Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis.
Kinetic stabilization of transthyretin (TTR) is established to prevent human neurodegeneration. Therefore, small molecule-mediated kinetic stabilization of the native state is an attractive strategy to prevent the misfolding and misassembly associated with TTR amyloid disease. Since the physiological microenvironment resulting in human TTR amyloidogenesis remains unclear, the conservative approach is to identify inhibitors that function under a variety of conditions. Small molecule kinetic stabilization of TTR has been established by concentration-dependent inhibition of acid-mediated amyloidogenesis and urea-induced tetramer dissociation. Since denaturing conditions reduce the binding affinity of inhibitors making it difficult to predict inhibitor efficacy under physiological conditions, we introduce a method for quantifying kinetic stabilization under physiological conditions. The rate of subunit exchange between wild-type TTR homotetramers and wild-type TTR homotetramers tagged with an N-terminal acidic flag tag is dictated by the rate of tetramer dissociation to its monomeric subunits prior to reassembly, rendering this method ideally suited for assessing the kinetic stabilization of TTR imparted by small molecule binding and evaluating small molecule binding constants. Addition of amyloidogenesis inhibitors to this exchange reaction slows tetramer dissociation in a concentration-dependent manner, stopping dissociation at concentrations where at least one inhibitor is bound to each tetramer in solution. Subunit exchange enables the rate of tetramer dissociation and the kinetic stabilization imparted by small molecule binding to be evaluated under physiological conditions in which the TTR concentration is not reduced by aggregation or irreversible dissociation.
Wiseman
R Luke
RL
The Department of Chemistry and the Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road BCC 265, La Jolla, California 92037, USA.
Green
Nora S
NS
Kelly
Jeffery W
JW
D000686:Amyloidosis / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D011228:Prealbumin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D011228:Prealbumin; D021122:Protein Subunits