the presence of SB are shown in Fig. 6. Wavelength maximum of HGD was not shifted on addition of SB. Therefore, it can be interpreted that HGD-SB interactions did not result any notable change around the Trp residues of HGD.
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Synchronous fluorescence
0 µM
600
SB concentration µM
300
0
300
320 340 Wavelength(nm)
Fig. 6: Synchronous fluorescence spectra of HGD in absence (top line) and the presence of SB at ∆λ=60 nm.
The effect of SB binding with HGD on the secondary structures of the protein was studied by Far-UV CD spectroscopy (Fig. 7). Only minor changes in the Far-UV CD spectra of HGD were detected in the presence of SB. This suggests that Schiff base binding did not cause any change in the overall secondary structure of HGD and the small changes are only due to conformational adjustment during binding of the ligand.
CD (mdeg)
20
0
-20 200 220 240 Wavelength (nm)
Fig. 7: Far-UV CD spectra of HGD (15 µM) in absence (black) and in presence (red) of SB (15 µM).
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Furthermore, docking studies were performed to corroborate experimental observations. Interactions between HGD and SB were predicted from the docking results and the docked conformation of SB with HGD is shown in Fig. 8. In that conformation, SB was placed inside the protein structure where the aromatic rings of SB may be involved in van der Waals interactions and the nitrogen and oxygen atoms of SB may undergo hydrogen bonding interactions. The polar atoms of the Schiff base (two phenolic –OH and the imine nitrogen) was found in close proximity of the polypeptide backbone nitrogen of Trp 131, Val 132, Leu 133, Leu 145, Leu 146 and Met 147 as well as backbone oxygen of Trp 131, Tyr 144 and Leu 146. The phenoilc –OH of ring B of SB was also predicted to interact with the side chain oxygen of Ser 130. The distances between the interacting residues of HGD and the polar atoms of SB are given in Table 3. The interactions between SB and Trp 131 can explain the observed static quenching of Trp fluorescence of HGD. Interestingly, some of these interacting residues from the C-terminal domain (C-td) of HGD were reported to constitute the aggregation site of HGD [35]. Another earlier report had also predicted Ser 130 as the hot spot of HGD aggregation [36]. As unfolding of the N-terminal domain (N-td) of HGD takes place prior to that of C-td [37], therefore, the unfolding of N-td exposes the above mentioned residues which were buried in the native conformation to initiate protein aggregation. Now, the predicted binding of SB at or near to a major aggregation site of the protein suggests that the Schiff base will be able to inhibit HGD aggregation due to favorable protein-ligand interactions in addition to its strong metal chelating ability. It was also reported that Cu(II)-mediated aggregation of HGD proceeds through partial unfolding of the protein [10]. In that condition, binding of SB in this region of HGD molecule will arrest homologous association of protein molecules leading to their aggregation.
13
Fig. 8: Docked conformation of SB with HGD. Interacting residues have been marked.
Table 3: Distances between the interacting residues of HGD and SB (A and B are the rings of SB marked in Fig. 1) Residue Ser 130 Oβ Trp 131 O Trp 131 N Val 132 N Leu 133 N Tyr 144 O Leu 145 N Leu 146 N Leu 146 O Met 147 N
Distance (Å) 2.03 [B(-OH)] 1.53 [A(-OH)] 1.74 [imine N] 2.42 [imine N] 2.78 [A(-OH)] 2.81 [imine N] 3.37 [A(-OH)] 2.48 [A(-OH)] 3.34 [A(-OH)] 2.41 [A(-OH)] 1.92 [B-(OH)] 2.76 [B(-OH)]
Conclusion A Schiff base was reported as an inhibitor of Cu(II)-mediated aggregation of HGD by virtue of its strong meal chelation ability. In addition to that, in the present work, the interactions between HGD and SB were studied. Ground state complexation between them had caused static
14
quenching of the Trp fluorescence of HGD. Hydrogen bonding and/or van der Waals interactions between HGD and SB were identified, which can contribute to the aggregation inhibition activity of SB.
Acknowledgement: Authors are grateful to the Director, NIT Hamirpur (H.P.) for providing the research facilities. KSG is very much thankful to Prof. D. Balasubramanian, L.V. Prasad Eye Hospital, Hyderabad for his kind gift of HGD clone. Support from Dr. Joy Debnath, SATRA University and his research group for the synthesis of the Schiff base is gratefully acknowledged. SR is grateful to Ms. Sheetal, NIT Hamirpur for her cooperation in some of the experimental studies. SR and KSG are also thankful to Dr. Subrata Ghosh, IIT Mandi and his research group for providing support in the CD experiments.
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•
Interactions between human γD-crystallin and its aggregation inhibitor Schiff base
•
Static quenching of Trp fluorescence of HGD by the Schiff base
•
Involvement of hydrogen bonding and/or van der Waals interactions
•
No notable conformational changes in HGD due to protein-small molecule interactions
•
Docking predicts binding of Schiff base at a major site of aggregation of HGD
The work was designed by KSG and SR. Experimental data were acquired by SR. Analysis and/or interpretation of the experimental results, drafting and further revision of the manuscript were done by both the authors.
Declaration of interest statement The authors declare that there is no conflict ofinterest.
Shiwani Rana, Department of Chemistry, NIT Hamirpur (lndia)
Dr. Kalyan Sundar Ghosh, Department of chemistry, NIT Hamirpur
(lndia) -{
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*
lLl
1-o
tl
S0022-2860(19)31665-5
DOI:
https://doi.org/10.1016/j.molstruc.2019.127556
Reference:
MOLSTR 127556
To appear in:
Journal of Molecular Structure
Received Date: 22 July 2019 Revised Date:
22 November 2019
Accepted Date: 7 December 2019
Please cite this article as: S. Rana, K.S. Ghosh, Explication of human γD-crystallin interactions with its aggregation inhibitor schiff base at molecular level, Journal of Molecular Structure (2020), doi: https:// doi.org/10.1016/j.molstruc.2019.127556. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
hγD-Crystallin
+
Schiff base aggregation inhibitor
OH
N HO
Explication of human γD-crystallin interactions with its aggregation inhibitor Schiff base at molecular level Shiwani Rana and Kalyan Sundar Ghosh* Department of Chemistry, National Institute of Technology Hamirpur, Himachal Pradesh
*Corresponding Author Tel: +91-1972-254104; Fax: +91-1972-223834; e-mail: [email protected]
1
Abstract Cu(II)-induced aggregation of human γD-crystallin (HGD) was reported to be inhibited by a Schiff base (SB). In this work, interactions between HGD and its aggregation inhibitor were probed by using different fluorescence based techniques, circular dichroism and molecular docking. Ground-state complexation between HGD and SB caused fluorescence quenching of tryptophan residues of HGD. Thermodynamic parameters suggest the involvement of hydrogen bonding and/or van der Waals interactions between the complexing species. From FRET, donor (tryptophan of HGD) to acceptor (SB) distance was calculated as 3.56 nm. Complexation of SB with HGD did not result any conformational alteration as confirmed by the synchronous fluorescence and CD spectroscopy. Molecular docking envisaged binding of SB in the Cterminal domain of HGD, which is presumably associated with the process of aggregation. Therefore, in addition to the reduction of free Cu(II) concentration in lens by virtue of the chelation ability of the Schiff base, the binding of the inhibitor with HGD can also inhibit its aggregation.
Key words: Human γD-crystallin; Schiff base; molecular interactions; fluorescence quenching.
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1 Introduction Opacification of eye lens due to cataract is the most prevalent cause of blindness. ~90% of the lens protein content is composed of crystallins. These proteins are reasonably stable with high solubility and responsible for the transparency of the lens. But with ageing, ocular β- and γcrystallins tend to aggregate due to their partial unfolding under stressful conditions. In this situation, α-crystallin acts as chaperone [1-3] through complexation with partially unfolded β/γcrystallins [4] and prevents the aggregation leading to cataract. Post-translational modifications such as oxidative damages in crystallin proteins increase the chance of their aggregation and hence the lens opacity gets enhanced [5, 6]. Generally, Cu(II) and Zn(II) ions play imperative roles in oxidative protein aggregation in Parkinson’s and Alzheimer’s diseases [7-9]. These metal ions were also found to induce amorphous aggregation of HGD [10]. In normal eye lens, tight binding between Cu(II) and α-crystallin prevents Cu(II)-mediated generation of reactive oxygen species [11] and assists α-crystallin to exhibit its cytoprotective effect [12]. Additionally, binding of Cu(II) and Zn(II) with α-crystallin enhances its chaperone activity [13, 14]. But in aged lens, Cu(II) binding capacity of α-crystallin is weakened due to complexation between αcrystallin and β/γ-crystallins [15]. So the free α-crystallin content as well as its chaperone activity is reduced in the aged lens. This will cause an increment in the free copper ion concentration in aged and cataractous lens [16, 17]. Under such circumstances, effective chelators of Cu(II) can open a new therapeutic window for the prevention of cataract. Antioxidant activity along with strong metal chelating ability of the Schiff bases was exploited for the inhibition of metal-mediated protein aggregation [18]. Inhibition of Cu(II)-mediated aggregation of γ-crystallins by mono and diimine Schiff bases was reported earlier by our group [19, 20]. The best Schiff base inhibitor (SB) of the monoamine series can also improve the
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chaperone function of human αA-crystallin in a cooperative manner possibly through its interactions with the chaperone site of αA-crystallin [21]. Mechanistically, strong Cu(II)complexation ability of the above mentioned inhibitor (Fig. 1) was accounted for its aggregation inhibition activity. The presence of two phenolic –OH groups in this inhibitor molecule seems to be crucial for demonstrating aggregation inhibition. Recently, we have also reported inhibition of oxidative aggregation and tryptophan (Trp) damages of HGD by two ployphenolic compounds rutin and hesperetin respectively [22, 23]. OH
A N
B HO
Fig. 1: Structure of Schiff base (SB)
In the present work, molecular interactions between HGD and SB have been studied using fluorescence quenching, synchronous fluorescence, circular dichroism and docking techniques to gain a deeper mechanistic insight about the mechanism of aggregation inhibition by SB. The quenching of fluorescence of a molecule can occur due to ground-state complextion, energy transfer, collision quenching, and excited-state reactions. Steady state fluorescence quenching of the Trp residues of a protein on addition of a small molecule has been exploited frequently for studying their interactions. The quenching studies provide information about quenching and binding constants, mode of binding, number of binding sites and quenching mechanism. Synchronous fluorescence spectroscopy offers information about the conformational changes of the Trp and tyrosine (Tyr) residues in the protein due to binding of small molecule. Förster
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resonance energy transfer (FRET) between a protein and small molecule is used generally to determine the interaction efficiency and the distance between the donor and the acceptor [24]. Circular dichroism (CD) spectroscopy is commonly used to probe the changes in the secondary structure of proteins in the presence of another molecule. Molecular docking predicts the preferred binding region of a molecule in a macromolecule like protein or nucleic acid. Binding affinity between them and the strength of association can also be predicted using docking studies.
2 Materials and methods The Schiff base (SB) used in this study was synthesized as reported by Chauhan et al. [19]. Experiments were performed in 10 mM phosphate buffer (pH 7.0) unless mentioned specifically. All consumables were procured from Himedia and SRL (India).
2.1 Overexpression and purification of HGD The protein was overexpressed and purified following the method described earlier by Chauhan et al. [19].
2.2 Fluorescence studies Emission spectra (300-450 nm) of HGD (4 µM) were recorded in Shimadzu RF 5301PC before and after successive addition of SB (0-26 µM) through excitation at 295 nm. Inner filter effect correction was made on recorded fluorescence intensity. Binding parameters for HGD-SB complexation were determined at four distinct temperatures by using double logarithmic plot as used elsewhere [25]. Quenching constant and fractional accessibility of fluorophores was
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determined using modified Stern-Volmer equation [26]. Considering enthalpy change as constant in this range of temperature, the change in enthalpy (∆H), entropy (∆S) and Gibbs free energy (∆G) were estimated using van’t Hoff plot.
Furthermore, synchronous fluorescence spectra of HGD (4 µM) were recorded on successive addition of SB (0-50 µM). The wavelength difference (∆λ) between emission and excitation monochromators was fixed at 60 nm.
2.3 Circular dichroism Far-UV CD spectra of HGD (15 µM) and its complex with SB (15 µM) were recorded from 190 to 250 nm in 5 mM phosphate buffer (pH 7.0) in JASCO J-1500 spectrophotometer.
2.4 Docking HGD crystal structure was extracted from protein data bank (Id: 1HK0). SB conformation with minimum energy was generated by applying AMMOS force field using FROG web-server [27]. This conformation of SB was used further for docking with HGD using Autodock 4.2. Docked conformations were visualized using PyMol.
3 Results and discussion HGD had shown intense emission at 328 nm due to the presence of multiple tryptophan residues in the protein. But, HGD intrinsic fluorescence at 328 nm was decreased on consequent addition of SB (Supplementary materials Fig. S1). Quenching of Trp emission of HGD pointed towards the possibility of binding interactions between HGD and SB. The emission λmax of HGD was
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unaltered, which suggests that Trp microenvironment remain unaffected during protein-ligand interactions.
Trp quenching of HGD by SB was studied at four different temperatures (294K, 299K, 304K and 310K). Using the quenching data, binding constant (K) and the number of binding sites (n) were determined by applying double logarithmic plots (Fig 2A). The quenching constant (Ksv) and fractional accessibility of fluorophore (fa) were also calculated using modified Stern-Volmer plots (Fig. 2B). The binding and quenching parameters for HGD-SB complexation were mentioned in Table 1. With increase in temperature, HGD-SB binding constant was found to be decreased suggesting the involvement of hydrogen bonding and/or van der Waals interactions between the protein and small molecule. The number of binding sites in HGD had been decreased at higher temperatures, which also indicates weaker binding of SB with HGD at higher temperatures.
0.0
log(F0-F/F)
294K 299K 304K 310K
9
(B)
294K 299K 304K 310K
F0/F0-F
-0.5
(A)
6
-1.0
3 -1.5 -6.0
-5.5 -5.0 log [Q]
-4.5
0.00
0.15 1/[Q] µM-1
0.30
Fig. 2: (A) Double logarithmic plots and (B) modified Stern-Volmer plots of HGD-SB system at 294, 299, 304 and 310 K. [HGD]: 4 µM; [SB]: 0 to 26 µM; λex: 295 nm.
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On the other hand, increase in quenching constant with rise in temperature, suggests the probability of collisional quenching by SB. This is in contrary to the formation of HGD-SB complex at ground state. To realize this abnormality, the bimolecular quenching constant (kq) was calculated at different temperatures (mentioned in Table 1) using the equation Ksv = kq x τ0. The excited state lifetime (τ0) of moderately fluorescent Trp residues of HGD (Trp 42 and Trp 131) in absence of the small molecule is 3 ns [28]. The calculated kq values ~were 1013 M-1s-1 at all experimental temperatures. Therefore, the chance of dynamic quenching was discarded as the maximum kq value is 1010 M-1s-1 in case of dynamic quenching [24]. To understand this anomalous observation, Arrhenius theory was also used. In static quenching, the quenching efficiency lowers down with increase in temperature due to the lower stability of fluorophorequencher ground state complex. But according to Arrhenius theory, efficiency of quenching increases with temperature and this effect predominated over the effect of temperature for decreasing KSV. kq is related to the energy of activation (Ea) as follows: = −
(1)
where, A is a pre-exponential factor and T is the temperature.
Ea was calculated as 19.85 kJ mol-1 from the Arrhenius plot (Fig. 3) and found higher as compare to its commonly observed values for protein-small molecule binding [29-31]. This observation suggests that the effect of temperature is significant on the quenching process and firmly discard the chances of dynamic quenching. A similar type phenomenon was also noticed earlier for the binding of chloramphenicol with β-lactoglobulin [32]. Finally, it may be concluded that quenching mechanism of Trp fluorescence of HGD by SB is static in nature.
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31.4
R2=0.933
ln kq
31.2 31.0 30.8 0.00321
0.00330 -1 0.00339 1/T (K )
Fig. 3: Arrhenius plot for the quenching of HGD fluorescence by SB.
Table 1: Parameters related to the binding and quenching of HGD by SB Temp (K) 294 299 304 310
Double logarithmic K (103 M-1) n 6.02±0.25 0.87±0.08 2.82±0.12 0.78±0.04 1.23±0.05 0.71±0.02 0.32±0.02 0.58±0.02
Modified Stern-Volmer Ksv (10 M-1) fa 7.70±0.21 0.54±0.04 8.60±0.18 0.56±0.05 10.80±0.31 0.48±0.02 11.36±0.22 0.51±0.03 4
kq (1013 M-1s-1) 2.57±0.09 2.87±0.12 3.60±0.11 3.79±0.13
Different types of intermolecular forces basically control the binding of small molecule with protein and they can be predicted on the basis of sign and magnitude of the changes in enthalpy and entropy [33]. These intermolecular forces include hydrogen bonding, van der Waals, electrostatic and hydrophobic interactions. Thermodynamic parameters were calculated using van’t Hoff plot (Fig. 4) for HGD-SB complexation and are given in Table 2. Negative change in free energy implies spontaneous binding. Decrease in the enthalpy and entropy was also noticed due to the binding of SB with HGD. During HGD-SB complexation, the change in enthalpy basically drove the process and contributed significantly to the free energy change (∆G).
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Negative ∆H and ∆S values infer the involvement of hydrogen bonding and/or van der Waals forces between HGD and SB.
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ln K
8 7 6 0.00328 0.00336 1/T (K-1) Fig. 4: van’t Hoff plot for the HGD-SB complexation. Table 2: Thermodynamic parameters for binding of SB with HGD Temp. (K)
-1
∆G (KJ mol )
294
-21.6±0.7
299
-19.6±0.5
304
-17.6±0.5
310
-15.2±0.6
-1
∆H (KJ mol )
-138.0±4.5
-1
-1
∆S (J mol K )
-396.2±14.7
Quenching of Trp fluorescence of HGD by SB indicates that the fluorophore of HGD and SB lies close to each other. Therefore, transfer of energy from HGD to SB is expected to occur. Fig. 6 demonstrates the overlap between the fluorescence spectrum of HGD (4 µM) and the absorption spectrum of SB (4 µM). This suggests the chances of efficient energy transfer between them. The Förster resonance energy transfer (FRET) parameters like overlap integral (J), Förster distance and (R0) and the distance between donor and acceptor (r) were calculated [24]. The overlap integral (J) was computed as 1.63×10-14 M-1cm3 by integrating the overlap area of two spectra in 10
Fig. 5. Then R0 was determined by using above calculated J value and the quantum yield of
400
0.03
Absorbance
Fluorescence intensity
HGD as 0.058 [34]. Furthermore, r was calculated as 3.56 nm.
200
0
400 Wavelength (nm)
0.00 500
Fig. 5: Overlap between HGD fluorescence (solid) and SB absorption spectra (dotted). [HGD] and [SB]: 4 µM each; λex: 295 nm.
Synchronous fluorescence spectroscopy can also provide indications regarding the changes in Trp and Tyr microenvironment within a protein due to ligand binding [26]. Information regarding Trp residues may be extracted from the synchronous spectra, if the wavelength difference (∆λ) between the excitation and emission monochromators is maintained at 60 nm. Changes in the synchronous fluorescence spectra of HGD at ∆λ=60 nm in the presence of SB are shown in Fig. 6. Wavelength maximum of HGD was not shifted on addition of SB. Therefore, it can be interpreted that HGD-SB interactions did not result any notable change around the Trp residues of HGD.
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Synchronous fluorescence
0 µM
600
SB concentration µM
300
0
300
320 340 Wavelength(nm)
Fig. 6: Synchronous fluorescence spectra of HGD in absence (top line) and the presence of SB at ∆λ=60 nm.
The effect of SB binding with HGD on the secondary structures of the protein was studied by Far-UV CD spectroscopy (Fig. 7). Only minor changes in the Far-UV CD spectra of HGD were detected in the presence of SB. This suggests that Schiff base binding did not cause any change in the overall secondary structure of HGD and the small changes are only due to conformational adjustment during binding of the ligand.
CD (mdeg)
20
0
-20 200 220 240 Wavelength (nm)
Fig. 7: Far-UV CD spectra of HGD (15 µM) in absence (black) and in presence (red) of SB (15 µM).
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Furthermore, docking studies were performed to corroborate experimental observations. Interactions between HGD and SB were predicted from the docking results and the docked conformation of SB with HGD is shown in Fig. 8. In that conformation, SB was placed inside the protein structure where the aromatic rings of SB may be involved in van der Waals interactions and the nitrogen and oxygen atoms of SB may undergo hydrogen bonding interactions. The polar atoms of the Schiff base (two phenolic –OH and the imine nitrogen) was found in close proximity of the polypeptide backbone nitrogen of Trp 131, Val 132, Leu 133, Leu 145, Leu 146 and Met 147 as well as backbone oxygen of Trp 131, Tyr 144 and Leu 146. The phenoilc –OH of ring B of SB was also predicted to interact with the side chain oxygen of Ser 130. The distances between the interacting residues of HGD and the polar atoms of SB are given in Table 3. The interactions between SB and Trp 131 can explain the observed static quenching of Trp fluorescence of HGD. Interestingly, some of these interacting residues from the C-terminal domain (C-td) of HGD were reported to constitute the aggregation site of HGD [35]. Another earlier report had also predicted Ser 130 as the hot spot of HGD aggregation [36]. As unfolding of the N-terminal domain (N-td) of HGD takes place prior to that of C-td [37], therefore, the unfolding of N-td exposes the above mentioned residues which were buried in the native conformation to initiate protein aggregation. Now, the predicted binding of SB at or near to a major aggregation site of the protein suggests that the Schiff base will be able to inhibit HGD aggregation due to favorable protein-ligand interactions in addition to its strong metal chelating ability. It was also reported that Cu(II)-mediated aggregation of HGD proceeds through partial unfolding of the protein [10]. In that condition, binding of SB in this region of HGD molecule will arrest homologous association of protein molecules leading to their aggregation.
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Fig. 8: Docked conformation of SB with HGD. Interacting residues have been marked.
Table 3: Distances between the interacting residues of HGD and SB (A and B are the rings of SB marked in Fig. 1) Residue Ser 130 Oβ Trp 131 O Trp 131 N Val 132 N Leu 133 N Tyr 144 O Leu 145 N Leu 146 N Leu 146 O Met 147 N
Distance (Å) 2.03 [B(-OH)] 1.53 [A(-OH)] 1.74 [imine N] 2.42 [imine N] 2.78 [A(-OH)] 2.81 [imine N] 3.37 [A(-OH)] 2.48 [A(-OH)] 3.34 [A(-OH)] 2.41 [A(-OH)] 1.92 [B-(OH)] 2.76 [B(-OH)]
Conclusion A Schiff base was reported as an inhibitor of Cu(II)-mediated aggregation of HGD by virtue of its strong meal chelation ability. In addition to that, in the present work, the interactions between HGD and SB were studied. Ground state complexation between them had caused static
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quenching of the Trp fluorescence of HGD. Hydrogen bonding and/or van der Waals interactions between HGD and SB were identified, which can contribute to the aggregation inhibition activity of SB.
Acknowledgement: Authors are grateful to the Director, NIT Hamirpur (H.P.) for providing the research facilities. KSG is very much thankful to Prof. D. Balasubramanian, L.V. Prasad Eye Hospital, Hyderabad for his kind gift of HGD clone. Support from Dr. Joy Debnath, SATRA University and his research group for the synthesis of the Schiff base is gratefully acknowledged. SR is grateful to Ms. Sheetal, NIT Hamirpur for her cooperation in some of the experimental studies. SR and KSG are also thankful to Dr. Subrata Ghosh, IIT Mandi and his research group for providing support in the CD experiments.
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•
Interactions between human γD-crystallin and its aggregation inhibitor Schiff base
•
Static quenching of Trp fluorescence of HGD by the Schiff base
•
Involvement of hydrogen bonding and/or van der Waals interactions
•
No notable conformational changes in HGD due to protein-small molecule interactions
•
Docking predicts binding of Schiff base at a major site of aggregation of HGD
The work was designed by KSG and SR. Experimental data were acquired by SR. Analysis and/or interpretation of the experimental results, drafting and further revision of the manuscript were done by both the authors.
Declaration of interest statement The authors declare that there is no conflict ofinterest.
Shiwani Rana, Department of Chemistry, NIT Hamirpur (lndia)
Dr. Kalyan Sundar Ghosh, Department of chemistry, NIT Hamirpur
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