Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

Abstract

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies.

Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

“…37,43−48 In particular, taking advantage of the statistical properties of c ( s ) analysis, and using Raman scattering of water as a meniscus marker, we have demonstrated low picomolar detection limits, allowing the determination of the binding energy of very high-affinity systems with K D as low as 20 pM. 47 Further, we and others have shown that signal linearity is usually not a concern at nM concentrations and below. 45,46 In fact, the intrinsic data quality is superb, leading to fits with models for macromolecular sedimentation and diffusion that rival that of the best conventional detection system, but only after characteristic data structure of this detection system is accounted for.…”

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…All samples were prepared in a freshly made working buffer of 25 mM Tris pH 7.6, 150 mM NaCl, and 5 mM DTT at 20°C. To enable work in the picomolar concentration range (Zhao et al , 2014), cell assemblies with double‐sector 12‐mm charcoal‐filled Epon centerpieces and quartz or sapphire windows were filled with working buffer and screened in an initial blank FDS‐SV run to ensure no obvious contamination with fluorescent signal. Buffer was then taken out, and SPOP samples were inserted into the cell assemblies.…”

“…Integration between 1 and 6 S was carried out to generate the isotherm analysis of signal‐weighted sedimentation coefficients, s w . The signal contribution of BSA was subtracted from the low concentration samples where its signal is significant, as described previously (Zhao et al , 2013c, 2014). The resulting s w isotherm was loaded into SEDPHAT and fit with the homo‐dimerization model for nonlinear least square analysis,$s_{w}false(c_{\mathrm{tot}}false)=\frac{c_1{s}_1+2K_{12}{c}_1^2{s}_2}{c_{\mathrm{tot}}}$in which s 1 and s 2 are the s ‐values for monomer and dimer, respectively, c 1 and c tot denote the molar concentration for monomer and protomer, respectively, and K 12 indicates the equilibrium association constant ( K 12  = 1/ K D ) (Zhao et al , 2013c).…”

Protein Assembly in Serum and the Differences from Assembly in Buffer

“…Since the total macromolecular concentration of serum is on the order of 60-80 mg/ml, making it a highly concentrated medium that nearly is optically opaque at the wavelengths for typical protein absorptions, the absorbance and interference optical systems are not amenable for detection. However, we and others have found that the fluorescence detection system (FDS), when coupled with the SV approach, is particularly well suited for studies with concentrated solutions (Kroe & Laue, 2009;Lyons, Lary, Husain, Correia, & Cole, 2013;MacGregor, Anderson, & Laue, 2004;Zhao, Mayer, & Schuck, 2014). Moreover, the sensitivity and wide dynamic range of the FDS allow for a broader and more biologically relevant binding affinity space to be explored; notably, pM-μM, and therefore, it can provide a more complete assessment of the biologically relevant distributions of assembled species that often occur over a broad concentration range, and in many different buffer compositions.…”