Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity

Abstract

Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid–liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun.2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer–dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately −9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data

“…Yang et al (2018) showed that increasing salt suppresses mAb association and allows k s values to increase and approach values of 10 ml/g. Recently Chaturvedi et al (2019) and Connolly et al (2012) reported k s values > 20 ml/g in low salt conditions. Many investigations vary salt concentration and observe a suppression of association and reduction in viscosity (Yadev et al 2012).…”

“…It is generally assumed that the correct concentration scale for high concentration work is volume fraction (Ross and Minton 1977) and recent AUC studies on high concentration use the volume fraction Φ scale (Chaturvedi et al 2018;Chaturvedi and Schuck 2019). The conversion is in principle a linear transformation, as simple as νc (Broide et al 1991), but more realistically V s c where the swollen or effective volume is used (Rowe 1992;Chaturvedi and Schuck 2019). At high volume fraction the shape and packing considerations may become important for mAbs (Garidel et al 2017).…”

Quantitative Analysis of Protein Self‐Association by Sedimentation Velocity

“…Use of detection at the >290 nm flank of the protein extinction should be avoided, since the extinction coefficient gradients are accompanied by degrading signal linearity (Schuck et al, 2015). Instead, one can exploit that the interference optical system offers a much wider linear concentration range and combine this with the use of shorter optical pathlength centerpieces (Chaturvedi, Sagar, Zhao, Wistow, & Schuck, 2019). 2As total protein weight concentrations exceed 1 mg/ml, nonideality has to be taken into account.…”

“…An advantage is that this absorbs nonideality effects, and the resulting s w -values do not need to be corrected for nonideality anymore. In the concentration series, the k S -value is typically determined from the data at the highest concentration and then fixed for all lower concentrations as described in Chaturvedi et al (2019). In this fashion, ultraweak interactions with millimolar K d -values can be studied (Chaturvedi et al, 2019).…”

Impact of soft protein interactions on the excretion, extent of receptor occupancy and tumor accumulation of ultrasmall metal nanoparticles: a compartmental model simulation

“…32,42,43 However, none of these simple experimental methodologies are appropriate for the characterization of transient, ultraweak interactions characterized by very high dissociation constants and short residence times. Analytical ultracentrifugation constitutes a more robust and sensitive tool to investigate ultraweak biomolecular interactions, [44][45][46] but this has yet to be explored in more detail in the study of NP interactions. 31,47 While ultraweak interactions with dissociation constants in the millimolar (mM) range usually have no major impact on conventional biochemical assays performed in vitro under dilute solution conditions, they cannot be ignored in macromolecular crowded media.…”