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Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to #-Lactoglobulin Narayani Ghosh, Ramakanta Mondal, and Saptarshi Mukherjee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02103 • Publication Date (Web): 04 Jul 2015 Downloaded from http://pubs.acs.org on July 11, 2015
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Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to β-Lactoglobulin
Narayani Ghosh, Ramakanta Mondal and Saptarshi Mukherjee*
Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Indore By-Pass Road, Bhauri, Bhopal 462 066, Madhya Pradesh, India
*To whom correspondence should be addressed:
[email protected]
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Abstract The binding interaction between the whey protein bovine β-Lactoglobulin (βLG) with the well-known antibiotic Chloramphenicol (Clp) is explored by monitoring the intrinsic fluorescence of βLG. Steady-state and time-resolved fluorescence spectral data reveal that quenching of βLG fluorescence proceed through ground state complex formation i.e., static quenching mechanism. However, the drug-protein binding constant is found to vary proportionately with temperature. This anomalous result is explained on the basis of the Arrhenius theory which states that the rate constant varies proportionally with temperature. Thermodynamic parameters like ∆H, ∆S, ∆G and the stoichiometry for the binding interaction have been estimated by Isothermal Titration Calorimetric (ITC) study. Thermodynamic data show that the binding phenomenon is mainly entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp-βLG binding. Constant pressure heat capacity change (∆Cp) has been calculated from enthalpy of binding at different temperatures which reveals that hydrophobic interaction is the major operating force. The inverse temperature dependence in static quenching is however resolved from ITC data which show that binding constant regularly decreases with increase in temperature. The modification of native protein conformation due to binding of drug has been monitored by Circular Dichroism (CD) spectroscopy. The probable binding location of Clp inside βLG is explored from AutoDock based blind docking simulation.
Keywords: Steady-state and time-resolved fluorescence; Static quenching; Arrhenius theory; Isothermal Titration Calorimetry; Constant pressure heat capacity change; Molecular docking. 2 ACS Paragon Plus Environment
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1. Introduction βLG (Scheme 1), a member of the lipocalin family, is the major protein in the whey fraction of milk of cows and sheep and some other mammalian species.1,2 βLG is a globular protein (molecular weight 18.3 kDa) containing 162 amino acid residues.1-3 The native structure of βLG is stabilized by two intramolecular disulfide bridges and is mainly characterized by β-sheet rich structure and consists of eight antiparallel β-strands and one αhelix which is located at the outer surface of the β-barrel.2-5 At physiological conditions it exists mainly as a dimer which dissociates into monomer at acidic pH.6 The protein βLG is known for its ability to bind and transport a wide variety of hydrophobic ligands including retinoids, lipids, vitamin D and fatty acids.2,5-11 βLG has two hydrophobic binding sites; the internal cavity (calyx) and the external hydrophobic surface between the α-helix and βbarrel,2-4,7,9-11 out of which the hydrophobic calyx is mostly argued to be the major binding site.2,5 For studying the interaction between protein and ligand, βLG is a very useful model protein as its conformation, function and physiological properties are well defined. βLG has two tryptophan amino acid residues (Trp19 and Trp61) which enable it to be studied using intrinsic fluorescence. Chloramphenicol (Clp, Scheme 1), originally extracted from the bacterium Streptomyces venezuela, has been reported to display a wide spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria.12-14 The antimicrobial property of Clp is attributed to its ability to prevent the peptidyl transferase activity of ribosome by binding at the 50S ribosomal subunit. This inhibits the protein chain elongation process of the bacterial ribosome.15 Clp also has several harmful side-effects in the humans such as gray baby syndrome, bone marrow suppression, fatal aplastic anaemia16 etc. The ability of the drug to bind with proteins (particularly transporting proteins) is believed to play key roles in governing the transportation, distribution, and physiological action within the body which
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underlines the importance of exploring its interactions with protein(s). The binding interaction of Clp with Human Serum Albumin and lysozyme has been reported previously,17,18 but the interaction with this important hydrophobic ligand binding and transporting protein, βLG is not yet studied. Here, the drug (Clp)-protein (βLG) binding interaction has been studied by spectroscopic and calorimetric techniques. It is interesting to note that our steady-state and time-resolved fluorescence spectroscopic results show apparently anomalous observations. Though spectroscopic results provide convincing evidence for a static mechanism of the Clpinduced quenching of the intrinsic fluorescence of βLG, the drug-protein binding constant is found to vary proportionately with temperature. This is in apparent contradiction to the usual expectation for a static quenching mechanism.19 This anomaly has been interpreted from the temperature dependence of the bimolecular quenching rate constant based on the Arrhenius relationship. The binding affinity of Clp to βLG and the thermodynamics of the interaction have been thoroughly investigated by Isothermal Titration Calorimetry (ITC). ITC study shows that the Clp-βLG interaction is mainly an entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp-βLG binding. From the temperature dependence of enthalpy change (∆H) a predominant role of the hydrophobic forces has been further established for the drug-protein binding process. The effect of drug binding on the native conformation of βLG has been determined by Circular Dichroism (CD) and the probable binding location of the drug within βLG has been rationalized on the basis of blind molecular docking study.
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Scheme 1. Schematic Structures of β-Lactoglobulin (βLG) and Chloramphenicol (Clp).
2. Experimental Section 2.1. Materials βLG, Clp, NaCl and phosphate buffer (0.01 M, pH = 7.40) were used as received from Sigma-Aldrich, USA. βLG (>90%) contains genetic variants β-Lactoglobulins A and B (βLG A and βLG B) and was used as received. Triply distilled deionized Milli pore water was used to prepare the required solutions. The concentration of βLG was 5 µM for the steady-state and time-resolved fluorescence experiments, 10 µM for CD spectroscopic measurements and 500 µM for ITC experiments.
2.2. Instruments and Methods Steady-State Spectral Measurements. The absorption and emission spectra were measured on a Cary 100 UV-Vis spectrophotometer and Fluorolog 3-111 fluorometer, respectively. The temperature was kept constant at a given value by a recycling water flow accurate up to ±0.1 °C. The fluorescence spectra were measured with a standard quartz cuvette of 1 cm path length. Clp has substantial absorbance at ~278 nm and hence in order to avoid inner filter effect, the fluorescence intensity was corrected according to the following equation:19,20
= × ⁄
(1)
where, I is the corrected fluorescence intensity and Iobs is the observed background-subtracted fluorescence intensity of the sample under investigation. Aex and Aem are the measured absorbance values at the excitation and emission wavelengths, respectively.
Fluorescence Lifetime Measurements. Fluorescence lifetimes were measured by the Time Correlated Single-Photon Counting (TCSPC) method (λex = 295 nm using a picosecond
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diode, IBH-NanoLED source N-295, FWHM ~800 ps,). The details of the instrumental setup are mentioned elsewhere.4 The multiexponential fluorescence decay function is given as:19
= ∑ ⁄
(2)
where, αi denotes the amplitude of ith lifetime, τi. Hence, the average lifetime 〈τ〉 is given as:19
〈〉 = ∑
(3)
Circular Dichroism (CD) Measurements. CD spectra were acquired on a JASCO J815 spectropolarimeter using a cylindrical cuvette of 0.1 cm path-length at 298 K. Each CD spectrum is an average of four consecutive scans at 50 nm/min scan rate with proper baseline correction.
Isothermal Titration Calorimetric (ITC) Measurements. The ITC experiments were performed with a Nano ITC, TA Instruments. All solutions of βLG and Clp were degassed before the experiments. The sample cell was loaded with βLG solution and was continuously stirred at 300 rpm by the injection syringe containing the drug solution ([Clp] = 4.5 mM, 2 µL titrant volume for each injection). The titration was conducted at different temperatures. The respective control experiments were carried out by injecting the titrant into the buffer solution in the absence of the protein to correct for the heat change due to mixing and dilution. The data were analyzed by NanoAnalyze software v2.4.1 and fitted with an independent site binding model. As per convention in the software used here for fitting the ITC data, the endothermic or exothermic processes are represented by “downward” or “upward” heat bursts, respectively. The sign of enthalpy change (∆H) obtained from fitting the experimental data provides the genuine thermodynamic signatures of the concerned processes and we have interpreted our data accordingly.
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Molecular Docking Study. The molecular docking simulation was carried out on AutoDock 4.2 software.21 The native βLG structure was taken from the Protein Data Bank (PDB entry: 1BSY22 (for βLG A), and 1BSQ23 (for βLG B)). The three-dimensional structure of Clp was prepared from the optimized geometry obtained from calculation on Gaussian 03W software (DFT/B3LYP/6-31G(d,p)).24 The AutoDocking parameters used were as follows: grid size: 106, 106 and 106 along X-, Y-, and Z-axes, respectively, grid spacing: 0.375 Å, GA population size = 150; maximum number of energy evaluations = 250000; GA crossover mode = two points. This promotes a blind docking investigation. The minimum energy docked conformation searched out of 10 different conformations was used for analysis. The docked conformation was examined using the PyMOL software package.25
3. Results and Discussion 3.1. Fluorescence Quenching of βLG in the Presence of Clp Upon interaction with Clp, the absorption spectra of βLG changes marginally; the absorbance values increase slightly along with a small red shift (figure not shown). However, a more dramatic change is observed on the emission profiles. Upon excitation at 295 nm, native βLG displays an emission maximum centered at ~334 nm,2,4 which can be attributed to the intrinsic fluorescence of the protein. It must also be mentioned here that Clp itself is nonfluorescent when excited at 295 nm (figure not shown). βLG has two Trp residues of which Trp19 is embedded in the hydrophobic protein cavity and Trp61 is partially exposed2-4 and it is believed that the Trp19 is the major contributor to the total fluorescence of βLG (Scheme 1).2-4 The modulation of the fluorescence property of the native protein due to interaction with exogenous ligands and drug molecules has been monitored to investigate the drugprotein interaction.2-4,7 Figure 1 displays a prominent quenching of the intrinsic fluorescence of βLG due to interaction with Clp.
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Figure 1. Emission spectra of βLG in presence of different concentrations of Clp at 293 K. Curves (i) to (xv) represent 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 72, 84, 96, 108 µM of Clp, respectively.
The fluorescence quenching is generally described by the well-known Stern-Volmer equation:19,26
=1
!"# $%&
(4)
where, I0 and I are the fluorescence intensities of βLG in the absence and presence of the quencher (Q), i.e., Clp, respectively, KSV is the Stern-Volmer quenching constant. Figure 2 depicts the representative Stern-Volmer plots for the fluorescence quenching of βLG at different temperatures.
Figure 2. Stern-Volmer plots of Clp induced quenching of intrinsic fluorescence of βLG at various concentrations of Clp monitored at different temperatures as indicated in the figure.
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It is interesting to note that the linear Stern-Volmer plots as expected from equation (4) are not obtained; rather the plots display an upward curvature. Usually upward curvature of Stern-Volmer plot may arise due to: (i) quenching due to static as well as dynamic mechanisms simultaneously and/or (ii) high extent of quenching at higher concentration region of quencher (in the present case, Clp).4,19,27 A linear Stern-Volmer plot indicates the operation of only one type of quenching mechanism, either static or dynamic4,19,27 and it is thus essential to classify the nature of quenching observed in this study. For this purpose, we resorted to the time-resolved fluorescence decay measurements. The fluorescence decay transients of βLG in the presence of increasing concentrations of Clp are displayed in Figure 3 and the fitted parameters are summarized in Table S1 of the Supporting Information (SI). The fluorescence decay of βLG in native form is biexponential in nature which corresponds well to literature reports.4,28 The data compiled in Table S1 of the SI show that the individual lifetime components (both τ1 and τ2) of βLG do not vary with increasing concentration of Clp. In case of dynamic quenching the individual decay components and average lifetime should decrease with increasing concentration of the quencher.4,19 Therefore, the possibility of dynamic quenching can be discarded thereby enabling us to conclude that the mechanism of quenching as a result of the interaction of Clp with βLG is static in nature. So the observed upward curvature of Stern-Volmer plot (deviation from linearity) may be ascribed to high extent of quenching with increasing drug (Clp) concentration.4,19
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Figure 3. Lifetime decay curves of βLG in the presence of various concentrations of Clp (0 µM, 18 µM, 90 µM). IRF represents the instrument response function. Inset shows the variation of average lifetime of βLG in the presence of various concentrations of the quencher, Clp.
3.2. Drug-Protein Binding Parameters Based on the quenching data, the binding constant (K) has been estimated using the following equation:19,29-31
'() * + = '()!
,'()$%&
(5)
where, I0 and I are the fluorescence intensities of βLG in the absence and presence of the drug, respectively, and n is the number of binding sites. The representative log[(I0 - I)/I] vs. log[Q] plots at different temperatures are displayed in Figure 4A and the binding constant values are summarized in Table 1 which show that the binding constant (K) increases with temperature. However, from the time-resolved data it can be concluded that the drug-induced intrinsic fluorescence quenching of βLG proceeds through a static mechanism.4,19 Conventionally, in case of static quenching the drug-protein binding constant should decrease with increasing temperature.19 This poses an apparent anomaly in the present case of Clp induced quenching of intrinsic fluorescence of βLG. Therefore, the modified Stern-Volmer plot is applied to determine the actual quenching mechanism:4,19,32
-
= .
-
.×/0
-
× $1&
(6)
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Here, f is the fractional maximum accessible protein fluorescence, KQ is the apparent quenching constant and other terms bear same meaning as mentioned above.4,19,32 Figure 4B depicts the representative modified Stern-Volmer plots for quenching of fluorescence of βLG due to addition of Clp at different temperatures. Following equation (6), the apparent quenching constant, KQ has been calculated from which the bimolecular quenching rate /
constant has been estimated as 23 = 〈〉0 (〈τ〉0 is the average fluorescence lifetime of βLG in
the absence of Clp).19 The value of kq is found to be on the order of 1012 M-1 s-1 at all the experimental temperatures (Table 1). This observation again establishes the static quenching mechanism in the present case as the bimolecular quenching constant (kq) is two orders of magnitude higher than the maximum threshold for a diffusion-controlled process (1010 M-1 s1 4,19
).
(a)
(b)
Figure 4. (a) Double logarithmic plots for the determination of binding constant of Clp-βLG interaction at different temperatures as marked in the figure. (b) Modified Stern-Volmer plots of Clp induced quenching of intrinsic fluorescence of βLG at different temperatures as marked in the figure.
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Table 1. Summary of the Binding Parameters for the Clp-βLG Interaction from Fluorescence Quenching Data Method Double log Temperature
KѰ (103 M-1)
(Kelvin)
Ѱ
Modified Stern-Volmer
Stoichiometry
KQѰ (103 M-1)
kq (1012 M-1)
(n)
283
7.17
1.20
5.59
3.65
288
8.26
1.16
6.58
4.30
293
9.20
1.17
8.25
5.39
298
11.22
1.12
9.18
6.00
303
12.88
1.10
9.90
6.47
308
13.86
1.09
11.40
7.45
313
14.80
1.07
12.42
8.12
±5% Thus, our results evidently demonstrate an unusual observation, i.e., an adverse
temperature dependence in a static quenching process. Here, we have used the Arrhenius theory to interpret the apparent anomaly. Generally, in static quenching mechanism, the quenching efficiency should decrease with increasing temperature due to destabilization of the fluorophore-quencher ground-state complex.19 On the contrary, the quenching efficiency should vary proportionately with temperature according to the Arrhenius theory. Thus, the increment of KQ with increasing temperature (Table 1) in the present case indicates that the effect of increase of KQ due to increasing temperature is greater than the decrease of KQ owing to the instability of the complex.33,34 The binding constant (K) determined from the quenching data is also found to vary proportionately with temperature (Table 1). According to the Arrhenius theory, the bimolecular rate constant of the quenching process (kq) should 12 ACS Paragon Plus Environment
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significantly depend on temperature. At higher temperature, the rate constant should also be higher. Further, the activation energy of the process (Ea) is also related to the rate constant and the temperature according to the Arrhenius relationship as:33,34 ln 23 = ln 6
/0
78
9 = ln : −
?
where, A is the pre-exponential factor. From the plot of lnkq vs. 1/T (Figure 5) the activation energy is estimated to be 19.4±1.2 kJ mol-1 which is higher than the magnitude for interactions of a host of organic compounds with proteins.33,34 This is a clear signature of a remarkable influence of temperature on the fluorescence quenching as encountered in the present case. This result strongly supports the static quenching mechanism though in our case the temperature dependence of the static quenching mechanism is apparently anomalous.
R2 = 0.98
Figure 5. Arrhenius plot for Clp induced quenching of intrinsic fluorescence of βLG. The goodness of the fit (R2) indicated in the figure.
3.3. Effect of Ionic Strength on the Interaction of Clp to βLG The strong electrolyte NaCl has been employed to determine the influence of electrostatic interaction between Clp and βLG. In order to study the effect of ionic strength on the binding interaction, the NaCl concentration has been varied from 0 to 100 mM. No 13 ACS Paragon Plus Environment
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significant change in binding constant was observed even in the presence of high concentration of NaCl (figure not shown) which probably indicates that electrostatic forces have no significant contribution in the drug-protein interaction.7 Thus, hydrophobic forces may be the major driving parameter for the Clp-βLG binding process7 which has also been substantiated in subsequent sections.
3.4. Isothermal Titration Calorimetry (ITC) Although van't Hoff relationship provides a well-known method to evaluate the thermodynamic parameters for drug-protein interaction process,4,19,35,36 it is not applied here as in our case anomalous results are obtained for the temperature dependent quenching experiment (a static quenching mechanism in which binding constant (K) increases with increasing temperature). Instead, ITC has been employed to determine the thermodynamics of Clp-βLG interaction, as this methodology offers a direct measurement of the parameters.7,37-41 Figure 6a shows the representative calorimetric profile during the titration of βLG with Clp at 298 K. The heat change during titration of βLG with incremental addition of Clp is fitted to an independent site binding model to estimate the thermodynamic parameters (Table 2). The value of binding stoichiometry, s (Table 2) indicates a 1:1 binding for the Clp-βLG interaction. It is interesting to note that the Clp-βLG binding constant obtained from ITC data decreases with increase in temperature (Table 2). This resolves the apparent contradiction in spectroscopic results (inverse temperature dependence of the static quenching). This is probably due to the fact that ITC provides a direct method to determine the binding constant taking the entire system into consideration in contrast to the fluorometric approaches which probes the immediate microenvironment in and around the fluorophore.38 The drug-protein interaction is found to be characterized by a large positive entropy change (∆S) and relatively small negative enthalpy change (∆H) clearly revealing entropy driven
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binding. A net negative free energy change (∆G < 0) of interaction indicates that the Clp-βLG interaction is thermodynamically favorable. The strong positive entropy term indicates that the complex formation is possibly driven by hydrophobic interaction forces.42 Based on the discussion in the previous section (Section 3.3) i.e., insignificant contribution of ionic strength on the interaction between Clp and βLG, it is further supported that hydrophobic interaction is the major force for the Clp-βLG binding interaction.43-45 To further support this conclusion, the thermodynamic parameters have been determined at different temperatures from ITC experiments and the relevant data are ∆C
comprised in Table 2. By using the thermodynamic relationship, ∆AB = 6 9 , the constant ∆? B
pressure heat capacity change (∆Cp) for the interaction has been estimated (Figure 6b) and is found to be -39.6±0.29 J mol-1. The value of ∆Cp in the protein-ligand interaction is of great importance as it gives fruitful information regarding the mechanism of interaction.7,46-49 According to literature reports,43-48 a negative value of ∆Cp is characteristic of a dominant hydrophobic effect in the binding process. The negative value of ∆Cp in the present study supports the conclusion that hydrophobic interaction is the major operating force in binding between Clp and βLG.7
(b)
(a)
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Figure 6. (a) Isothermal titration calorimetric profile for the titration of βLG with Clp at 298 K. The upper panel shows the raw data for the integrated heat after correction for heat of dilution. The lower panel shows the integrated heat data against molar ratio of [Clp]/[βLG] at 288 K (blue circle), 298 K (olive square) and 308 K (purple triangle) and the solid lines are the fitted curves. (b) Plot of enthalpy of binding as a function of temperature. Power sign convention is mentioned in Instrument and Method Section (Section 2.2).
Table 2: Summary of the ITC Results for Interaction of Clp with βLG at Different Temperatures Temperature
∆H
∆S
∆G
(K)
(kJ mol-1)
(J mol-1 K-1)
(kJ mol-1)
288
-2.81±0.11
59.32±1.5
-19.89
298
-3.21±0.11
56.45±1.6
308
-3.60±0.12
52.36±2.1
s
Ka
∆Cp
(103 M-1)
(J mol-1)
1.13±0.08
4.05±0.4
-39.6±0.29
-20.02
1.12±0.08
3.22±0.3
-19.73
0.86±0.08
1.97±0.5
3.5. Circular Dichroism (CD) Study The far-UV CD spectrum of native βLG exhibits a broad negative minimum at ~216 nm (Figure S1) which is a clear signature of β-sheet rich secondary structure of the protein.4,7 Following interaction with Clp, a marginal decrease in CD signal of the far-UV CD spectra of βLG is observed. This indicates conformational change in secondary structure of βLG upon interaction with Clp. The decrement of ellipticity signifies the reduction in β-sheet structure which indicates the unfolding of the peptide strand of the native protein upon interaction with the drug.4,7
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3.6. Molecular Docking Study AutoDock based blind docking simulation study has been applied to explore the binding site of the drug (Clp) in βLG. The resultant lowest energy docked conformation revealing the probable binding location of Clp within the protein scaffold is displayed in Figure 7. As seen from the figure, the drug is located in the internal hydrophobic cavity of βLG. Thus it may be logical to assume that hydrophobic forces predominate in βLG-Clp binding. This seems to corroborate the ITC results discussed previously (Section 3.4). Further, the protein residues in the vicinity (around 6 Å) of the drug binding site are shown in Figure 7. From docking result it is found that the aromatic rings of both Clp and PHE105 residue of βLG are almost parallel to each other (Figure S2 of SI). Therefore, the possibility of aromatic stacking cannot be discarded. Here, the docked pose of Clp (ligand) to βLG (receptor) correlates to the best-energy solutions in the most populated cluster as obtained from the docking analysis (figure not shown).
Figure 7. Minimum energy docked pose (left) for blind docking simulation of Clp (in CPK model) with βLG A (PDB ID: 1BSY). The protein residues in near vicinity of the drug binding site are shown in the right.
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We have also done molecular docking study of Clp with the genetic variant B of βLG, as the protein used herein contains the genetic variants, β-Lactoglobulins A and B. The resultant lowest energy docked conformation is shown in Figure S3 in SI. As shown in the figure, Clp is located in the internal hydrophobic cavity of βLG, in agreement with the genetic variant A of βLG. The negative free energy change from docking study in both βLG A (∆GDock = -3.8 kcal mol-1) and βLG B (∆GDock = -3.03 kcal mol-1) indicates spontaneity of the interaction.
4. Conclusions In the present report, we have studied the binding interaction of an antibacterial drug Clp with the protein βLG. Steady-state spectroscopy reveals that the fluorescence intensity of βLG decreases with increasing concentration of Clp and the Stern-Volmer plots exhibited an upward curvature. The time-resolved fluorescence decay results did not show any appreciable change due to the interaction of Clp with βLG which indicates that the mechanism of quenching is static in nature. The most interesting observation in this context is the increase of the drug-protein binding constant with temperature which is an apparent anomaly when static quenching is encountered. This contradiction has been explained from the Arrhenius theory. Isothermal titration calorimetric study shows that the Clp-βLG interaction is mainly an entropy-driven process indicating the key role hydrophobic interaction forces. A negative value of ∆Cp strongly supports this inference. This is again reiterated by our Molecular Docking studies. High concentrations of NaCl have almost no effect on the binding interactions which conclusively established the fact that electrostatic forces have no major role in this binding process.
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Acknowledgments NG acknowledges a research fellowship from the Govt. of India through CSIR-NET. RM acknowledges IISER Bhopal for a research fellowship. SM sincerely thanks DST, Govt. of India for financial support. The authors also thank Dr. Bijan Kumar Paul for many stimulating discussions. Supporting Information Lifetime decay parameters, circular dichroism spectra and docked conformations. This material is available free of charge via the Internet at http://pubs.acs.org
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