pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl

Article pubs.acs.org/JPCB

pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl Sulfate with Bovine Serum Fetuin: A Biophysical Insight Nida Zaidi, Saima Nusrat, Fatima Kamal Zaidi, and Rizwan H. Khan* Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India S Supporting Information *

ABSTRACT: Sodium dodecyl sulfate (SDS)-glycoprotein interaction serves as a model for a biological membrane. To get mechanistic insight into the interaction of SDS and glycoprotein, the effect of SDS on bovine serum fetuin (BSF) was studied in subcritical micellar concentrations at pH 7.4 and pH 2 using multiple approaches. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically via its alkyl chains at pH 7.4 up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2, as confirmed by enhanced ThT fluorescence, β-sheet formation, and TEM microscopy, whereas BSF undergoes induction of an α-helical structure in the presence of higher SDS concentration at pH 7.4. The increase in αhelical content with increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of tryptophan conformers decreases, resulting in a decrement of the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

1. INTRODUCTION Sodium dodecyl sulfate (SDS) is an anionic surfactant with a 12-carbon hydrophobic tail and a negatively charged sulfate hydrophilic headgroup. The interaction of SDS with glycoprotein has great significance in that it mimics the native hydrophobic milieu of the phospholipids bilayer in vivo and thus serves as a model for the interaction of membrane proteins and lipids.1,2 SDS is known to interact with globular proteins and, in general, causes denaturation at concentrations almost 1000 times lower than is generally needed for other chemical denaturants, such as urea.3−6 It can also act as an inducer of protein self-oligomerization or amyloid fibril formation that is associated with several neurodegenerative disorders. Previous studies have reported that the interaction of SDS with proteins depends on the charge on the headgroup and length of the hydrophobic tail.7 Consequently, SDS induced amyloid fibrillogenesis in proteins at a pH below their respective isoelectric points, whereas no such process was observed above it. In addition, it has also been reported that critical micellar concentration of SDS also decides the mechanism of interaction. At subcritical micellar concentration (sub-CMC), SDS monomers bind to proteins by predominantly hydrophobic interactions causing unfolding of the tertiary structure, whereas at concentrations higher than CMC, the micelles nucleate on the hydrophobic patches of the protein chain, driving it to expand.8 Although considerable progress has been made in studying the interaction of protein with SDS, the exact mechanism and forces responsible for the interaction of negatively charged SDS to glycoprotein under conditions when it carries net positive and net negative charges is still not fully deciphered. Thus, to © 2014 American Chemical Society

determine the mechanism, interaction of SDS with a model glycoprotein, bovine serum fetuin (BSF),9−12 was studied at pH above (net negative charge) and below (net positive charge) its isoelectric point (pI), that is, at pH 7.4 and 2. The glycoprotein chosen for the present study, BSF, contains six carbohydrate chains, three N-linked glycosylation sites, and three O-linked glycosylation sites, which comprise 24% of its total molecular weight (48 585), along with 8% sialic acid.13 It contains 341 amino acids, and their sequence shows over 70% similarity to human α2-HS glycoprotein.14 It is a member of the cystatin superfamily of cysteine protease inhibitors.15,16 In the present study, we attempted to monitor the effect of submicellar concentrations of SDS on the BSF at pH 7.4 (net negative charge) and pH 2 (net positive charge) by steady state, time-resolved fluorescence spectroscopy and isothermal titration calorimetry. The changes in secondary structures were monitored by far-UV circular dichroism spectroscopy. The thioflavin-T (ThT) binding, turbidity measurements, and transmission electron microscopy were used to analyze the nature of aggregates. The interacting forces between SDS and glycoprotein were also determined.

2. MATERIALS AND METHODS 2.1. Materials and Sample Preparation. Fetuin from fetal bovine serum (F3004) and sodium dodecyl sulfate (L3771) were procured from Sigma-Aldrich. All other reagents and buffer components used were of analytical grade. The Received: February 12, 2014 Revised: September 22, 2014 Published: October 22, 2014 13025

dx.doi.org/10.1021/jp501515g | J. Phys. Chem. B 2014, 118, 13025−13036

The Journal of Physical Chemistry B

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database having accession number CAA34596.1. The solventaccessible surface area was calculated by InterProSurf,21 and the structure was visualized with Chimera 1.7.22 2.7. Protein Charge Determination. The charge on BSF at pH 2 and pH 7.4 was calculated using the program Protein Calculator v3.4. 2.8. Turbidity Measurements. Turbidity measurements of BSF (5 μM) in the presence of SDS at submicellar concentrations in 20 mM buffer of pH 2 and 7.4 after the overnight incubation were taken. The measurements were carried out at 350 nm on a PerkinElmer Lambda 25 double beam spectrometer using a sample cell of path length 10 mm at 25 °C. 2.9. Rayleigh Scattering Measurements. Rayleigh scattering measurements of BSF (5 μM) in the presence of SDS at submicellar concentrations in 20 mM buffer of pH 2 and 7.4 after the overnight incubation were taken. The measurements were carried at 350 nm using a Shimadzu 5301PC fluorescence spectrophotometer equipped with a water circulator (Julabo Eyela) at 25 °C. 2.10. Steady State Fluorescence Spectroscopic Measurements. The fluorescence measurements of BSF (5 μM) in the presence of SDS at submicellar concentrations in 20 mM buffer of pH 2 and 7.4 were taken after overnight incubation. All fluorescence measurements were done using the Shimadzu 5301PC fluorescence spectrophotometer equipped with a water circulator (Julabo Eyela) at 25 °C. The excitation and emission slits were set at 5 nm for ThT and intrinsic fluorescence measurements. Samples were excited at 295 and 440 nm for intrinsic fluorescence and ThT fluorescence measurements, respectively. The emission spectra were recorded in the range of 300−500 nm for intrinsic fluorescence and 460−600 nm for ThT measurements. A stock solution of ThT was prepared in distilled water, and its concentration was determined using a molar extinction coefficient of εM = 36 000 M−1 cm−1 at 412 nm.23 ThT was added to the protein samples in a molar ratio of 1:2. 2.11. Transmission Electron Microscopy (TEM). Transmission electron micrographs of BSF in the presence 0.75 mM SDS in 20 mM buffers of pH 2 and 7.4 were collected on a transmission electron microscope operating at an accelerating voltage of 200 kV. The samples were diluted appropriately and applied to copper grids (200 mesh), blotted, and air-dried. The samples were then negatively stained with 2% (w/v) uranyl acetate prior to imaging. 2.12. Isothermal Titration Calorimetric Measurements. The energetics of the binding of SDS to BSF at pH 2 and 7.4 was measured using a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA) maintained at 25 °C. Prior to the titration experiment, all samples were degassed properly on a thermovac. The sample and reference cell of the calorimeter were loaded with protein solution (17 μM) and the respective buffers, respectively, then multiple injections of 10 μL of SDS solution (1.68 mM) were made into the sample cell containing protein at pH 2 and 7.4. Each injection was made over 20 s with an interval of 180 s between successive injections. The reference power and stirring speed were set at 16 μcal−1s and 307 rpm, respectively. The heats of dilution for the protein and the heat of micellization of SDS were subtracted from the integrated data before curve-fitting. The ITC of BSF and SDS were also performed in the presence of 0.34 mM NaCl at pH 2 and pH 7.4.

protein was dialyzed, and its concentration was estimated 17 SDS solutions spectrophotometerically using E1% 278nm = 4.5. were prepared by weight/volume (w/v) in 20 mM buffer of pH 2 (glycine−HCl) and 7.4 (sodium phosphate). The pH measurements were carried out on a Mettler Toledo pH meter (Seven Easy S20-K) model using an Expert “‘Pro3 in 1’” type electrode. 2.2. SDS−PAGE. The stability of BSF at pH 2 and 7.4 over the lifetime of the experiment has been analyzed by performing SDS−PAGE according to Laemmli18 in 10% gel with electrophoresis buffer containing 0.1% SDS in Tris glycine buffer (pH 8.8). 2.3. Electron Spray Ionization Mass Spectrometry (ESI-MS) of SDS. The mass spectra of SDS at pH 2 and pH 7.4 after overnight incubation were recorded with a SYNAPT G2-S HD mass spectrometer (Waters, Milford, MA) in positive ion mode with a mass scan from m/z 80 to 400 to check the mass of the SDS molecule. 2.4. LC−MALDI-MS−MS of BSF. (a). Trypsin Digestion. BSF after overnight incubation at pH 2 was digested with trypsin at 30 °C with a BSF-to-trypsin ratio of 10:1 (w/w) in 50 mM ammonium bicarbonate buffer solution at pH 8.5. Prior to tryptic digestion, BSF was reduced, followed by alkylation with dithiothreitol and iodoacetamide, respectively, according to the procedure modified from a Pierce in-solution trypsin digestion kit. (b). LC−MALDI-MS−MS. The 0.5 μL of digested protein sample was spotted on 0.5 μL of α cyano-4-hydroxycinnamic acid matrix (matrix in 50% HPLC grade acetonitrile, 0.1% trifluoroacetic acid) on a MALDI sample plate. After the spot dried, MALDI-MS−MS was performed using an ABI SCIEX 5800 TOF/TOF system with LC−MALDI. The MS−MS was operated in positive ion mode with a mass scan from m/z 700 to 4000. (c). Data Analysis. Mass spectral data were processed using the paragon algorithm for accurate identification of peptides and proteins. Raw data files were processed using protein pilot 3.0 (AB SCIEX) paragon algorithm19 against a target protein, fetuin [Bos taurus], gi|344 accession number from the NCBInr databases. Data were searched with and without deamidation (N and Q). 2.5. Critical Micellar Concentration Measurements (CMC). The CMC of SDS was determined at pH 2 and 7.4 by isothermal titration calorimetry and conductivity measurements. For CMC determination by ITC, concentrated surfactant solutions of SDS micelle systems (40 mM) were titrated into 20 mM buffers of pH 2 and 7.4 in a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA) at 25 °C. For conductivity measurements at pH 2 and 7.4, the SDS solution was added to the buffer solution, and conductance was measured using an Elico (type CM 82T) bridge equipped with platinized electrodes. The measurements were carried out in a thermostated water bath at 25 °C. The CMC value corresponds to the SDS concentration, at which break point was found in the plot between conductance and SDS concentration. 2.6. Molecular Modeling and Solvent Accessible Surface Analysis. The protein molecule was modeled by Protein Homology/analogY Recognition Engine (Phyre2).20 Phyre2 takes a sequence, builds a profile using PSI-BLASTs, and automatically compares it with templates deposited in the structural classification of proteins database and PDB. The amino acid sequence of BSF was directly taken from the protein 13026



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2.13. Circular Dichroism Spectroscopic Measurements. To monitor the secondary structural changes of BSF (5 μM) in the presence of SDS at submicellar concentrations in 20 mM buffers of pH 2 and 7.4 after the overnight incubation, CD spectra were collected in far (200−250 nm) using a JascoJ815 spectropolarimeter equipped with a Peltier-type temperature controller at 25 °C with a cuvette of 0.1 cm path length. The scan speed and response time were set as 20 nm/min and 2 s, respectively. To monitor the secondary structural change of protein upon interaction with SDS, results are expressed as MRE (mean residue ellipticity) in deg cm2 dmol−1, which is given by MRE =

Θobs(mdeg) 10 × n × c × l

(1)

where θobs is the observed ellipticity in millidegrees, c is the concentration of protein in mol/L, l is the length of the light path in centimeters, and n is the number of peptide bonds. 2.14. Time-Resolved Fluorescence Measurements. Fluorescence lifetime measurements were performed using the time-correlated single photon counting (TCSPC) model 5000 U (Horiba Jobin Yvon) equipped with a water circulator at 25 °C. The BSF (5 μM) in the presence of varying concentrations of SDS (0−3.5 mM) after overnight incubation were excited at 295 nm using a NanoLED pulsed laser. The instrument response function (IRF) was obtained using a Ludox suspension. The full width at half-maxima (fwhm) of the IRF was 750 ps. The emission decay data at 340 nm were analyzed using the software, DAS6, provided with the instrument.

Figure 1. Electron spray ionization mass spectra of SDS at (A) pH 7.4 and (B) pH 2. Inset shows the structure of dodecyl sulfate ion.

3. RESULTS AND DISCUSSION 3.1. SDS−PAGE Analysis. The BSF incubated overnight in pH 7.4 (lane 1) and pH 2 (lane 3), moved as a single band, as shown in Supporting Information (SI) Figure S1. It confirms that BSF remains stable during the lifetime of the experiment in buffer of both pH values. The multiple bands in lane 2 are of molecular weight markers. 3.2. ESI-MS of SDS. The SDS generates dodecyl sulfate ion (Figure 1 inset) in buffer having a theoretical molecular mass of 265.411 Da. To check the degradation of SDS at pH 2, ESI-MS spectra of dodecyl sulfate after overnight incubation at pH 2 were collected and compared with that at pH 7.4 (Figure 2A,B). Similar peaks observed at m/z 265.9287 and 265.9381 at pH 2 and pH 7.4 respectively, surely negate the chances of degradation of SDS at pH 2. 3.3. LC−MALDI-MS−MS of BSF. The focus of the present study is on electrostatics, thus negating the chances of deamidation of asparagines (N) and glutamine (Q) side chains in BSF at pH 2 are necessary. For assessment of deamidation, LC−MALDI-MS−MS was performed. BSF was subjected to tryptic digestion after incubation overnight at pH 2. The employed method for trypsin digestion of BSF resulted in peptide generation that has a sequence coverage of 68%. The peptides containing asparagines and glutamine obtained after tryptic digest are listed in Table 1. As can be seen from the table, there is almost no difference in the theoretical and the observed mass, and thus, no deamidation occurs, which otherwise creates a difference of 1 Da. Furthermore, the results that are obtained directly from the protein pilot 3.0 paragon algorithm do not show deamidation modification in the listed tryptic peptides.

3.4. Critical Micelle Concentration Determination. To understand the mode of interaction of monomeric SDS with BSF, it is necessary to determine the CMC value because proteins interact differently with the monomeric and the micellar forms of SDS.24 The CMC can be determined in a single titration experiment by ITC. To illustrate this, concentrated surfactant solutions of SDS micelle systems were titrated into 20 mM buffer of pH 2 and 7.4. At concentrations below the CMC, the reaction is

micelles → monomers

(2)

and at concentrations above the CMC, the reaction is concentrated micelles → dilute micelles

(3)

At 25 °C, titration of concentrated SDS into water elicits a titration profile with endothermic peaks (Figure 2A,B). The endothermic peaks represent the dispersion of the concentrated micelle solution (demicellization) upon titration into buffer.25 As the surfactant concentration increases, the process of demicellization comes to a halt, and the reaction enthalpy approaches zero, since CMC is not seen as a sharp “phase boundary” but is defined as the midpoint of a rather broad transition range.26 Therefore, for a more precise quantitation of the midpoint and the width of the micellization process, it is advantageous to plot the first derivative of the enthalpy change (ΔH) vs the [SDS] curve,27 as shown in the bottom panels of Figure 2A,B. Thus, the CMC value of SDS in pH 2 and 7.4 corresponding to the midpoint of inflection was found to be 2.16 and 3.10 mM, which agrees well with the reported literature.25,28,29 Furthermore, the CMC is also determined by conductivity measurements at pH 2 and 7.4 and is found to be 13027



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Figure 3. Molecular structure of BSF as obtained from molecular modeling by Phyre2. (A) The molecular structure of BSF showing its secondary structure. (B) molecular surface of BSF colored by amino acid hydrophobicity according to the Kyte and Doolittle scale. The most hydrophobic is orange-red; the most hydrophilic, blue. Tryptophan residue is shown in red.

3.5. Molecular Modeling. The BSF is modeled by the Protein Homology/analogY Recognition Engine (Phyre2) and visualized by Chimera1.7, as shown in Figure 3A,B. The obtained molecular structure and spatial location of Trp of BSF is utilized to understand the steady state and time-resolved fluorescence spectroscopic results. It is clearly shown that the Trp 51 is located on the outer region, that is, in a polar and nonconstrained environment, as in monellin, another member of cystatin family,14 and thus, it may show similar characteristic features in solution. Furthermore, to get an idea of the surface occupied by nonpolar amino acid residues, the solvent

Figure 2. Enthalpy changes determined by isothermal titration calorimetry to predict critical micellar concentration of SDS at (A) pH 7.4 and (B) pH 2. The upper panel in each figure represents the integrated heat per injection, normalized with respect to the injected number of moles of SDS (0−7 mM). The bottom panel represents the first derivative of the above transition curve.

2.22 and 3.16 mM, as shown in SI Figure S2, and is in agreement with the values obtained from ITC results.

Table 1. Assessment of Asparagine (N) and glutamine (Q) Deamidation in Tryptic Digested BSF after Incubation at pH 2 by LC−MALDI position of N and Q

amino acid sequence of peptides

theoretical mass

observed mass

deamidation

N31, Q21 N43, Q44 N43, Q44 N43, Q44 Q86, Q87, Q89, Q103, Q106 Q86, Q87, Q89 N202 N202, Q208 N202, Q208 N202, Q208, Q218 Q321 Q321

IPLDPVAGYKEPACDDPDTEQAALAAVDYINKHLPR HTLNQIDSVKVWPR GYKHTLNQIDSVKVWPR GYKHTLNQIDSVK QQTQHAVEGDCDIHVLKQDGQFSVLFTK QQTQHAVEGDCDIHVLK EVVDPTKCNLLAEK CNLLAEKQYGFCK EVVDPTKCNLLAEKQYGFCK EVVDPTKCNLLAEKQYGFCKGSVIQK HTFSGVASVESSSGEAFHVGKTPIVGQPSIPGGPVR TPIVGQPSIPGGPVR

3961.952 1691.911 2040.091 1501.789 3227.567 1976.937 1614.829 1629.764 2398.166 3010.526 3574.817 1473.83

3962.45 1692.097 2040.358 1501.954 3227.915 1977.184 1614.988 1629.931 2398.447 3010.959 3575.237 1474.029

no no no no no no no no no no no no

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Figure 4. Effect of SDS (0−2 mM) on BSF. (A) Turbidity profile of BSF at pH 2 (■) and pH 7.4 (●). (B) Rayleigh scattering profile of BSF at pH 2 (■) and pH 7.4 (●). (C) ThT spectra of BSF in the presence of varying concentration of SDS at pH 2. (D) ThT fluorescence intensity profile of BSF in the presence of varying concentration of SDS at pH 2 (■) and pH 7.4 (●).

presence of protein aggregation, Rayleigh scattering measurements were performed. 3.8. Rayleigh Scattering Measurements. Figure 4B shows the Rayleigh scattering profile of BSF at pH 2 and 7.4 in the presence of SDS. There is a prominent increase in the Rayleigh scattering at pH 2, whereas at pH 7.4, no significant increase was observed with an increase in the SDS concentration. It indicates the formation of protein aggregates at pH 2. The nature of the aggregates is further determined by ThT fluorescence and far UV−CD measurements. 3.9. Thioflavin-T Binding with Aggregates. A standard fluorescent dye, ThT, which experiences a marked enhancement in its fluorescence intensity upon binding to amyloid structures, was introduced as a regular characteristic molecular probe to detect the formation of amyloid fibrils.23 ThT has a low fluorescence quantum yield in solution, which increases considerably when bound to amyloid fibrils, displaying its high propensity to interact with amyloid fibrils.34 Figure 4C,D show ThT fluorescence spectra of BSF and the change in ThT fluorescence intensity at 484 nm in the presence of SDS (0−2 mM) at pH 2 and pH 7.4. As shown in Figure 4D, BSF has negligible ThT fluorescence intensity in the absence of SDS, but on addition of SDS, an increase in ThT fluorescence intensity occurs until 0.75 mM at pH 2, whereas no significant increase was observed at pH 7.4. It may be due to the fact that at pH 2, the protein exists in a partially unfolded form with exposed hydrophobic patches and positively charge residues.35 These charged residues exert the electrostatic repulsion between protein molecules and thus prevent aggregation. But when SDS interacts with BSF through its negatively charged head groups, then the hydrophobic tails of SDS on the protein−SDS complex become free. As a result, the protein− SDS complex interacts with each other and forms amyloid

accessible surface area was calculated from InterProSurf. It was found that of the total solvent accessible surface area of 19649.13 Å2, nonpolar amino acid residues occupy a maximal area (12895.87 Å2). The molecular surface of BSF colored by amino acid hydrophobicity according to the Kyte and Doolittle scale30 is shown in Figure 3B. Thus, it can be said that hydrophobic regions were distributed all over the protein molecule. 3.6. Charge on BSF. The total charges on BSF at pH 2 and pH 7.4 were found to be +37.8 and −14.5 as determined by Protein Calculator v3.4. The nature of the charge (negative or positive) agrees well with the result expected as an outcome of the fact that BSF below its isoelectric point (pI ∼ 5) acquires a net positive charge, whereas above it, a net negative charge, as all other proteins possess. 3.7. Turbidity Measurements. The turbidity of samples/ precipitates at a nonabsorbing wavelength can be used to rapidly detect insoluble protein aggregates.31 Thus, optical density at 350 nm (OD350) of BSF in the presence of submicellar concentrations of SDS in buffer of pH 2 (pH below pI) and 7.4 (pH above pI) was taken and is summarized in Figure 4A. It was observed that the solution of BSF in the presence of submicellar SDS concentrations has significant values of OD350 for pH 2, whereas they are insignificant for pH 7.4. Furthermore, it was observed that at pH 2, the turbidity increases in the presence of SDS up to 0.75 mM. It may be due to aggregation that occurs as a result of interaction of negatively charged SDS with positively charged BSF. Aggregation of the protein−SDS complex at pH values below the isoelectric point at low surfactant concentration is common and has been reported for the lysozyme−SDS system32 as well as for the bovine serum albumin−SDS system.33 However, to confirm the 13029



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fibrils, as indicated by the increase in the ThT fluorescence intensity. Conversely, at pH 7.4, BSF has a net negative charge and, thus, has less probability of interaction via a negatively charged headgroup and less propensity of the SDS hydrophobic tails to interact with each other and form aggregates. However, to confirm the nature of the forces in the interaction of SDS and BSF at pH 2 and 7.4, isothermal titration calorimetric measurements were performed. 3.10. Transmission Electron Microscopic Measurements. In parallel with ThT fluorescence and far UV−CD measurements, amyloid fibrils of BSF in the presence of SDS at pH 2 were clearly visible in transmission electron microscopic images; however, no such structure was observed at pH 7.4, as shown in Figure 5A,B respectively.

Figure 6. Isothermal titration binding isotherms at pH 2 of (A) BSF with SDS at a molar ratio of 1:20. (B) BSF in the presence of NaCl with SDS at molar ratio 1:20:20.

sites, to one with high affinity (K1 ∼ 105) and to other with low affinity (K1∼ 103). More SDS molecules interacts with the lower affinity binding sites and fewer with the higher affinity sites of BSF. Furthermore, from the thermodynamic parameters, enthalpy changes (ΔH°), and entropy changes (ΔS°), the forces involved in binding of SDS to proteins can be predicted.36 It can be seen from Table 2 that negative values of ΔH° for both sites are indicative of the electrostatic interaction for binding SDS to BSF at pH 2. Furthermore, the positive ΔS° also confirms the involvement of electrostatics interaction as it occurs at the expense of disruption of the hydrogen-bonding network between water molecules by the SDS molecule.37 The decrease observed in the binding constants on titration of SDS to BSF in the presence of equimolar NaCl to SDS, keeping other conditions similar, also confirms the involvement of electrostatic interaction at pH 2 because the ions of NaCl also compete for binding to the protein (Figure 6B and Table2). Figure 7A−C shows the binding isotherms of BSF to SDS up to a molar ratio of 1:20 in the absence and presence of NaCl and 1:180 in the absence of NaCl at pH 7.4. The titration of SDS to BSF up to a molar ratio of 1:20 leads to a binding profile that fits well to the two-independent-binding-sites models with high (K1 ∼ 105) and low (K1 ∼ 103) affinity sites (Figure 7A). In addition, positive values of ΔH° and ΔS° are indicative of hydrophobic interaction. However, by increasing the molar ratio of the titration of BSF to SDS to 1:180, the initial portion of the binding isotherm reduces. The binding isotherm fits best to a two-independent-binding-sites model, with each site binding to more than one molecule of SDS, as shown in Figure 7B. The parameters obtained after fitting are summarized in Table 2. The generated titration profile can be divided into three characteristic regions: (A) at low SDS concentrations, a small endothermic heat flow is observed until 0.48 mM with a inclination toward exothermic effects, followed by (B) a significant shift in heat flow from the endothermic to exothermic. (C) The exothermic heat flow then levels out at an intermediate concentration range of a molar ratio of 107 (approximately 2.14 mM SDS), and this ends with an upward bend. At still higher concentrations of SDS, the endothermic contribution increases, and the inflection point signifies that

Figure 5. Transmission electron microscopic images of BSF in the presence of 0.75 mM SDS (A) at pH 2 and (B) at pH 7.4.

3.11. Isothermal Titration Calorimetric Measurements. The thermodynamic parameters and binding affinity of SDS to BSF at pH 2 and 7.4 were measured using ITC and are presented in Figures 6 and 7, respectively. Figure 6 shows the ITC binding isotherms of SDS with BSF at pH 2, 25 °C in which each peak in the top panel represents a single injection of the SDS into the protein solution. The bottom panel of each figure shows an integrated plot of the amount of heat liberated per injection as a function of the molar ratio of the SDS to protein. These profiles are best fitted for the integrated heats having the lowest χ2 using a two independent binding sites model, with each site binding to more than one molecule of SDS. The binding and thermodynamic parameters of SDS with BSF that were obtained after fitting are summarized in Table 2. The results shows that at pH 2, the SDS binds to BSF at two 13030



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Figure 7. Isothermal titration binding isotherms at pH 7.4 of (A) BSF with SDS at molar ratio of 1:20. (B) BSF with SDS at molar ratio of 1:180. (C) BSF in the presence of NaCl with SDS at molar ratio 1:20:20.

Table 2. Thermodynamic Parameters Obtained by Isothermal Titration Calorimetric Measurements of BSF with SDS in the Absence and Presence of NaCl at pH 2 and pH 7.4a pH 2

a

pH 7.4

BSF: SDS: NaCl

1:20:0

1:20:20

1:20:0

1:20:20

1:180:0

N1 K1 (M−1) ΔH1° TΔS1° ΔG1° N2 K2 (M−1) ΔH2° TΔS2° ΔG2°

1 ± 0.02 (4.18 ± 0.3) × 105 −1.97 ± 0.07 5.62 −7.66 27 ± 1.70 (2.54 ± 0.02) × 104 −2.57 ± 0.02 3.42 −5.99

1.16 ± 0.3 (3.30 ± 0.52) × 105 −1.47 ± 0.02 6.12 −7.59 24 ± 1.61 (1.91 ± 0.17) × 104 −1.73 ± 0.02 4.02 −5.75

2 ± 0.2 (1.18 ± 0.39) × 105 2.28 ± 0.11 9.20 −6.92 17 ± 3 (8.75 ± 0.72) × 103 1.84 ± 0.11 7.21 −5.37

2.4 ± 0.3 (2.76 ± 0.25) × 105 4.72 ± 1.02 12.12 −7.48 21 ± 2 (8.91 ± 0.11) × 103 2.10 ± 0.04 7.47 −5.38

71 ± 3.0 (4.54 ± 0.92) × 104 0.11 ± 0.01 6.03 −5.92 104 ± 10 (1.98 ± 0.73) × 103 −0.91 ± 0.16 3.36 −4.27

Units of ΔH°, TΔS and ΔG° are expressed in kcal mol−1.

secondary structure changes occur in BSF, as shown in Table 3. Consequently, the obtained thermodynamic parameters are

there is a reduction in the protein−surfactant interactions. Results shows that the SDS binds to BSF at two sites, one with high affinity (K1 ∼ 104) and the other with low affinity (K1∼ 103). More SDS molecules interact with the lower affinity binding sites, whereas fewer interact with the higher affinity sites of BSF. The values of ΔH° and ΔS° were zero/slightly negative and positive, respectively, at both sites and may indicate the involvement of hydrophobic interaction or conformational alteration. Furthermore, the values of ΔGo were negative, so the interactions of SDS and BSF were spontaneous under all the conditions studied. Thus, it can be said that initially, SDS monomers bind to proteins predominantly by hydrophobic interactions at pH 7.4. Furthermore, to negate the role of electrostatic interaction at pH 7.4, the binding of SDS to BSF was studied by performing titration of SDS to BSF in the presence of equimolar NaCl to SDS, under similar experimental conditions. The binding constants for low- and high-affinity sites slightly change at pH 7.4 in the presence of NaCl, as shown in Figure 7C and Table 2. This behavior indicates that the SDS binding to BSF does not involve electrostatic interaction at pH 7.4 because the ions of NaCl do not compete for binding to the protein. The result that SDS interaction with BSF is hydrophobic in nature draws further support from the fact that at pH 7.4, BSF has a net negative charge and, thus, has fewer chances to interact with the negatively charged headgroup of SDS via electrostatic interaction.8 Furthermore, to confirm that the titration of BSF to SDS up to a molar ratio of 1:20 at pH 2 and pH 7.4 is not accompanied by structural changes, far-UV CD measurements were performed at the same molar ratio of BSF to SDS, that is, 0.1 mM SDS for 5 μM protein. Under these conditions, no

Table 3. Percent Secondary Structure Content of BSF in the Presence of SDS at pH 2 and 7.4 As Obtained by K2D3 Software of Secondary Structure Analysis pH 2

a

pH 7.4

SDS (mM)

% α-helix

% β-sheet

% α-helix

% β-sheet

0 0.1 0.25 0.5 0.75 1 1.5 2 2.5 3 3.5

6.45 6.45 5.53 5.28 3.61 4.18 4.63 6.08

34.51 34.52 38.58 42.35 43.59 43.26 41.99 35.78

10.00 10.05 11.51 13.00 15.52 17.07 19.51 21.84 24.28 26.88 28.24

34.00 34.00 32.40 30.00 28.03 26.95 25.17 23.10 21.56 20.22 19.61

Error is under 0.45.

associated only with the type of interaction involved. However, the thermodynamic parameters obtained on titration of BSF to SDS up to a molar ratio of 1:180 at pH 7.4 may contribute to conformational changes. 3.12. Far-UV Circular Dichroism Measurements. Circular dichroism spectroscopy was used to analyze the protein secondary structures in solution. Figure 8A,B shows the far-UV CD spectra of the SDS-induced secondary structural changes of BSF in submicellar concentrations of SDS at pH 2 13031



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Figure 8. Far-UV CD measurements. (A) Far-UV CD spectra of BSF in the presence of SDS (0−2 mM) at pH 2. (B) Far-UV CD spectra of BSF in the presence of SDS (0−3.5 mM) at pH 7.4. (C) Percent secondary structure content of BSF in the presence of SDS at pH 2. (D) Percent secondary structure content of BSF in the presence of SDS at pH 7.4.

Figure 9. Steady state fluorescence results of BSF in the presence of SDS (0−3.5 mM) at pH 7.4. (A) Fluorescence spectra of BSF in the presence of SDS. (B) Changes in emission maximum of BSF in the presence of SDS. (C) Fluorescence intensity profile of BSF in the presence of SDS.

and 7.4, respectively. The secondary structural contents of BSF in the presence of varying concentration of SDS at pH 2 and 7.4 were obtained by K2D3 software38 and are summarized in Table 3. The spectra of BSF at pH 2 and 7.4 in the absence of SDS have a minimum at 208 nm and a small shoulder at 222 nm. The essential absence of the proper 222 nm minimum suggests that the protein has a low α-helix content that agrees well with the literature reports12 as well as the K2D3 findings represented in Table 3. As shown in Figure 8A,B and Table 3, the spectral characteristic of BSF in the presence of 0.1 mM SDS (protein-to-SDS molar ratio of 1:20) remains the same at both pH values, an indication of no secondary structural alteration. Consequently, the thermodynamic parameters obtained from the titration of the protein to SDS up to a molar ratio of 1:20 by ITC truly predict the type of interaction. However, in the presence of higher SDS concentrations, the change occurs in the secondary structure of BSF at both pH 2 and 7.4. The BSF at pH 2 shows an increase in the β-sheet

content on increasing the SDS concentration until 0.75 mM that may be attributed to the formation of amyloid fibrils.34 These findings are in agreement with the results obtained from ThT and TEM measurements. However, on increasing the SDS concentration to 2 mM ∼ CMC of SDS, amyloid formation is suppressed as is evident from the decrease observed in the βsheet content. The same findings have been reported for the lysozyme− SDS system.32 Conversely, at pH 7.4, on higher SDS concentrations, the minima at 208 and 222 nm of BSF deepen with an increase in the percent of α-helical content and concomitant loss in β-sheet contents. However, the increase in the percent of α-helical content is steep up to 1 mM, followed by a gradual rise until 2 mM and beyond, at which point almost no change was observed, as shown in Figure 8C and summarized in Table 3. The increase in percent helicity might be contributed as a result of the hydrophobic environment generated as a result of the SDS alkyl chain 13032



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interaction around Trp 51of BSF, as reported earlier for other protein system.39−43 Therefore, to confirm it, intrinsic fluorescence spectroscopic measurements were performed. 3.13. Intrinsic Fluorescence Spectroscopic Measurements. Fluorescence spectroscopy is widely employed to study proteins and peptides.44 Among the three aromatic amino acids Trp, Tyr, and Phe, Trp has the highest fluorescence yield, which is influenced by the microenvironment due to the presence of an indole group.24 Thus, to get the information on the effect of SDS on BSF at pH 7.4, Trp was excited, and fluorescence intensities were taken at 340 nm. Furthermore, the changes in the wavelength of the emission maximum (λmax), a parameter sensitive to protein conformation, were also taken for analysis because the λmax depends on the environment of the tryptophanyl residues.45 BSF has a single Trp at position 51,12 located on the outer region as evident from molecular modeling results. The fluorescence emission spectra, changes in the wavelength of the emission maximum, and fluorescence intensity at 340 nm of BSF in the presence of SDS (0−3.5 mM) are shown in Figure 9A−C, respectively. As shown, on increasing the SDS concentration, there occurs a sharp decrease in the fluorescence intensity up to 1 mM, followed by a gradual decrease until 2 mM and thereafter, almost no change was observed. Furthermore, a decrease in the fluorescence intensity was accompanied by concomitant blue shift of 5 nm in λmax. It is noticeable that the changes in λmax also show the same pattern as that of fluorescence intensities on increasing the SDS concentration. The observed decrease in fluorescence intensities might be due to the interaction of SDS to Trp51, whereas a blue shift in λmax occurs as a result of the nonpolar environment around Trp and may be generated as a result of the alkyl chain of the SDS molecules. The pattern observed in the increment of the percent helical content has a direct correlation with the steady state fluorescence results, and thus, the hydrophobic environment generated around Trp 51 may induce secondary structural changes in BSF, as reported earlier for other protein systems.39−43 3.14. Time-Resolved Fluorescence Spectroscopic Measurements. To understand the interaction between SDS and protein, the local environment around the fluorophore (Trp) can be explored by measurement of the fluorescence lifetime.46 It is observed that at pH 7.4, in an aqueous solution, Trp exhibits multiple exponential decay, which has been attributed to the existence of conformers (conformational isomers) in determining lifetimes.47 Consequently, the timeresolved decay profiles of BSF at submicellar concentrations of SDS are fitted with biexponential fitting, as shown in Figure 10A. All the experimental decay curves are well fitted, as evidenced by the statistical parameters such as chi-square (χ2), which was close to 1, and the distribution of the weighted residuals. When using the biexponential decay law, it is often useful to determine the average lifetime (τavg), which is given by44,48

τ = α1τ1 + α2τ2

Figure 10. (A) Time-resolved fluorescence decay profile of BSF and (B) average fluorescence lifetime as a function of SDS. IRF indicates the instrument response function.

Table 4. Time-Resolved Fluorescence Decay Parameters of BSF in the Presence of SDS at pH 7.4 SDS (mM)

α1

τ1 (ns)

α2

τ2 (ns)

τav (ns)

0 0.5 1 1.5 2 2.5 3 3.5

27.16 30.21 40.10 40.72 43.18 43.91 43.97 45.22

1.48 1.46 1.36 1.26 1.20 1.14 1.14 1.14

72.84 69.79 59.90 59.28 56.82 56.09 56.03 54.78

4.91 4.88 4.86 4.64 4.61 4.38 4.27 4.31

3.97 3.84 3.45 3.26 3.13 2.95 2.89 2.87

fluorescence lifetime to another protein, monellin, of the same cystatin family.49 However, on addition of SDS, the average fluorescence lifetime first decreases until 2.5 mM, and thereafter almost no change was observed, as shown in Figure 10B and Table 4. The decrease in the fluorescence lifetime may be due to the transition among Trp conformers as well as the charge transfer process from the indole ring of Trp51 to a nearby substituent, which was enhanced on binding with SDS. Thus, to understand the contribution of an individual lifetime in multiexponential decay, an effort has been made by observing the relative amplitudes of lifetime. It is believed that Trp has three conformers: A, B, and C.47 Of these, conformer C is rather stable, and conversion from C to either A or B is difficult on a nanosecond time scale. Because Trp exhibits multiexponential decays in aqueous solutions, the faster component can be attributed to the conversion of rapidly interconverting A and B conformers into conformer C, whereas the longer lifetime mainly arises as a result of the conversion of

(5)

The decay parameters obtained are plotted against the SDS concentration, as shown in Figure 10B and summarized in Table 4. Trp51 of BSF has a fluorescence lifetime value of 3.97 ns, which indicates it is present in both polar and nonconstrained environment, which agrees well with molecular modeling results. That the Trp of BSF is exposed to a solvent environment is further confirmed by comparable values of its 13033



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Figure 11. Mode of interaction of SDS with BSF at pH 2 and 7.4.

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conformer C into rapidly interconverting A and B conformers.50 As evident from the relative amplitude values from Table 4, the contribution from relatively stable conformer C (shorter lifetime) and rapidly interconverting conformer A and B (longer lifetime) of BSF are 27.16% and 72.84%, respectively. But because of an increase in the secondary structural content on increasing SDS concentrations to 2.5 mM, Trp51 experiences a nonpolar constrained environment. As a consequence, the conversion from conformers C to A/B decreases the increase of the relative amplitude of the shorter lifetime. Furthermore, beyond 2.5 mM, not much change in the secondary structure was observed; thus, no change is seen in the relative amplitude. Thus, the change in the secondary structure may be associated with the transition among Trp conformers. Furthermore, a small contribution of dynamic quenching cannot be neglected in the decrease of the fluorescence lifetime in the presence of SDS.

ASSOCIATED CONTENT

S Supporting Information *

Figures showing SDS−PAGE and conductivity profiles for critical micellar concentration determination. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91-571-2720388. Fax: + 91-571-2721776. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University is acknowledged for providing mass spectrometry facilities. The comments of reviewers that enabled us to improve the manuscript are also highly appreciated. N. Zaidi acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing financial assistance in the form of a Senior Research Fellowship (SRF).

4. CONCLUSIONS The mode of interaction of anionic detergent, SDS with BSF in subcritical micellar concentrations is different at pH 7.4 and pH 2 and is shown in Figure 11. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically through its alkyl chains at pH 7.4 up to 1:20 molar ratio of BSF to SDS. No conformational alterations were observed in BSF at both pHs up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2 but induction of an α-helical structure at pH 7.4. The increase in the α-helical content on increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of conformers decreases and results in a decrease in the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

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pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl

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