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Protein-Protein Interaction Probed by Label-Free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study Kamini Mishra, Abhijit Chakrabarti, and Puspendu Kumar Das J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04503 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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The Journal of Physical Chemistry

Protein-protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study Kamini Mishra,a Abhijit Chakrabarti b and Puspendu K. Das*,a a

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India b

Crystallography & Molecular Biology Div., Saha Institute of Nuclear Physics, HBNI, 1/AF Bidhannagar, Kolkata 700 064, India.

ABSTRACT In this article, we have studied the binding of different naturally occurring hemoglobin (Hb) variants on erythrocyte skeletal protein, spectrin surface using the label free non-destructive second harmonic light scattering (SHLS) technique in aqueous buffer. Hemoglobin variants like sickle hemoglobin (HbS) and hemoglobin E (HbE) were chosen as they associate with sickle cell disease and HbEβ-thalassemia, respectively and their interaction with spectrin is compared with normal adult hemoglobin (HbA). The concentration dependent change in the second harmonic light intensity from nanomolar spectrin solution has been measured after addition of small aliquots of hemoglobins. From the second harmonic titration data, the binding constant is calculated using a modified Langmuir adsorption model of hemoglobin binding to the spectrin surface. Interestingly, it is found that the binding constant for HbE (13.8x108 M-1) is one order of magnitude higher than that of HbS (1.6x108 M-1) or HbA (2.1x108 M-1) which indicates higher affinity of HbE for spectrin compared to HbA and HbS. The number of the Hb molecules bound to the spectrin surface was estimated to be of the order of hundred’s which is determined for the first time.

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INTRODUCTION Many biological processes involve specific protein-protein interaction and protein’s functions are often modified by this interaction.1 For identification and characterisation of these interactions, researchers have used spectral method derived from graph theory to analyse hidden topological structures in evaluating protein-protein interactions. This spectral method has also been used to evaluate the interaction of the cytoskeleton protein spectrin with hemoglobin in erythrocytes, the red blood cells of the circulating blood.2 However, not much insight on the nature of the interaction or physical properties of the spectrin-Hb binding could be obtained from this work. The bioinformatics method is far from perfect and produces false positives. Several other reports indicated the association of spectrin with hemoglobin under different experimental and physiological conditions.3-8 Spectrin membrane skeleton offers elasticity and flexibility to the red blood cells so that it passes through the narrow vessels and capillaries. Hemoglobin (Hb), an iron-containing oxygen-transport protein, is the most abundant protein in the human erythrocytes over 90% of the cellular dry weight. Hemoglobin is water soluble, globular, heterotetrameric protein consisting of two α and two β type globin chains, having molecular mass of 64.5 kDa. Hb variants are abnormal forms of hemoglobin which occurs due to mutation in the globin chains. These changes affect the structure of Hb, its behaviour, its production rate, and/or stability. As one of the most important proteins of the living systems, the interactions of Hb with spectrin become an obvious subject of investigation. Hemoglobin E is the most common Hb variant in the world which is caused by single mutation in the β-globin gene (β 26 Glu →Lys) and associates with β-thalassemia giving rise to one of the most prevalent hemoglobin disease, HbEβ-thalassemia.9 In sickle cell disease (SCD), a point mutation occurs in the β-globin gene on chromosome 11 replacing thymine with an adenine causing a single amino acid substitution from β 6 Val → Glu.10 Spectrin is composed of two large worms like non identical subunits α and β having 2 ACS Paragon Plus Environment

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molecular masses 240 kDa and 220 kDa, respectively. It is a double stranded fibre like heterodimer, formed by the lateral association of α and β monomers which then associate in a head-to-head fashion to form a tetramer giving mechanical flexibility to the erythrocytes. 10, 11 Binding of hemoglobin to spectrin was reported from gel filtration data of spectrin hemoglobin mixture but it was contradicted by experiments of spectrin fluorescence quenching by hemoglobin. Recent fluorescence quenching experiments by Chakrabarti et al. 12

established that both HbA and HbE bind to spectrin with different binding constants. Van

gestel et al.13 used gel electrophoresis and LC-MS/MS to determine erythrocyte spectrin levels in hemolytic anemia patients and found that spectrin level was dramatically decreased but the binding constant or parameters were not reported. Until now, mainly in silico protein– protein interactions inside erythrocyte have been predicted and only a small number of these have been validated by large scale experiments.14, 15 In this article, we have used label free second harmonic light scattering (SHLS) or hyperRayleigh scattering (HRS) to probe the binding of spectrin dimer and tetramer with different Hb variants. Proteins give the large second harmonic signal due to their charge anisotropy within the length scale. Incoherent SHLS arises from the instantaneous fluctuation of molecular dipoles within the time and space window. The intensity of SH signal depends on the nature of the anisotropic charge distribution and the degree of asymmetry. If a solute dissolved in a noninteracting solvent scatters SH light from solution (I2ω), the observed SH signal can be written in terms of the molecular hyperpolarizabilities of the solute and the solvent as I 2ω Iω 2

= g  N1β 21 +N 2 β 2 2 



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Where N's are the number densities of the solute and the solvent, respectively and β's their molecular hyperpolarizabilities, g is the instrument factor and Iω is the incident light intensity. However, when a protein molecule interacts with another protein molecule in solution, the second harmonic light generated by the protein-protein complex could be either higher or lower than that of the sum of the individual responses in the same solvent. If they are all noninetracting the SH signal should be the sum of the contributions from them separately. Thus the absence of additivity of the SH signal generated from them could indirectly indicate that they are interacting. In case of two proteins interacting with each other in solution, the interaction leads either to more anisotropic or isotropic charge distributions within the protein-protein complex. We have earlier used SHLS to study two types of binding between HSA and bilirubin.16 Most recently, Ma et al. reported large hyperpolarizability from bacterial light harvesting complexes.17 But till date, there is no report on application of this technique to study protein-protein interaction. We have used modified Langmuir model to extract the number of Hb molecules which bind to the dimeric and tetrameric spectrin. EXPERIMENTAL Materials Blood samples collected in EDTA vials (BD Biosciences), from normal healthy volunteers were used for purification of HbA. Blood samples from homozygous HbE and HbS patients were obtained from Ramakrishna Mission Seva Pratisthan Hospital, Kolkata, India with informed written consent of the patients following the guidelines of the Institutional Ethical Committee, elaborated earlier.18 Blood samples were analyzed by BioRad Variant HPLC system and the hemoglobin variants, HbE and HbS were characterized and were used for purification of HbE and HbS. Erythrocytes after removal of the buffy coat and plasma, were extensively washed with phosphate buffered saline (5 mM phosphate, 0.15 M NaCl, pH 7.4). 4 ACS Paragon Plus Environment

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Hemoglobin was isolated from packed erythrocytes by osmotic lysis using three volumes of 1 mM Tris, pH 8.0, at 4°C for 1 h and further purified by gel filtration on Sephadex G-100 column (30×1 cm) in a buffer containing 5 mM Tris, 50 mM KCl, pH 7.4, stored in oxy-form at -70°C for less than 7 days and characterized by the measurements of absorption at 415 nm and 541 nm, respectively. Hb concentrations were determined spectrophotometrically using molar extinction coefficient of 125000 M-1cm-1 at 415 nm and 13500 M-1cm-1 at 541 nm respectively.19 Both dimeric and tetrameric spectrin were isolated and purified from white erythrocyte ghosts from ovine blood by hypotonic lysis in 5 mM phosphate, 1.0 mM EDTA containing 20 g/ml PMSF at pH 8.0. Dimeric spectrin was isolated at 37°C in presence of 20 mM KCl and tetrameric spectrin at 4°C in presence of 150 mM KCl. Spectrin concentrations were determined spectrophotometrically using an ε1% value of 10.7 at 280 nm i.e., an absorbance of 1.07 at 280 nm for a solution of 1mg spectrin per ml.20 Methods Spectrin solutions were prepared in 10 mM Tris-HCl buffer (pH 8, 20 mM KCl) and Hb variants were prepared in 1mM Tris-HCl buffer (pH 8, 10 mM NaCl). MilliQ water was used for measurement throughout. Hb concentration was always kept above 10-5 M since above this concentration Hb is present mostly in its tetrameric form at 20-27°C.21 Spectrin concentrations were kept at 20 nM for dimeric spectrin experiments and at 10 nM with tetrameric spectrin in 150 mM KCl. Absorption spectra of spectrin and Hb variants were obtained by using Perkin-elmer lambda-750 UV-Vis spectrophotometer with a slit width of 2 nm. Spectrin showed an absorption peak at 280 nm due to π-π* transition which is characteristic of the 84 tryptophan residues. In case of Hb two peaks were observed at 415



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(not shown) and 500-600 nm which are the characteristic of the Soret band and the Q band (which generally appears as a doublet), respectively of the porphyrin present in hemoglobin. Second harmonic light scattering measurements (SHLS) SHLS experiments were done by using the fundamental wavelength (1064 nm) from a Qswitched Nd-YAG laser as the incident source having 10 ns pulse-width and 10 Hz repetition rate. The incident laser power was kept below 10 mJ/pulse to prevent degradation of proteins. The incident beam power was varied by using a polarizer and simultaneously measuring the intensity through a photo diode. The beam steered by several optics was focused onto a cylindrical sample vial (diameter 2.5 cm) of volume 20 ml, using a converging lens of focal length 20 cm ahead from the centre of the vial. The incoherent second harmonic light (532 nm) was collected by a visible photomultiplier tube (PMT) at a perpendicular direction after passing through a set of collection optics and an interference filter. The output signal from the PMT was digitized and fed into a 500 MHz digital storage oscilloscope which stores the signal after averaging over 512 shots.

Initially the experiment was done with a

monochromator and wavelength scan showed that there was no two-photon or multiphoton fluorescence from either spectrin or hemoglobins in solution. Then we switched to the filter set-up where it was much easier to do the experiments at low protein concentrations. A quadratic power dependence of the SH signal was done with para-nitroaniline in water as a standard and it is found to be quadratic. Spectrin concentrations were fixed at 20 nM in all SH titrations for dimeric and at 10 nM for tetrameric spectrin. Small aliquots (20 µL) of freshly prepared Hb variants were added to spectrin solution. RESULTS AND DISCUSSION The large second harmonic signal observed from rod like spectrin dimer is due to charge anisotropy in the protein. On the other hand Hb is spherically symmetric and thus in the 6 ACS Paragon Plus Environment

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electric dipole approximation, the SH signal from Hb is expected to be zero and that is what is found. As we are looking at the SH signal, a quadratic power dependence check was done with para nitroaniline in water and then with spectrin (see Supporting Information). After the power dependence check, 20 nM spectrin was taken in a glass vial and small aliquots of 20 µL of Hb were added to the dimeric spectrin solution. A considerable decrease in the SH intensity of spectrin was observed until it reached a point when no further quenching of SH signal was seen. The decrease must be due to the spectrin-Hb interaction which indicates the formation of a protein-protein complex having a more symmetric charge distribution leading to a decrease in the SH scattering intensity compared to spectrin. Spectrin has a rod like, highly anisotropic structure that is 100 nm long in its dimeric form and 200 nm long in its tetrameric form. When hemoglobin binds to spectrin, the structural anisotropy is reduced and the cylindrical structure becomes more spherical in nature which leads ultimately to the reduction of the SH signal. The titrations were done with the other two Hb variants in the same way and we found that HbE and HbS quenched the SH signal but with different efficiencies. The observed SH intensity decreased by 75% with HbA and HbS but only by 50% with HbE.

Figure 1. Absorption spectra of Hb Variants in Tris-HCl buffer at pH 8 Since Hb variants have absorption due to the Q band of porphyrin (500-660 nm) (Fig. 1) around the second harmonic wavelength, absorption correction to the second harmonic signal 7 ACS Paragon Plus Environment


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was done. We carried out the titration in micro molar or submicromolar concentrations where there is very little absorption as shown in Fig. 1. Absorption correction of the SH data was done by using the Beer-Lambert law, with I cor = I 2ω 10ε 2ω cl where ε2ω is the extinction coefficient of Hb at the second harmonic wavelength.22 Fig. 2 shows the SH titration plot after absorption correction. It is clear from the plot that there is no significant change in the absorption corrected and uncorrected SH signal.

Figure 2. Plot showing the normalized SH intensity of spectrin uncorrected (black) and corrected (red) for HbA absorption at 532 nm. The decrease in the SH intensity of spectrin is due to adsorption of Hb molecules on the surface of spectrin. Both spectrin and Hb have negative charges at pH 8 but HbE has more positive potential only at the mutation site compared to HbA.23 So, interaction between spectrin and HbE could be electrostatic in nature and hence stronger than HbA or HbS. Recently, adsorption of organic molecules and proteins on the surface of metal nanoparticle 24-27

has been studied using the label free SHLS in solution by using a modified Langmuir

model where decrease in second harmonic signal is related to the concentration of Hb variant as,

I SH

 55.5 55.5 2  (C + N max + ) − (C + N max + ) − 4CN max K K  = 1−Q '  2 N max  

     

2

(1)


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Where ISH is the normalized SH scattered light intensity, Q' is a quenching factor that represents the extent of quenching of the signal by the Hb variants, Nmax is the maximum number of binding sites per unit volume, C is the total concentration of the Hb molecules per unit volume, K, the equilibrium binding constant and 55.5 is the molarity of water. The experimental data points were fitted by using eq 1 for all the variants (shown in Fig 3 (a)) with three adjustable parameters. From the fit we extracted the equilibrium binding constant, K, the free energy change, ∆G° and the number of Hb molecules bound to the spectrin surface (Nmax /Nspectrin ) which are listed in Table 1.

(a) 20 nM Spectrin dimer

(b) 10 nM Spectrin tetramer

Figure 3. Normalized SH intensity at 532 nm from (a) 20 nM spectrin dimer, (b) 10 nM spectrin tetramer in 10 mM, 20 mM KCl pH 8 Tris-HCl buffer as a function of addition of Hb variants. Each data point was recorded after 4-7 minutes of Hb variants addition when the second harmonic signal became invariant with time. The SH intensity was normalized with respect to the SH intensity from a pure spectrin solution before addition of Hb variants. Table 1. Thermodynamic parameter for binding of spectrin dimer with different Hb variants where subscript ‘d’ refers to the dimer. (Data in parenthesis shows the same for the tetrameric spectrin) Hb

Kd (108 M-1)

∆Gd° (kcal/mol)

Ndmax (10-6 M)

ndsat

ned (emprical)



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HbE

13.4±0.01

-12.4 ± 0.01

2.2 ± 0.16

110 ± 16

(16.0± 0.01)

(-12.5 ± 0.01)

(2.2 ± 0.17)

(220 ± 17)

-11.5 ± 0.4

3.5 ± 0.20

175 ± 20

(2.4 ± 1.7)

(-11.4 ± 0.2)

(3.0 ± 0.27)

(300 ± 27)

1.6 ± 0.6

-11.2 ± 0.3

2.5 ± 0.23

125 ± 23

(1.8 ± 1.0)

(-11.3 ± 0.4)

(2.0 ± 0.31)

(200 ± 31)

HbA 2.1 ± 0.8

HbS

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56 (110)

65 (128)

-

From the binding data listed in Table 1, it is interesting to note that the binding constant of HbE is one order of magnitude higher than that of HbA or HbS indicating stronger binding of HbE. The standard Gibb’s free energy change for the binding of Hb variant with spectrin was calculated by using thermodynamic equation (∆G° = - RTlnK). The number of Hb molecules bound to spectrin at saturation (nsat=Nmax/ Nspectrin) is shown in Table 1. The data imply that there is less number of binding sites for HbE on dimeric spectrin than either HbA or HbS indicating a probable increase in size and distortion in shape of HbE from that of HbA. This is surprising since all three variants, HbA, HbS and HbE are structurally identical, as found from the crystal structures. Structural comparison of adult human HbA, HbS and HbE do not show considerable differences in their overall dimensions. Therefore, observed changes in the binding parameters could not be explained merely on the basis of their size. However, surface charge distribution of these Hbs clearly shows an extra positive patch on HbE due to its mutation from Glu → Lys, compared to HbA or HbS.23 Spectrin surface at pH 8.0 is predominantly negative. The additional positive patch of HbE is perhaps responsible for stronger binding to spectrin observed in our experiments. However, structural changes accompanied with such binding has prevented more HbE molecules to be adsorbed on the spectrin surface leading to less quenching of the SH signal. In other words, the strength of the


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binding is determined by the strength of the electrostatic interaction between the various Hbs while the decrease in the SH signal indicates the number that is accommodated on the spectrin surface. In order to test our binding model further we carried out the same set of experiments with tetrameric spectrin. From dimer to tetramer the surface area is expected to double and as a consequence the number of hemoglobins that will bind to the spectrin surface should also double. To verify this, SH scattering signal from tetrameric spectrin was followed as small aliquots of Hb were added to the solution of the tetramer. The experimental data shown in Fig. 3b were fitted to the modified Langmuir adsorption isotherm model described in equation (1). The values of K, Nmax, and ∆G° for the tetramer were obtained from the fit and are also listed in Table 1. The number of Hb molecules bound to the spectrin tetramer surface became double which is expected from the basis of surface area consideration alone. Considering spectrin dimer as a cylinder of length 100 nm and diameter 5 nm

10

if we calculate the number of Hb molecules

which can physically be accommodated on the surface of spectrin dimer or tetramer using a close-packed arrangement as shown in Fig. 4, we obtain the numbers (See supporting information) of HbE and HbA adsorbed on the spectrin surface. Considering that normal hemoglobin variant HbA having a diameter of 5 nm 28, 56 units of HbA are adsorbed on the spectrin dimer and 110 on the spectrin tetramer.



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Figure 4. Schematic showing the adsorption of Hb on the surface of tetrameric spectrin having 200 nm length and 5 nm diameter. We have taken the average diameter of HbE as 5.4 nm

23

and obtained the numbers of HbE

adsorbed on the spectrin dimer and tetramer, as 65 and 128, respectively. Therefore, the number of Hb molecules that are estimated to be adsorbed on the spectrin surface obtained from a crude geometrical consideration falls in the range obtained from the SHLS experiments using a modified Langmuir adsorption model. This agreement points out that the number of Hb variants interacting with spectrin is realistic and differential binding of the Hb variants points out the sensitivity of the SHLS technique in detecting such interactions which from the measured free energy change appear to be weak.

CONCLUSION The binding constant and free energy of spectrin-Hb interaction have been studied by label free SHLS. The consistency of the number of Hb molecules adsorbed on spectrin surface as estimated from the measurements in dimeric and tetrameric forms of spectrin and a empirical geometrical estimate of the same based on close-packing provides the confidence that the useful in deciphering protein-protein interaction. Although we have studied one pair of protein-protein interaction by this method, the application of the technique in other such systems should be straight forward and simple. Differential association of HbE with spectrin observed in our experiments indicates toward functional implications on hemoglobin interactome29 and pathophysiology of HbEβ-thalassemia, particularly in determining the severity of the disease where a tighter binding between HbE and spectrin could cause differential sequestration of HbE in the erythrocyte membrane leading to hemolysis. Specific interaction between the two proteins at the interaction site on the surface and its local


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environment definitely play important roles in manifestation of the malfunctioning of the mutated hemoglobin variants.

ASSOCIATED CONTENT Supporting information The Supporting information is available free of charge on the ACS publication website at DOI: Power dependence plots of standard pNA in water and spectrin dimer in water are shown in the supporting information (figure S1). A description and comparison of Langmuir and Modified Langmuir model for adsorption of HbE on spectrin dimer is shown in figure S2. Also description of number of Hb molecules which are adsorbed on the surface of spectrin were calculated geometrically (figure S3).

AUTHOR INFORMATION Corresponding author * Email: [email protected] Phone number: +918022932662

fax: +918023600416

ACKNOWLEDGMENTS PKD thanks the Ministry of Communication and Information Technology for the Centre of Excellence in Nanoelectronics, Phase II grant for generous funding of this work.

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Hemoglobin Adsorption on Spectrin Surface a - ACS Publications

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