Quantification of QuaternaryStructure Stability inAggregation-Prone Proteins under Physiological Conditions: The TransthyretinCase

摘要

The quaternary structure stability of proteins is typically studied under conditions that accelerate their aggregation/unfolding processes on convenient laboratory time scales. Such conditions include high temperature or pressure, chaotrope-mediated unfolding, or low or high pH. These approaches have the limitation of being nonphysiological and that the concentration of the protein in solution is changing as the reactions proceed. We describe a methodology to define the quaternary structure stability of the amyloidogenic homotetrameric protein transthyretin (TTR) under physiological conditions. This methodology expands from a described approach based on the measurement of the rate of subunit exchange of TTR with a tandem flag-tagged (FT2) TTR counterpart. We demonstrate that subunit exchange of TTR with FT2·TTR can be analyzed and quantified using a semi-native polyacrylamide gel electrophoresis technique. In addition, we biophysically characterized two FT2·TTR variants derived from wild-type and the amyloidogenic variant Val122Ile TTR, both of which are associated with cardiac amyloid deposition latein life. The FT2·TTR variants have similar amyloidogenicpotential and similar thermodynamic and kinetic stabilities comparedto those of their nontagged counterparts. We utilized the methodologyto study the potential of the small molecule SOM0226, a repurposeddrug under clinical development for the prevention and treatment ofthe TTR amyloidoses, to stabilize TTR. The results enabled us to characterizethe binding energetics of SOM0226 to TTR. The described techniqueis well-suited to study the quaternary structure of other human aggregation-proneproteins under physiological conditions.