Study Small Molecule-Membrane Protein Binding Kinetics with. Nanodisc and Charge Sensitive Optical Detection

Transcription

1 Support Information Study Small Molecule-Membrane Protein Binding Kinetics with Nanodisc and Charge Sensitive Optical Detection Guangzhong Ma 1,2, Yan Guan 1,3, Shaopeng Wang 1*, Han Xu 4*, Nongjian Tao 1,2,3* 1. Center for Bioelectronics and Biosensors, The Biodesign Institute at Arizona State University; 2. Department of Chemistry and Biochemistry, Arizona State University; 3. School of Electrical, computer and energy engineering, Arizona State University; 4. Therapeutic Discovery, Amgen Inc. Contents 1. Performance of the humidity control chamber... S-2 2. Nanodisc assembly and characterization... S-2 3. Characterization of fiber modification... S-3 4. PEG-ShK - KcsA-Kv1.3 nanodisc interaction... S-6 5. Data processing and analysis... S-7 6. Specificity of Kv1.3 ligands binding... S-8 7. DMSO effect on KcsA-Kv1.3 nanodisc... S-9 8. References... S-10 S- 1

2 1. Performance of the humidity control chamber Figure S1. The temperature and humidity in the humidity control chamber were stable over at least 40 min, sufficient for completing the binding experiment. 2. Nanodisc assembly and characterization The nanodisc assembly and characterization protocols were described in detail previously 1. Briefly, we grafted selected residues of the S5-S6 domain of human Kv1.3 channel onto the M1- M2 region of KcsA to get the chimeric KcsA-Kv1.3, which was expressed in E. coli. The cells were lysed and membranes were collected after centrifugation. Then the membranes were solubilized in lysis buffer followed by centrifugation. KscA-Kv1.3 was purified from the supernatant. The KcsA-Kv1.3 nanodisc was assembled by mixing KcsA-Kv1.3, biotin-msp1d1, and DMPC (1, 2-dimyristoyl-sn-glycero-3-phosphocholine) at 1:8:320 molar ratio in assembly buffer containing sodium cholate. Sodium cholate was removed using XAD-2 bio-beads (Sigma) after incubating the mixture at room temperature for 1 hour. KcsA-Kv1.3 nanodisc was separated S- 2

3 from empty nanodisc with Ni-NTA affinity chromatography. The components of KcsA-Kv1.3 nanodisc were confirmed with SDS-PAGE. The Stokes diameters of KcsA-Kv1.3 nanodisc and empty nanodisc were determined as 11.0 nm and 9.5 nm, respectively by size exclusion chromatography. We also used analytical ultracentrifugation (AUC) to determine stoichiometry in KcsA-Kv1.3 nanodisc and empty nanodisc. The results showed KcsA-Kv1.3 nanodisc was composed of 144 DMPC molecules, four KcsA-Kv1.3 proteins, and two MSPs, and empty nanodisc was composed of 144 DMPC molecules and two MSPs. 3. Characterization of fiber modification To verify the modification of APTES on the optical fiber probe, FITC (fluorescein isothiocyanate) was used to characterize the presence of primary amine on the surface. FITC is a fluorescence dye that is reactive to primary amine groups. An APTES modified optical fiber tip was treated in 50 µm FITC solution (prepared in PBS buffer, ph = 8.3) for 30 min, followed by washing three times with 1X PBS. An Olympus IX81 microscope with 20X objective was used to record fluorescence images (excitation = 495 nm, emission = 525 nm). Both APTES coated fiber and bare fiber were treated with FITC, and the fluorescence images (Figure S2) showed that only the APTES coated fiber gave obvious fluorescent signal. S- 3

4 Figure S2. APTES characterization. a) and b) are bright field and fluorescence images of bare fiber, c) and d) are bright field and fluorescence images of APTES functionalized fiber, respectively. The scale bar in all images represents 20 µm. The immobilization of biotin, streptavidin and KcsA-Kv1.3 nanodisc on the same fiber were monitored by CSOD in real time. An APTES coated optical fiber was dipped in a well of a microplate, and NHS-biotin was added (as indicated by the red arrow) to reach a final concentration of 16 µg/ml. The process is shown in Figure S3a. APTES is positively charged at ph 7.4 due to the amino group, so that the fiber oscillation was in phase with the applied voltage (0 phase shift). The addition of biotin (no charge) reduced positive charges on the fiber, which led to a decrease in the oscillation amplitude. The amplitude dropped to 0, and the phase suddenly jumped to 180. This was because deprotonated silanol (negatively charged) background became dominant. Finally, the amplitude became stabilized when all the amino groups were coupled with biotin. The biotinylated fiber was then rinsed and dipped in another well containing 14 nm streptavidin. Since streptavidin is negatively charged at ph 7.4, the S- 4

5 immobilization of streptavidin added more negative charges onto the surface and induced an increase in oscillation amplitude (Figure S3b). The KcsA-Kv1.3 nanodisc immobilization was done in 40 times diluted nanodisc buffer (Figure S3c). At the red arrow, 10 nm KcsA-Kv1.3 nanodisc was added and induced amplitude increase due to the negatively charged KcsA-Kv1.3 nanodiscs. These binding curves validated the surface modification. We also studied the variation of the oscillation amplitude of KcsA-Kv1.3 nanodisc fibers among different batches (Figure S4), and the result showed the surface chemistry was repeatable. Figure S3. Real time monitoring of surface modification of the optical fibers with CSOD. a) Biotinylation of an APTES coated fiber. Inset shows oscillation phase change during biotinylation, + and indicate the fiber is positively and negatively charged respectively. b) Immobilization of streptavidin on biotin. The red curve is after 10 points smoothing. c) Immobilization of KcsA-Kv1.3 nanodisc on streptavidin. 40 times diluted 1X PBS was used in biotin and streptavidin modification, and 40 times diluted nanodisc buffer was used in nanodisc immobilization. S- 5

6 Figure S4. Repeatability of surface chemistry. We functionalized fibers with K-Kv1.3 nanodisc and measured the oscillation amplitude of each fiber in the same batch. Four batches were measured. The number of fibers in each batch was 12, 12, 7, and 10, respectively. All experiments were done in 40 times diluted nanodisc buffer with % BSA blocker, ph PEG-ShK - KcsA-Kv1.3 nanodisc interaction PEG-ShK is ShK incorporated with a poly(ethylene glycol) (PEG) moiety, which has improved potency and selectivity than ShK. In Figure S5a, 4 different concentrations of PEG- ShK were measured with KcsA-Kv1.3 nanodisc-coated fiber tip, and the binding kinetic constants were found to be k " = * M,- s,-, k / = ,0 s,-, and K 2 = 3.4 pm (Res sd = 0.66), respectively, from the global fitting (solid curves) of the data with the first order kinetics model. The oscillation amplitude change due to PEG-ShK binding was smaller compared to ShK. This is because PEG is negatively charged, which partially neutralizes the positive charge on ShK. By fitting the equilibrium curve (Figure S5b), the affinity was determined to be K 2 = 2.3 pm (R ; = 0.74). A control experiment was also performed with an empty nanodisc modified fiber dipping into an 11.4 nm PEG-ShK microplate well (Figure S5c), S- 6

7 and no response was observed. Our result is consistent with the previously reported results (27 pm). 1 Figure S5. Kinetics of PEG-ShK - KcsA-Kv1.3 nanodisc interaction. a) CSOD Response to PEG-ShK at different concentrations (0.12 pm, 1.2 pm, 3.8 pm, and 34.2 pm). Solid lines are global fitting of the data with the first order kinetics model. b) Equilibrium response vs. concentration, with each concentration measured 3 times. c) Negative control experiment using empty nanodisc modified optical fiber in 11 nm PEG-ShK, where the red and blue arrows mark the points the fiber was dipped into the PEG-ShK solution, and switched back to the buffer. All experiments were performed in 40 times diluted nanodisc buffer with 0.01% BSA and ph Data processing and analysis In CSOD measurement, the oscillation amplitude of the fiber probe was extracted by the differential detection method from the fiber tip image recorded by the CCD camera at 247 frames per second. The noises at frequencies different from that of the electric field were removed by a Fast Fourier Transform (FFT) filter. The signal was integrated over one second to generate one raw data point of the sensor response curve. For clarity, the plotted response curves were smoothed with a 3 point moving average for clarity. Then the data is globally fitted using response curves of different ligand concentrations to obtain kinetic constants. The dissociation S- 7

8 phase was fitted first to obtain k d, and then the association phase was fitted with the obtained k d to get k a. We attribute the noises to Brownian motion of the fiber probe, which was discussed in our previous work. 2 The Brownian motion leads to thermal noise in surface charge density, which is given by σ >?@ABCD = ;E FGHI ;J K LM, where k N is the Boltzmann constant, T is temperature, c is damping coefficient, ω is the bandwidth of the detection, and E is electric field strength. For a fiber with r = 7.5 µμm and l = 8.0 mm, σ >?@ABCD is about 0.04 electron charges per µμm 2. The damping coefficient of serum is greater than that of buffer solution, therefore a greater noise level is expected in serum. 6. Specificity of Kv1.3 ligands binding We also performed two negative control experiments to examine the specificity of ligands binding to KcsA-Kv1.3 nanodisc using Immunoglobulin G (IgG) and imatinib. IgG is monoclonal anti-human IgG, and purchased from Sigma-Aldrich. Imatinib is a small molecule anti-cancer drug, which is not expected to bind KcsA-Kv1.3 nanodisc. Indeed, no detectable binding was observed at 10 mg/l of IgG as expected (Figure S6a). Similarly, no response was observed for 2.8 µm imatinib either (Figure S6b). S- 8

9 Figure S6. Negative controls with a) 10 mg/l IgG and b) 2.8 µm imatinib. The red and blue arrows mark the moments when the fiber was dipped into the sample well, and switched to the buffer well, respectively. The fiber was coated KcsA-Kv1.3 nanodisc and blocked with 0.01% casein. 7. DMSO effect on KcsA-Kv1.3 nanodisc We tested the effect of DMSO on KcsA-Kv1.3 nanodisc coated fiber (Figure S7). This is important because compound 1 was initially dissolved in DMSO and diluted with buffer prior to the measurement, and DMSO could affect the binding kinetics. The inset table in Figure S7 lists the corresponding amount of DMSO in compound 1 samples used in kinetics experiments. The amplitude was stable in up to 3.9% (191 µm) DMSO. S- 9

10 Figure S7. Effect of DMSO on CSOD signal. Each arrow indicates an addition and the final concentration of DMSO. The inset table shows the concentrations of compound 1 measured with CSOD in Figure 3b, and their corresponding DMSO concentrations. 8. References (1) Xu, H.; Hill, J. J.; Michelsen, K.; Yamane, H.; Kurzeja, R. J. M.; Tam, T.; Isaacs, R. J.; Shen, F.; Tagari, P. Biochimica et Biophysica Acta (BBA) - Biomembranes 2015, 1848, (2) Guan, Y.; Shan, X.; Wang, S.; Zhang, P.; Tao, N. Chemical Science 2014, 5, S- 10