Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions


Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein–protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein–antibody complexes with picomolar KD values. Using an EGFP–nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein–protein interactions in the micromolar KD value range. Sedimentation experiments typically take 3 h of preparation and 6–12 h of run time, followed by data analysis (typically taking 1–3 h).

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Figure 1: Recorded sedimentation boundaries and fit.
Figure 2: GUSSI overlay of sedimentation coefficient distributions showing the EGFP control (black) and mixtures with 0.1, 0.3, and 20 nM nanobody.
Figure 3: Screenshot of the isotherm text file created using Windows Notepad.
Figure 4: SEDPHAT screenshots showing experimental and global parameters.
Figure 5: GUSSI display of the signal-average sedimentation coefficients.


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This work was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health.

Author information




S.K.C., J.M., H.Z., and P.S. collected the data and developed the protocol. S.K.C., H.Z., and P.S. analyzed the data. S.K.C., H.Z., and P.S. prepared the manuscript.

Corresponding authors

Correspondence to Huaying Zhao or Peter Schuck.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Surface Plasmon Resonance Data

SPR experiments were conducted in a Biacore 3000 instrument (GE Healthcare, Piscataway, NJ), at a temperature of 25 °C). The nanobody was chemically coupled to the surface on a CM3 sensor chip associated with one of the flow cells by amine coupling using the standard procedures. Immobilization was carried out with nanobody solution of 1 μg/mL at pH 5.5. PBS buffer with 0.005% v/v surfactant P20 was used as the working buffer for all the binding experiments. At a flow rate of 5 μL/min, cycles of 1200 sec surface binding of 0.30, 1, 3, 10, 30, and 100 nM EGFP were each followed by 2300 sec dissociation. The binding surface was regenerated with 1 min injection of glycine/HCl at pH 1.50 after the dissociation in each cycle. Analysis of SPR binding data was carried out with the program EVILFIT, which fits the data with a distribution of sites at different KD and different koff. Shown in Panel A is the family of binding traces at the different EGFP concentrations (blue to green solid lines) and global best-fit traces (red lines). Appended below is an overlay of the residuals of the fit, which have a root-mean-square deviation of 0.36 RU. Panel B shows the best-fit surface site distribution. The major peak (red) resulted in an average KD of 23 pM and koff of 3.0×10-5 sec-1. The total signal contribution from this peak is 37 RU from a total estimated binding capacity of 81 RU.

Supplementary Figure 2 Final SEDPHAT Screenshot

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Supplementary Figures 1 and 2, and Supplementary Table 1. (PDF 549 kb)

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Chaturvedi, S., Ma, J., Zhao, H. et al. Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions. Nat Protoc 12, 1777–1791 (2017).

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