Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT

Materials and Reagents

1. Bare gold sensor disk (Metrohm Autolab, catalog number: 1-04-04-000)
2. 384-well Clear Flat Bottom Polystyrene Not Treated Microplate, with Lid, Sterile (Corning, catalog number: 3680)
3. 6-well Clear Not Treated Multiple Well Plates (Corning Costar, catalog number: 3736)
4. Immersion oil
5. Demineralized (demi) water (Sigma-Aldrich, catalog number: 38796)
6. Mercaptoundecanoic acid (11-MUA) (Sigma-Aldrich, catalog number: 450561, MW 218.36 g/Mol)
7. N-Hydroxy Succinimide (NHS) (Sigma-Aldrich, catalog number: 130672, MW 115.09 g/Mol)
8. Dimethylaminopropyl-N′Ethylcarbodiimide N-3-hydrochloride (EDC) (Sigma-Aldrich, catalog number: 03450, MW 191.70 g/Mol)
9. Ethanolamine (Sigma-Aldrich, catalog number: E6133, MW 97.54 g/Mol)
10. Sodium acetate trihydrate (Sigma-Aldrich, catalog number: S8625, MW 136.08 g/Mol)
11. Acetic acid (Sigma-Aldrich, catalog number: A6283)
12. HEPES sodium salt (Sigma-Aldrich, catalog number: H7006, MW 260.29 g/Mol)
13. Ethanol, ≥ 99.9% (Merck, catalog number: 100983)
14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653, MW 58.44 g/Mol)
15. Sodium hydroxide (NaOH) (Merck, catalog number: 1.06498, MW 40.00 g/Mol)
16. Hydrochloric acid (HCl), 37% (Merck, catalog number: 100317)
17. Tween 20 (Sigma-Aldrich, catalog number: P1379)
18. Nitrogen gas (Sigma-Aldrich, catalog number: 295574)
19. 11-MUA solution (see Recipes)
20. 400 mM EDC (see Recipes)
21. 100 mM NHS (see Recipes)
22. 1 M Ethanolamine (see Recipes)
23. 1 M Acetate buffer (see Recipes)
24. 1 M HEPES buffer (see Recipes)
25. 5 M NaCl (see Recipes)
26. 1 M NaOH (see Recipes)
27. 10% Tween 20 (see Recipes)
28. Buffer 1: Coupling buffer (see Recipes)
29. Buffer 2: Association buffer (see Recipes)
30. Buffer 3: Regeneration buffer (see Recipes)
31. Protein solutions (see Recipes)
    

Equipment

1. Tweezers
2. Pipettes
3. Autolab SPR ESPRIT (Metrohm, model: Autolab ESPRIT)
4. Tabletop high-speed centrifuge (Eppendorf, model: 5430R)
5. Filtration unit (Milipore, model: XX1514700, and simple Syringe filters)
   

Software

1. Autolab Data Acquisition program
2. Autolab Kinetic Evaluation program
   

Procedure

1. Preparation of the gold disk sensor (Figure 5)
   1. Place the bare gold disk carefully in a 6-well plate using a pair of tweezers, keeping the gold surface facing upwards.
   2. Add the solution of 11-MUA (Recipe 1) and cover the plate carefully.
   3. Incubate the disk overnight in this solution to obtain a uniform layer of thiols over the gold surface. The plate is covered with foil and incubated at room temperature.
   4. Next day, wash three times with ethanol to remove excess thiol groups. For this, pipette out the 11-MUA solution and pipette in ethanol. The volume of the ethanol should be just enough to completely immerse the disk. Gently shake the plate to rinse the disk.
   5. To remove ethanol, wash thrice with demineralized water, while gently shaking the plate for rinsing the gold disk.
   6. After thorough washing, blow-dry the disk by flushing the well with nitrogen gas.
   7. These disks can now be stored up to 2 months in their container at room temperature.
      
      
      Figure 5. Preparation of the gold sensor disk. The diagram explains all the steps for the Steps A1 to A7 as mentioned in the text.
      
      
2. Setting up the sensor disk (Figure 6)
   1. Put a small drop of the immersion oil on the outer edge of the hemi-cylinder.
   2. Place the 11-MUA coated gold disk on the other end using a pair of tweezers. Now, simply push the disk carefully by sliding till it is correctly set on the hemi-cylinder.
   3. Make sure the gold surface is facing upwards.
   4. No air bubble should be present between the disk and the immersion oil on the hemi-cylinder.
      
      
      Figure 6. Setting up the gold disk on the hemi-cylinder. B1. The hemi-cylinder is prepared by adding a drop of immersion oil on it. B2. The coated gold disk is placed on the oil drop and slid through using a pair of tweezers.
      
      
      
3. Immobilization of the protein (Figure 7)
   1. Pass Buffer 1 (Coupling Buffer) over the modified gold disk. This hydrates the gold surface and stabilizes the thiol layer (Recipe 10).
   2. In the 384-well microplate, dispense out all the solutions required for the immobilization procedure into the wells of the column 1 as marked in Figure 7.
   3. Add 80 μl EDC (400 mM) in wells 1A and 1B, followed by 80 μl of NHS (100 mM) in both the wells 1C and 1D.
   4. Leave 1E and 1F blank for mixing of EDC and NHS. This mixture will activate the gold sensor disk for ligand protein immobilization.
   5. Add 80 μl of the ligand protein (example: 5 mg/ml of EhP3) in both 1G and 1H wells.
   6. Finally, add 80 μl of Ethanolamine (1 M) in 1I and 1J, to be used for blocking the remaining unbound area on the sensor.
   7. Place the 384-well plate onto the rack as shown in Figure 1.
   8. Use the following steps for setting the data acquisition times in the Data Acquisition Software:
      

      Steps	Time
      Baseline (Coupling buffer)	120 s
      Activation (EDC/NHS)	300 s
      Coupling (Ligand protein)	900 s
      Blocking (Ethanolamine)	600 s
      Regeneration (coupling buffer)	120 s

      
      
      Figure 7. 384-well plate used in this protocol. Each well used in the plate is marked here.
      
      
4. Interaction studies
   1. In column 2 of the 384-well microplate, add different dilutions of the Analyte, i.e., protein (or different proteins) to be used for determining their association kinetics.
   2. In well 2A, add 80 μl of Analyte 1 (example: Filamin) [or one dilution (1 μM) of a single protein (example: EhCoactosin)] followed by 80 μl of Buffer 2 (Association Buffer) (Recipe 11) in well 2B as reference.
   3. Similarly, add 80 μl of all the Analytes (examples: Actophorin, Profilin A, Profilin B, Twinfilin, EhCoactosin) or (examples: 2 μM, 4 μM, 8 μM, 10 μM of EhCoactosin) into alternative wells, followed by Buffer 2 (Association buffer) in each of the consecutive wells.
   4. Use Buffer 2 as the running buffer for the assay.
   5. Use the following incubation times for interaction studies:
      

      Steps	Time
      Baseline	120 s
      Association	300 s
      Dissociation	300 s
      Regeneration	120 s

   6. The SPR assays also use controls like any other protein-protein interaction study. Bovine Serum Albumin generally serves as the negative control, i.e., unless the protein in focus binds to it specifically. BSA normally does not bind majority of the proteins. Similarly, if any known interacting protein is known, then it can be used as the positive control. Further, the buffers used in the study should be compatible to all the proteins. This can be checked by diluting a small amount of the proteins in all the buffers to be used, and check them for any precipitation.

Data analysis

Use the AutoLab Kinetic Evaluation software to obtain the ‘Differential curves’ (Figure 8). Use these to determine the K_on and K_off, along with with the affinity constant KD. The strength of interaction between two molecules is said to be stronger if the display a smaller KD value. The higher value of KD signifies weaker attraction between the interacting molecules. K_on represents the association constant with units in min^-1 multiplied by concentration^-1. K_off is the dissociation constant with units in min^-1. Affinity constant is determined by the ratio of K_off/K_on in Molar units.
The Video 1 can be seen for data analysis steps.

1. Open the Kinetic Evaluation Software.
2. Follow the steps shown in the Video 1.
   
   
   
   Video 1. Steps to use the Kinetic Evaluation Software
   
   
3. Select the folder where the files from the Data Acquisition software have been saved.
4. In the folder, select all the series of injections recorded for the protein interaction study.
5. On the left hand top corner, under the heading ‘Create Files’, click New Overlay and select all the injections to be used for the kinetic analysis.
6. After this click on Signal Processing tab on the left hand bottom corner. Click on zoom and select the region of the sensogram before the association curve. Select Synchronize under the drop down tab of ‘Actions’. Next, click on ‘Normalize’ under the same tab and apply.
7. To remove the regeneration curves, click on ‘Delete Selection’ and select the region after the dissociation curve.
8. Again click on Normalize.
9. Close the tab, and open New project under ‘Create Files’ and save the data with appropriate name.
10. Next, open the Analysis wizard, select Equilibrium, click next. After this feed in the respective concentration.
11. Finally, click next and the calculations for the kinetic parameters can be performed successfully.
    
    
    Figure 8. SPR analyses of cytoskeletal proteins to immobilized EhP3. A. Surface Plasmon Resonance was used to determine the interaction between EhP3 and cytoskeletal proteins like Filamin, Actophorin, Profilin A and B, Twinfilin, and EhCoactosin. EhP3 was immobilized covalently through amide coupling onto the gold sensor. Shown here are the sensorgrams of various proteins injected at 200 nM concentrations. B. Based on the sensograms observed in (A), we have depicted the interaction in this cartoon model, in which, only EhCoactosin showed binding to EhP3. The other proteins could not bind to the immobilized protein EhP3. C. Shown here are sensorgrams of EhCoactosin injections of increasing concentration. The equilibrium binding curves were used to derive the affinity constant KD. The concentrations of recombinant His₆-Ehcoactosin are indicated alongside the binding curves

Notes

1. The ideal buffer for proteins to be used for SPR studies would be HEPES, but if the protein stability is an issue, then keep the concentration of any other buffer to a maximum of 50 mM.
2. EDC and NHS should be preferably freshly prepared as they are not very stable in solution. If prepared in larger quantity, one should store them as 300 μl aliquots in -20 °C for as long as thawed next time. However, it is not recommended to store them after thawing once.
3. Preparation of the ligand in acetate buffer should always be done fresh. Moreover, the same acetate buffer stock should be maintained during the entire immobilization procedure.
4. The pH of the coupling buffer can vary in case of different proteins. A general equation can be used to determine the pH of the coupling buffer: pH (buffer) = pI (ligand protein) - 0.5. pI is the isoelectric pH of the protein at which all its negative charges are equal to all its positive charges, making it electrically neutral. It can be theoretically calculated for any protein with known amino acid composition.
5. There exists a linear relationship between the shift in SPR angle with the amount of the bound material. 120 m° change represents a change in surface protein mass of ~1 ng/mm².
6. Most kinds of organic solvents are not recommended for this machine, kindly refrain from using those in any of the sample preparations.
7. Acetate buffer, with its lowest refractive index, serves as the best buffer for the immobilization process, as it gives the smallest SPR angle. Ethanolamine solution records the largest SPR angle.
8. For any new protein-protein interaction, normally one can decide the concentration by referring to the available data on those proteins, if any. Otherwise, one can begin by taking a wide range of concentrations, example 50 nM-500 nM. If this range gives no information, one can increase the range up to 2 μM. Based on these reaction curves, ideal concentrations can be chosen for the particular set of proteins.
9. Maintenance of the instrument:
   1. Hemi-cylinder should be regularly cleaned due to inevitable spillage of the immersion oil. As shown in Figure 2, M3X3 screw should be carefully removed and the hemi-cylinder should be cleaned with either ethanol or in an ultrasonic water bath. To dry the apparatus, ONLY lens tissue must be used. After the cleaning and drying, the hemi-cylinder must be placed and screwed back on the slider.
   2. In order to avoid any evaporation of solution from the cuvette, cover it with Para film.
   3. Always clean the instrument with distilled water. Use of any kind of buffer will lead to salt deposition and thus rendering the coating useless.
   4. The syringe pump seals should be changed on a yearly basis for optimum performance of the instrument.

Recipes

1. 11-MUA solution
   11 mg mercaptoundecanoic acid
   50 ml ethanol
   Filter sterilize the solution for optimum results
2. 400 mM EDC (2 ml)
   153.4 mg EDC
   2 ml demi water
3. 100 mM NHS
   23 mg NHS
   2 ml demi water
4. 1 M Ethanolamine
   0.97 g salt
   9 ml demi water
   Use HCl to adjust its pH to 8.5
   Finally, make up the volume to 10 ml
5. 1 M Acetate buffer pH 4.5
   1.36 g salt
   9 ml demi water
   Use acetic acid to adjust the pH to 4.5
   Finally, make up the volume to 10 ml (Abdul Rehman et al., 2013)
6. 1 M HEPES buffer pH 7.5
   26.03 g salt
   80 ml demi water
   Use HCl to adjust the pH to 7.5
   Finally, make up the volume to 100 ml
7. 5 M NaCl
   29.22 g salt
   100 ml demi water
8. 1 M NaOH
   0.4 g of NaOH
   10 ml demi water
9. 10% Tween 20
   Prepared with demi water
10. Buffer 1 (Coupling buffer)
    Dilute 0.5 ml of Acetate buffer stock to 50 ml solution using demi water
11. Buffer 2 (Association buffer) (1 L)
    10 ml 1 M HEPES buffer pH 7.5
    30 ml NaCl from its stock solution
    0.5 ml 10% Tween 20
    Make up the final volume to 1 L with demi water
    Filter and use
12. Buffer 3 (Regeneration buffer)
    Take 2.5 ml from NaOH stock solution
    Make up the volum
13. Protein solutions
    1. Convert mg/ml protein concentration to molarity. Use the following formula:
       μM of protein = (μg/μl of protein)/(MW in Da)
    2. Next, dilute the protein stock in Buffer 2 to prepare 100 μM of working stock.
    3. Use the following calculations to prepare the dilution in Buffer 2 (Association buffer):
       

       Sample name	Volume of protein working stock	Volume of Buffer 2
       Dilution 1 (1 μM)	1 μl	99 μl
       Dilution 2 (2 μM)	2 μl	98 μl
       Dilution 3 (4 μM)	4 μl	96 μl
       Dilution 4 (8 μM)	8 μl	92 μl
       Dilution 5 (10 μM)	10 μl	90 μl

    4. When using different proteins against one protein, use the same dilution (made in Buffer 2) for all the proteins. Make 1 μM dilution of all the proteins to be used as working stock. Take 20 μl of this stock and add 80 μl of Buffer 2 to prepare 200 nM solutions.
       

       Protein name	Concentration
       Analyte 1 (example: EhCoactosin)	200 nM
       Analyte 2 (example: Profilin A)	200 nM
       Analyte 3 (example: Profilin B)	200 nM
       Analyte 4 (example: Filamin)	200 nM
       Analyte 5 (example: Twinfilin)	200 nM
       Analyte 6 (example: Actophorin)	200 nM

Acknowledgments

We acknowledge Advanced Instrument Research Facility, JNU and Manu Vashistha for SPR studies. We would also like to thank Prof. Alok Bhattacharya (Ashoka University, New Delhi, India) and Prof. Samudrala Gourinath (Jawaharlal Nehru University, New Delhi, India) for providing us the necessary help during the study. SA thanks the Department of Science and Technology for DST Inspire Faculty Award and Grant. PPR thanks the Council of Scientific and Industrial Research (CSIR) for SRF Fellowship.

Competing interests

The authors have declared that no competing interests exist.

References

1. Abdul Rehman, S. A., Verma, V., Mazumder, M., Dhar, S. K. and Gourinath, S. (2013). Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori. J Bacteriol 195(12): 2826-2838.
2. Agarwal, S., Anand, G., Sharma, S., Parimita Rath, P., Gourinath, S. and Bhattacharya, A. (2019). EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica. PLoS Pathog 15(5): e1007789.
3. Davis, T. M. and Wilson, W. D. (2000). Determination of the Refractive Index Increments of Small Molecules for Correction of Surface Plasmon Resonance Data. Anal Biochem 284: 348-353.
4. Kumar, N., Somlata, Mazumder, M., Dutta, P., Maiti, S. and Gourinath, S. (2014). EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica. PLoS Pathog 10(9): e1004362.
5. Nguyen, H. H., Park, J., Kang, S. and Kim, M. (2015). Surface plasmon resonance: a versatile technique for biosensor applications. Sensors (Basel) 15(5): 10481-10510.