Integrative biophysical characterization of molecular interactions: A case study with the sfGFP-nanobody complex

Abstract

This study compares several analytical biophysical methods for investigating protein-protein interactions (PPIs) in solution, using the interaction between superfolder green fluorescent protein (sfGFP) and its anti-sfGFP nanobody enhancer as a model system. Techniques evaluated include microscale thermophoresis, fluorescence correlation spectroscopy, analytical ultracentrifugation with multi-wavelength and fluorescence detection, isothermal titration calorimetry, and analytical size exclusion chromatography coupled to multi-angle static light scattering and dynamic light scattering. Each method was assessed for information content, dynamic range, precision, and complementarity. The results consistently indicate a single-digit nanomolar dissociation constant and 1:1 stoichiometry for the interaction. While each technique offers unique insights into binding affinity, thermodynamics, and stoichiometry of the interaction, the multi-method approach provides a more complete and reliable characterization of PPIs. The study demonstrates how combining multiple complementary techniques enhances the robustness of PPI analysis in solution-phase conditions.

Fig. 1.

Molar extinction coefficient spectral profiles for sfGFP (green), nanobody (orange), combined 1:1 M ratio mixture (blue), and 1:2 M ratio mixture (sfGFP:nanobody, purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.

Spectral decomposition of sfGFP:nanobody mixtures. A: 1:1 M (sfGFP:nanobody) mixture where the target spectrum (blue) is the intrinsic extinction coefficient spectrum of the mixture, the basis spectra are the intrinsic extinction coefficient spectra of sfGFP (green) and nanobody (orange) and the fit of the target spectrum (magenta) provides the solution of the decomposition (top). The decomposition indicates a molar composition of 44.27 ± 0.99 % of sfGFP and 55.73 ± 1.26 % of nanobody. The residuals are shown in yellow (bottom). The relatively large deviations are an indication of hyper- and hypo-chromic shifts in the absorbance spectrum upon complex formation. B: 1:2 M (sfGFP:nanobody) mixture where the target spectrum (blue) is the intrinsic extinction coefficient spectrum of the mixture, the basis spectra are the intrinsic extinction coefficient spectra of sfGFP (green) and nanobody (orange) and the fit of the target spectrum (magenta) provides the solution of the decomposition (top). The decomposition indicates a molar composition of 32.48 ± 0.99 % of sfGFP and 67.52 ± 2.06 % of nanobody. The residuals are shown in yellow (bottom). The relatively large deviations are an indication of hyper- and hypo-chromic shifts in the absorbance spectrum upon complex formation, which show a nearly identical pattern as the 1:1 M ratio fit shown in A. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3.

Microscale thermophoresis measurement for the interaction between sfGFP and nanobody. The nanobody was titrated up to a maximum concentration of 10 μM into a constant amount of sfGFP at a concentration of 37.7 nM and was measured after a minimum of a 20 min incubation time at room temperature (25 °C). The dissociation constant was determined to be 1.53 ± 3.98 nM (reduced X² = 1.548, Std. error of regression = 0.459). The error bars are based on triplicate measurements.

Fig. 4.

Isothermal titration calorimetry measurement for the interaction between sfGFP and nanobody. Exothermic enthalpy plots showing (top) the raw data of 32 titrations with a 120 s interval of 96 μM nanobody to 15 μM sfGFP and (bottom) the integrated enthalpy using an independent model as a function of the molar ratio of the nanobody with the derived thermodynamic parameters, including dissociation constant $\left(k_D\right)$, stoichiometry $\left(\mathrm{n}\right)$, enthalpy $\left(\mathrm{\Delta }\mathrm{H}\right)$, and entropy $\left(\mathrm{\Delta }\mathrm{S}\right)$.

Fig. 5.

Multi-wavelength AUC results, integral representation: Van Holde Weischet distributions of sedimentation velocity 2DSA-IT fitted data of the nanobody control in red (RMSD = 2.11e-3), and sfGFP in green (RMSD = 2.53e-3), and the multi-wavelength deconvolution signals of sfGFP in the 1:1 M mixture (purple), sfGFP in the 1:2 mixture (cyan), nanobody in the 1:1 M mixture (orange), and nanobody in the 1:2 M mixture (pink). The lines in this figure are for visualization purposes while the dots are representative of the measured sedimentation coefficients for each part of the boundary fraction. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6.

Multi-wavelength AUC results, differential representation: A: Sedimentation velocity 2DSA-Monte Carlo controls of nanobody in red and sfGFP in green with RMSDs of 2.03e-3 and 2.47e-3 respectively. B: Deconvolution of the 1:1 M mixture of nanobody (orange) and sfGFP (purple). C: Deconvolution of the 1:2 M mixture of nanobody (pink) and sfGFP (cyan). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7.

Fluorescence AUC analysis: A: Sigmoidal fit to weight-averaged sedimentation coefficients from 100 iterations of the 2DSA-Monte Carlo analysis for individual titration points plotted against the molar fraction of nanobody to sfGFP. B: Sigmoidal fit of fraction bound nanobody to sfGFP plotted against the concentration of titrated nanobody in nM.

Fig. 8.

Autocorrelation curves acquired by fluorescence correlation spectroscopy. A: sfGFP without enhancer nanobody. B: sfGFP with enhancer nanobody. C: sfGFP with and without nanobody, normalized and plotted on the same graph. All curves and fits are from one representative measurement. The black lines are a one-component pure diffusion model.

Fig. 9.

SEC-MALS data showing UV in green, refractive index (RI) in blue, and light scattering (LS) in red, with the calculated molecular weight in black for A nanobody, B sfGFP, and C 1:1 M ratio for the complex. D shows the refractive index traces for the complex (purple), sfGFP (pink), and nanobody (orange), depicting the shifts in elution volume for each species. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)