Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST)

Materials and reagents

Reagents

1. IPTG (isopropyl β-D-1-thiogalactopyranoside) (Inalco, catalog number: 1758-1400)

2. Glutathione (GE Healthcare, catalog number: 17-5132-02)

3. Kanamycin (Inalco, catalog number: 1758-9316)

4. Ampicillin (Inalco, catalog number: 1758-9314)

5. Lysozyme (Solarbio, catalog number: L8120)

6. DNase I (Beijing DingGuoChangSheng, catalog number: DH113-5)

7. Glutathione-Sepharose 4 Fast Flow (GE Healthcare, catalog number: 17513201)

8. Ni-nitrilotriacetic acid agarose (QIAGEN, catalog number: 30210)

9. ATTO-488 NHS ester (ATTO-TEC GmbH, catalog number: AD488-35)

10. Coomassie brilliant blue G250 (Amresco, catalog number: M140-50G)

11. Tryptone (QiSong biology, catalog number: BQS133120)

12. Yeast extract (Oxiod, catalog number: LP0021)

13. Glucose (QiSong biology, catalog number: BQS119483)

14. Tris-base (Sigma, catalog number: T1387)

15. EDTA (Amresco, catalog number: 0105)

16. DTT (Amresco, catalog number: 0281)

17. β-Mercaptoethanol (Macklin, catalog number: M6230)

18. Imidazole (Sigma-Aldrich, catalog number: 0664)

19. Ethanol (Beijing Yili Fine Chemicals Co., Ltd)

20. Tween-20 (DingGuoChangSheng, catalog number: DH358-3)

21. DMSO (Leagene, catalog number: CC0118)

22. NaCl (Beijing Yili Fine Chemicals Co., Ltd)

23. MgCl₂ (Beijing Yili Fine Chemicals Co., Ltd)

24. KCl (Beijing Yili Fine Chemicals Co., Ltd)

25. NaHCO₃ (Beijing Yili Fine Chemicals Co., Ltd)

26. H₃PO₄ (Beijing Yili Fine Chemicals Co., Ltd)

Solutions

1. SOC (used for E. coli transformation) (see Recipes)

2. LB (used for E. coli culture) (see Recipes)

3. 10× TBS (used for equilibration of Ni-nitrilotriacetic acid-agarose or Glutathione-Sepharose 4 Fast Flow) (see Recipes)

4. GST-tag protein lysis buffer (used for GST-tagged protein purification) (see Recipes)

5. GST-tag protein washing buffer (used for GST-tagged protein purification) (see Recipes)

6. GST-tag protein elution buffer (used for GST-tagged protein purification) (see Recipes)

7. GST-tag protein dialysis buffer (used for GST-tagged protein purification) (see Recipes)

8. His-tag protein lysis buffer (used for His-tagged protein purification) (see Recipes)

9. His-tag protein washing buffer (used for His-tagged protein purification) (see Recipes)

10. His-tag protein elution buffer (used for His-tagged protein purification) (see Recipes)

11. His-tag protein dialysis buffer (used for His-tagged protein purification) (see Recipes)

12. Bradford (used for protein detection) (see Recipes)

13. MST buffer (used for dilution of fluorescently labeled proteins in the MST reaction) (see Recipes)

14. ATTO-488 NHS-ester dye (see Recipes)

Recipes

1. SOC

Adjust the final volume to 1 L with distilled water.

2. LB

Adjust the final volume to 1 L with distilled water.

3. 10× TBS

Adjust the final volume to 1 L with distilled water. Adjust pH to 7.5 with concentrated HCl.

4. GST-tag protein lysis buffer

Adjust the final volume to 90 mL with distilled water.

5. GST-tag protein washing buffer

Adjust the final volume to 500 mL with distilled water.

6. GST-tag protein elution buffer

Adjust the final volume to 20 mL with distilled water.

7. GST-tag protein dialysis buffer

Adjust the final volume to 1 L with distilled water. Adjust pH to 8.2–8.3 with concentrated HCl.

8. His-tag protein lysis buffer

Adjust the final volume to 90 mL with distilled water.

9. His-tag protein washing buffer

Adjust the final volume to 500 mL with distilled water.

10. His-tag protein elution buffer

Adjust the final volume to 20 mL with distilled water.

11. His-tag protein dialysis buffer

Adjust the final volume to 1 L with distilled water.

12. Bradford

Adjust the final volume to 1 L with distilled water.

13. MST buffer

Adjust the final volume to 50 mL with distilled water.

14. ATTO-488 NHS-ester dye

Dissolve 5 mg of ATTO-488 NHS-ester dye powder in 510 μL of DMSO. Aliquot into 20 μL per tube, lyophilize, and store at -80 °C. Each tube contains 0.196 mg of ATTO-488 NHS-ester.

Laboratory supplies

1. General-purpose dialysis bags 8000–14000 (for protein purification ) (ShangHai Yuye, catalog number: MD1425)

2. Gravity flow column B (for column chromatography) (NanoTemper Technologies GmbH, catalog number: L001)

3. 200 μL PCR tubes (for MST experiments) (Axygen, catalog number: PCR-02-C)

4. Monolith capillaries (for MST experiments) (NanoTemper Technologies GmbH, catalog number: MO-K002)

Equipment

1. Ultrasonic cell pulverizer (for cell lysis) (NingBo XinZhi Biotechnology Co., LTD, model: JY92-II)

2. Ultracentrifuge (for rapid sample processing and preservation) (Beckman Coulter, model: Optima XPN-80)

3. Benchtop centrifuge (for precipitating insoluble proteins) (Eppendorf, model: 5415R)

4. Nanodrop-1000 (for measuring nucleic acid and protein concentrations) (Thermo Fisher)

5. NanoTemper^® Monolith NT.115 (for MST measurements) (NanoTemper Technologies GmbH)

Software and datasets

1. Nanodrop-1000 4.64.0.0 (for determination of protein concentration) (Thermo Fisher Scientific)

2. Monolith^® NT.115 (for analyzing MST data) (NanoTemper)

3. KaleidaGraph 4.0 (for data analysis and graphing) (Synergy)

4. Illustrator CS6 (for drawing images) (Adobe)

5. Mo.Control software (for controlling MST experiments) (NanoTemper)

Procedure

A. Protein expression

Expression of GST-Mlph-ABD

1. Transformation:

a. Transfer 100 ng of plasmids (1 μL) GST-Mlph-ABD/pGEX4T2 into 100 μL of BL-21(DE3) E. coli competent cells.

b. Keep the mixture on ice for 5 min, heat-shock at 42 °C for 45 s, and then immediately place back on ice for 2 min.

c. Add 200 μL of SOC medium to each sample and incubate at 37 °C with shaking for 45 min.

d. Spread 100 μL of the above culture to an ampicillin-resistant agar plate (LB agar with ampicillin) and incubate the plate at 37 °C for 12–16 h.

2. Culture of E. coli:

a. When the colonies on the plate grow to the appropriate size, pick up a single colony to inoculate 4 mL of the ampicillin-resistant LB medium (at room temperature) and incubate at 37 °C with shaking at 200 rpm for 6 h.

b. Use the above culture (~4 mL) to inoculate 250 mL of the ampicillin-resistant LB medium (room temperature) and incubate at 37 °C with shaking at 200 rpm for 3–4 h until the OD₆₀₀ reaches 0.8–1.

c. Add 50 μL of 1 M IPTG (final concentration 0.2 mM) to the above culture to induce protein expression and incubate with shaking at 200 rpm at 37 °C for 3 h or at 17 °C for 12 h (General note 1).

3. Collect E. coli:

a. Harvest the induced E. coli by centrifugation at 4,000 rpm (~3,000× g) for 10 min at room temperature.

b. Resuspend the E. coli pellets with 1× TBS and precipitate the E. coli by centrifugation again at 4,000 rpm (~3,000× g) for 10 min at room temperature. Discard the supernatant and use the pellets for purification directly or store them at -80 °C for later use.

Expression of His-tagged Myo5a tail (His-Myo5a-MTD and His-Myo5a-MTDΔG)

1. Transformation:

a. Transfer 100 ng of bacterial expression plasmids (1 μL) His-Myo5a-MTD/pET30a or His-Myo5a-MTDΔG/pET30a into 100 μL of BL-21(DE3) E. coli competent cells (Myo5a-MTD can interact with Mlph, whereas Myo5a-MTDΔG cannot).

b. Keep the mixture on ice for 5 min, heat-shock at 42 °C for 45 s, and then immediately place back on ice for 2 min.

c. Add 200 μL of SOC medium to each sample and incubate at 37 °C with shaking for 45 min.

d. Spread 100 μL of the mixture to a kanamycin-resistant agar plate (LB agar with kanamycin) and incubate the plate at 37 for 12–16 h.

2. Culture of E. coli:

a. When the colonies on the plate grow to the appropriate size, pick up a single colony to inoculate 4 mL of the kanamycin-resistant LB medium (at room temperature) and incubate at 37 °C with shaking at 200 rpm for 6 h.

b. Use the above culture (~4 mL) to inoculate 250 mL of the kanamycin-resistant LB medium (room temperature) and incubate at 37 °C with shaking at 200 rpm for 3–4 h until the OD₆₀₀ reaches 0.8–1.

c. Add 50 μL of 1 M IPTG (final concentration 0.2 mM) to the above culture to induce protein expression and incubate with shaking at 200 rpm at 37 °C for 3 h or at 17 °C for 12 h (General note 1).

3. Collect E. coli:

a. Harvest the induced E. coli by centrifugation at 4,000 rpm (~3,000× g) for 10 min at room temperature.

b. Resuspend the E. coli pellets with 1× TBS and precipitate the E. coli again by centrifugation at 4,000 rpm (~3,000× g) for 10 min at room temperature. Save the pellets and store at -80 °C or use them immediately for purification.

B. Protein purification

The purification of proteins requires several sequential steps, as shown in Figure 1.

Figure 1. Flowchart for protein purification

For the purification of GST-Mlph-ABD

1. Lysis:

a. Thaw the E. coli. pellet of GST-Mlph-ABD, which was collected from 250 mL of culture and stored at -80 °C.

b. Resuspend the E. coli pellets in 25 mL of GST-tag protein lysis buffer by pipetting up and down repeatedly and then stand on ice for 20 min.

c. Add 10⁴ U/mL DNase I to achieve a final concentration of 10 U/mL, 1 M MgCl₂ to a final concentration of 3 mM, and 4 M NaCl to a final concentration of 0.2 M. Mix by inverting and leave on ice for 10 min.

2. Sonication: Set the power of the ultrasonic cell pulverizer to 200 W, with a cycle of 3 s on and 7 s off, repeating this process 80 times for complete lysis of E. coli (General note 2).

3. Centrifugation: Centrifuge the lysate at 20,000 rpm (~40,000× g) using an ultracentrifuge for 40 min at 4 °C and save the supernatant of the lysate in a 50 mL conical tube.

4. Binding to GSH-Sepharose 4 Fast Flow beads (GSH-Sepharose beads):

a. Equilibrate GSH-Sepharose 4 beads: Suspend 1 mL of GSH-Sepharose 4 beads in 10 mL of 1× TBS; then, let the beads settle down and discard the supernatant.

b. Transfer the GSH-Sepharose 4 beads to the 50 mL conical tube containing the supernatant of the lysate and rotate the conical tube at 4 °C for 2 h.

5. Wash:

a. Centrifuge the 50 mL conical tube at 2,000 rpm (~800× g) for 10 min at 4 °C using a multifunctional tabletop centrifuge.

b. Discard the supernatant, then suspend the GSH-Sepharose 4 beads with ~50 mL of GST-tag protein washing buffer and centrifuge again at 2,000 rpm (~800× g) for 10 min. Discard the supernatant.

c. Transfer the GSH-Sepharose 4 beads to a disposal chromatography column. Rinse the column with GST-tag protein wash buffer and detect protein in the eluate by mixing 5 μL of eluate with 45 μL of Bradford staining solution. The color of the Bradford staining solution changes from brown to blue when it reacts with protein. Stop rinsing the column when the eluate does not change the color of Bradford staining solution.

6. Elution: Elute the target proteins using GST-tag elution buffer by gravity flow. Detect the proteins in the eluate by mixing 5 μL of eluate with 45 μL of Bradford staining solution. The color of the Bradford staining solution changes from brown to blue when it reacts with protein. Combine the eluate fractions containing high-concentration proteins.

7. Dialysis: Cut an appropriate length of the dialysis tube based on the estimated volume of the eluted protein. Place the dialysis tube in deionized water and boil at 100 °C for 3 min. Transfer the eluted proteins into the dialysis tube and dialyze against 1 L of GST-tag protein dialysis buffer overnight at 4 °C. This step is essential for labeling GST-Mlph-ABD with ATTO-488 NHS-ester, as it removes free amines.

8. Concentration:

a. Transfer the dialyzed proteins into ultrafiltration tubes and centrifuge at 4,000 rpm (~3,000× g) at 4 °C using a multifunctional tabletop centrifuge until the appropriate concentration is reached.

b. Aliquot the concentrated protein into a small volume (20–200 μL), quickly freeze in liquid nitrogen, and store at -80 °C.

c. Measure the protein concentration using Nanodrop-1000 and detect protein purity by SDS-PAGE (Figure 2). The purified GST-Mlph-ABD can be used for subsequent fluorescent labeling.

Figure 2. SDS-PAGE of purified GST-Mlph-ABD, His-Myo5a-MTD, and His-Myo5a-MTDΔG

Purification of His-tagged Myo5a tail (His-Myo5a-MTD and His-Myo5a-MTDΔG)

1. Lysis:

a. Thaw E. coli pellets of His-Myo5a-MTD or His-Myo5a-MTDΔG, which were collected from 250 mL of culture and stored at -80 °C.

b. Resuspend the E. coli pellets in 25 mL of His-tag protein lysis buffer by pipetting up and down repeatedly and then stand on ice for 20 min.

c. Add 10⁴ U/mL DNase I to achieve a final concentration of 10 U/mL, 1 M MgCl₂ to a final concentration of 3 mM, and 4 M NaCl to a final concentration of 0.2 M. Mix by inverting and leave on ice for 10 min.

2. Sonication: Set the power of the ultrasonic cell pulverizer to 200 W, with a cycle of 3 s on and 7 s off, repeating this process 80 times to fully lyse the organisms (General note 2).

3. Centrifugation: Centrifuge the lysate at 20,000 rpm (~40,000× g) using an ultracentrifuge for 40 min at 4 °C and save the supernatant of the lysate in a 50 mL conical tube.

4. Binding to Ni-nitrilotriacetic acid-agarose (Ni-agarose):

a. Suspend 1 mL of Ni-agarose in 10 mL of 1× TBS, let the beads settle down, and discard the supernatant.

b. Transfer the GSH-Sepharose 4 beads to the 50 mL conical tube containing the supernatant of the lysate and spin the conical tube on a rotary shaker at 4 °C for 2 h.

5. Wash:

a. Centrifuge the conical tube at 2,000 rpm (~800× g) for 10 min at 4 °C using a multifunctional tabletop centrifuge.

b. Discard the supernatant, then suspend the Ni-agarose with ~50 mL of His-tag protein washing buffer and centrifuge again at 2,000 rpm (~800× g) for 10 min. Discard the supernatant.

c. Transfer the Ni-agarose to a disposal chromatography column. Rinse the column with His-tag protein washing buffer and detect protein in the eluate by mixing 5 μL of eluate with 45 μL of Bradford staining solution. The color of Bradford staining solution changes from brown to blue when it reacts with protein. Stop rinsing the column when the eluate does not change the color of the Bradford staining solution.

6. Elution: Elute the target proteins using His-tag elution buffer by gravity flow. Detect the proteins in the eluate by mixing 5 μL of eluate with 45 μL of Bradford staining solution. The color of Bradford staining solution changes from brown to blue when it reacts with protein. Combine the eluate fractions containing high-concentration proteins.

7. Dialysis: Estimate the volume of the eluted protein and cut an appropriate length of the dialysis tube. Place the dialysis tube in deionized water and boil at 100 °C for 3 min. Transfer the eluted proteins into the dialysis tube and dialyze overnight at 4 °C in 1 L of His-tag protein dialysis buffer.

8. Concentration:

a. Transfer the dialyzed proteins into ultrafiltration tubes and centrifuge at 4,000 rpm (~3,000× g) at 4 °C using a multifunctional tabletop centrifuge until the appropriate concentration is reached.

b. Aliquot the concentrated protein into small volumes (20–200 μL), quickly freeze in liquid nitrogen, and store at -80 °C.

c. Measure the protein concentration using a Nanodrop-1000 and detect the protein purity by SDS-PAGE (Figure 2). The purified His-Myo5a-MTD and His-Myo5a-MTDΔG are directly used for MST experiments.

C. Labeling of GST-Mlph-ABD with ATTO-488 NHS-ester

1. Preparation of fluorescent dye: Dissolve 0.196 mg of ATTO-488 NHS-ester with 20 μL of DMSO to make a 10 mM solution, and then dilute to 1 mM by mixing 2 μL of ATTO-488 NHS-ester with 18 μL of GST-tag protein dialysis buffer.

2. Labeling:

a. Prepare 90 μL of 40 μM GST-Mlph-ABD protein by diluting GST-Mlph-ABD protein stock with GST-tag protein dialysis buffer.

b. Add 10.8 μL of 1 mM ATTO-488 NHS-ester dye to 90 μL of 40 μM GST-Mlph-ABD protein, resulting in a 3:1 molar ratio of ATTO-488 NHS-ester dye to GST-Mlph-ABD. Mix well.

c. Incubate for 1 h at room temperature in the dark (General notes 3–6).

3. Purification of the labeled protein:

a. Equilibrate the gravity flow column B with 3 mL of MST buffer three times.

b. Add a maximum of 500 μL of labeling reaction to the center of column B. Let the sample enter the column completely (when using less than 500 μL, adjust the volume to 500 μL using MST buffer after the sample has entered the column) and discard the flowthrough.

c. Add 2 mL of MST buffer to the center of column B and collect in 200–250 μL fractions.

4. Measure ATTO-488-labeled GST-Mlph-ABD concentration in effluent fractions using Nanodrop-1000:

a. Add 3 μL of deionized water dropwise to the Nanodrop's detection probe. Open the software Nanodrop-1000 and select the module Proteins & Labels.

b. Select the excitation light Alexa Fluor 488 for the fluorescent dye ATTO-488 NHS ester, add 3 μL of GST-tag protein dialysis buffer to the probe of the Nanodrop-1000, and click Blank.

c. Add 3 μL of effluent fractions to the Nanodrop-1000 probe and click Measure. The two blue highlight boxes in Figure 3 show the concentration of the fluorescent dye and protein.

d. Save the fractions containing high concentrations of ATTO-488-labeled GST-Mlph-ABD with a dye/protein molar ratio below 1.5 for subsequent MST experiments.

Figure 3. Measurement of the labeling efficiency of ATTO-488-labeled GST-Mlph-ABD using Nanodrop-1000

D. MST measurement of the interaction between His-Myo5a-MTD and GST-Mlph-ABD

1. Pre-test:

a. Dilute ATTO-488-labeled GST-Mlph-ABD to 40 nm with MST buffer.

b. Mix 10 μL of 40 nm ATTO-488-labeled GST-Mlph-ABD with 10 μL of His-tag protein dialysis buffer in a PCR tube, pipetting up and down several times to ensure adequate mixing, and then aspirate the mixture using Monolith capillaries (General note 7).

c. Put the capillaries in the slots on the sample tray, not touching the optical measurement section of the capillary.

d. Place the sample tray in the instrument by pushing it into the instrument tray slot as far as possible. The Monolith NT.115 instrument scans the tray and automatically determines the position of the capillaries on the tray.

e. Start the Mo.Control software and select Start New Session.

f. Select the green excitation filter for the upcoming experiment (General note 8).

g. Select the mode Pretest before performing a binding experiment and perform a pre-test experiment to detect the fluorescence intensity of the labeled GST-Mlph-ABD (see red highlight 1 in Figure 4). The fluorescence intensity of the target protein needs to be between 200 and 1,000, with a value between 400 and 800 being optimal.

Figure 4. Initial setup of an experiment

2. Sample loading:

a. Take 16 clean PCR tubes, labeled 1–16, and arrange them in order on the PCR tube holder.

b. Add 10 μL of His-Myo5a-MTD dialysis buffer to tubes 2–16 and 20 μL of 40 μM His-Myo5a-MTD to tube 1.

c. Dispense 10 μL of His-Myo5a-MTD from tube 1 and transfer it into tube 2, mixing thoroughly. Then, using the same tip, transfer 10 μL from tube 2 into tube 3. Repeat this process until tube 16. After thoroughly mixing the sample in the final tube 16, remove 10 μL from that tube and discard it (General note 9).

d. Add 10 μL of ATTO-488-labeled GST-Mlph-ABD to each of the 16 tubes, mix well, and centrifuge the samples at 13,000× g for 5 min. Transfer the sample from the PCR tubes into 16 clean Monolith capillaries sequentially.

e. Put the capillaries in the slots on the sample tray (General note 10).

f. Place the sample tray in the instrument by pushing it into the instrument tray slot.

3. Binding affinity measurement:

a. Return to the homepage of the Mo. Control software and select Binding Affinity (see red highlight 2 in Figure 4).

b. Enter the Plan interface and set the following parameters: the reaction temperature of the MST experiment, the initial and final concentrations of the target and ligand proteins, the estimated Kd, the type of capillary, the excitation power and the MST power (see red highlight in Figure 5A).

c. Click Go to Instructions (see blue highlight in Figure 5A) to enter the Instruction interface, which allows you to dilute the target and ligand proteins to the appropriate concentrations according to the protocol provided.

d. Click Start Measurement to enter the Results interface (see blue highlight in Figure 5B) and wait for the system to automatically measure the MST traces and the dissociation constant Kd.

Figure 5. Procedure for measuring binding affinity experiments using Mo.Control software. (A) Plan interface for setting the temperature of the reaction, initial and final concentrations of the target and ligand proteins, excitation power, and MST power. (B) Instruction interface that provides a protocol on how to dilute the target and ligand proteins to the desired concentration for binding.

Data analysis

The MST data can be analyzed with Monolith^® NT.115, which comes with the MST machine, or using third-party software. Here, we show how to analyze MST data using the KaleidaGraph 4.0 software.

1. Input data:

a. Open the KaleidaGraph software and select File in the first icon from the top row of the KaleidaGraph software (Figure 6).

b. Select New under the File menu to enter the MST experiment data separately into the Data 1 table. Dose values should be entered in column A. The mean values of response obtained from three repetitions of MST experiments for two different sets of interacting proteins are entered in columns B and D. Columns C and E correspond to the standard deviation of the mean values in B and D over three experiments, respectively.

Figure 6. Screenshot of the KaleidaGraph version 4.0 software showing the location of the Data window under the File tab

2. Analysis data:

a. Select Gallery in the third icon from the top row of the KaleidaGraph software (Figure 7).

Figure 7. Screenshot of the KaleidaGraph version 4.0 software showing the submenu Scatter under the Linear menu under the Gallery tab

b. Select Scatter under the Linear menu to generate a dot plot.

c. Select Curve Fit in the sixth icon from the top row of the KaleidaGraph software and choose fit1 under the General menu (Figure 8).

Figure 8. Screenshot of the KaleidaGraph version 4.0 software showing the submenu fit1 under the General menu under the Curve Fit tab

d. Select the data in column B in the pop-up Curve Fitting Selection window. Click Define in the upper-right corner and enter the MST curve fitting formula (Figure 9):

m1 - m2/0.02/2*((m0 + 0.02 + m3) - ((m0 + 0.02 + m3) ^ 2 - 4*m0*0.02) ^ 0.5)/2; m1 = 953; m2 = 8; m3 = 0.5.

Figure 9. Screenshot of KaleidaGraph version 4.0 software showing the Curve Fitting Selection sub-window under the Curve Fitting Selection window

Assign the value of m1 as the maximum MST value, m2 as the maximum MST change, and m3 as the estimated K_d value in μM, which is the x-value corresponding to the vertical coordinate reaching half of the maximum of MST change (Figure 10) (General note 11).

Figure 10. Analysis of MST data using KaleidaGraph. Top, MST data plots and curve fits using KaleidaGraph. Middle, curve fitting equation and interpretation of related parameters. Bottom, the final figure used in publication (Pan et al. [9], https://elifesciences.org/articles/93662).

e. Click File and then save Graph as to save it in TIF format for editing in Illustrator CS6.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

• Pan et al. [9]. Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes. eLife (Figure 3A).

We monitored the interaction between His-Myo5a-MTD and GST-Mlph-ABD using MST and obtained the dissociation constant (Kd) of 562 ± 169 nM of Myo5a-MTD for binding to Mlph-ABD. Consistent with the GST pulldown assay, we found that deletion of the C-terminal half of exon-G (His-Myo5a-MTDΔG) greatly decreased MST signaling.

General notes and troubleshooting

1. The optimal conditions for IPTG to induce the expression of different proteins vary, and factors such as the concentration of IPTG, E. coli culture temperature, E. coli culture time, and stain of E. coli need to be taken into account. In general, a lower IPTG concentration can reduce the burden of protein expression on host cells, while a higher IPTG concentration can induce protein expression more rapidly. Low-temperature induction can prolong the time of protein expression so that the protein can be folded sufficiently, but it may also cause protein degradation, while the opposite is true for high-temperature induction. In practice, the optimal conditions should be determined based on experimental requirements to achieve the best protein expression results. We routinely use 0.2 mM IPTG to induce protein expression.

2. After sonication, it can be observed that the bacterial solution becomes transparent; if it is still turbid, it may be that the lysis is not sufficient, and the volume of lysis buffer or the number of sonication needs to be increased.

3. Avoid using buffers containing primary amines (e.g., ammonium ions, tris, glycine, ethanolamine, glutathione) or imidazole for labeling fluorescent proteins, which compete with the labeled proteins to reduce labeling efficiency.

4. Concerning reducing agents, DTT and β-mercaptoethanol interfere with the labeling reaction and, therefore, need to be avoided. If a reducing agent is required during the labeling reaction, use TCEP. However, DTT and β-mercaptoethanol are better suited than TCEP for the subsequent MST experiment, since TCEP may in some cases reduce reproducibility.

5. Purified proteins containing carriers like BSA will not be labeled properly and should not be used.

6. Labeled protein concentration: If the protein concentration is too low, the labeling efficiency will be greatly affected, and the general concentration will not be lower than 0.5 mg/mL.

7. Place the capillary horizontally into the reaction tube to aspirate the sample. Do not touch the capillary in the middle section where the optical measurement will be performed.

8. The excitation color can be changed at the start of each new experiment.

9. Thorough mixing ensures consistent fluorescence intensity of fluorescent proteins in each PCR tube.

10. Note the order of the capillaries. The highest concentration is placed in the front of the tray. This position is denoted as “1” on the sample tray and in the control software.

11. To fit the equations correctly, it is necessary to make approximately correct guesses about m1, m2, and m3.

Acknowledgments

We are grateful to the core facility platform of the State Key Laboratory of Integrated Management of Pest Insects and Rodents at the Institute of Zoology for providing the laboratory equipment. This work was supported by the National Natural Science Foundation of China (31970657).

This protocol was adapted and modified from Pan et al. [4].

Competing interests

The authors declare that there are no competing financial interests.

References

1. Li, X. D., Ikebe, R. and Ikebe, M. (2005). Activation of myosin Va function by melanophilin, a specific docking partner of myosin Va. J Biol Chem. 280(18): 17815–17822.
2. Yao, L. L., Cao, Q. J., Zhang, H. M., Zhang, J., Cao, Y. and Li, X. D. (2015). Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a. Sci Rep. 510874.
3. Fukuda, M. and Itoh, T. (2004). Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitiveto proteolysis. J Biol Chem. 279(21): 22314–22321.
4. Geething, N. C. and Spudich, J. A. (2007). Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin. J Biol Chem. 282(29): 21518–21528.
5. Wu, X., Wang, F., Rao, K., Sellers, J. R. and Hammer, J. A. (2002). Rab27a is an essential component of melanosome receptor for myosin Va. Mol Biol Cell. 13(5): 1735–1749.
6. Nowak, P. M. and Woźniakiewicz, M. (2022). The Acid-Base/Deprotonation Equilibrium Can Be Studied with a MicroScale Thermophoresis(MST). Molecules. 27(3): 685.
7. Seidel, S. A., Dijkman, P. M., Lea, W. A., van den Bogaart, G., Jerabek-Willemsen, M., Lazic, A. and Duhr, S. (2013). Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 59(3): 301–315.
8. Magnez, R., Thiroux, B., Taront, S., Segaoula, Z., Quesnel, B. and Thuru, X. (2017). PD-1/PD-L1 binding studies using microscale thermophoresis. Sci Rep. 7(1): 17623.
9. Pan, J., Zhou, R., Yao, L. L., Zhang, J., Zhang, N., Cao, Q. J., Sun, S. and Li, X. D. (2024). Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes. eLife. 13e93662.