Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

Abstract

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins.

The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms

“…GroEL was purified following protocols outlined previously (Voziyan and Fisher, 2000 ; Lea et al, 2016 ). GroEL stock solutions were stored in GroEL buffer (50 mM Tris, 50 mM KCl, 10 mM MgCl 2 , 0.5 mM EDTA, pH 7.5) at 50 μM tetradecamer with 50% glycerol at 4°C.…”

“…If the sample standard deviations are equal, a score of 0.5 indicates there are 12 standard deviations separating the sample means. This data was collected on the single-channel BLItz unit and the assay sensitivity and reproducibility can be improved if the eight-channel Octet is utilized (Lea et al, 2016 ).…”

“…The vWF denaturant pulse assays were performed on an automated eight-channel Octet RED96 instrument ( forté BIO) shaking at 1,000 rpm, 25°C. For detailed programming of the denaturant pulse system, see our previous publication (Lea et al, 2016 ). The programmed steps were as follows with the urea range from 0 to 7 M by 1 M step:…”

“… Generation of a Kinetically Controlled Denaturant Pulse Isotherm for the Wild-type von Willebrand Factor A1-A2-A3 Triple Domain Fragment. (A) Protein is partially denatured during automated urea pulse steps, and (B) the GroEL binding amplitude was (C) plotted as a function of urea concentration to generate a kinetically controlled denaturation isotherm (Lea et al, 2016 ). …”

“…This ATP dependent reversal of protein binding to GroEL results in a return to the baseline before capture indicating that the GroEL-protein interaction specific for the GroEL binding site (Naik et al, 2014 ; Pace et al, 2018 ). If stabilization of the protein is achieved either through the alteration of the formulation solution, the addition of stabilizing ligand, or genetic engineering, the capture by the GroEL biosensor is diminished, sometimes quite dramatically (Naik et al, 2014 ; Lea et al, 2016 ; Pace et al, 2018 ).…”