Alzheimer's-disease-associated conformation of intrinsically disordered tau protein studied by intrinsically disordered protein liquid-phase competitive enzyme-linked immunosorbent assay.
Tau protein, the major constituent of paired helical filaments in Alzheimer's disease, belongs to the intrinsically disordered proteins (IDPs). IDPs are an emerging group in the protein kingdom characterized by the absence of a rigid three-dimensional structure. Disordered proteins usually acquire a "functional fold" upon binding to their interaction partner(s). This property of IDPs implies the need for innovative approaches to measure their binding affinity. We have mapped and measured the Alzheimer's-disease-associated epitope on intrinsically disordered tau protein with a novel two-step sandwich competitive enzyme-linked immunosorbent assay (ELISA). This approach allowed us to determine the binding affinity of disordered tau protein in liquid phase without any disturbance to the competitive equilibrium and without any need for covalent or noncovalent modification of tau protein. Furthermore, the global fitting method, used for the reconstruction of tau binding curves, significantly improved the assay readout. The proposed novel competitive ELISA allowed us to determine the changes in the standard Gibbs energy of binding, thus enabling measurement of tau protein conformation in the core of paired helical filaments. IDP competitive ELISA results showed, for the first time, that the tau protein C terminus of the Alzheimer's-disease-derived paired helical filaments core subunit adopts beta-turn type I' fold and is accessible from solution.
Skrabana
Rostislav
R
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 845 10 Bratislava, Slovakia.
Skrabanova-Khuebachova
Michaela
M
Kontsek
Peter
P
Novak
Michal
M
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D008968:Molecular Conformation; D016874:Neurofibrillary Tangles / Q000378:metabolism; D011487:Protein Conformation; D016875:tau Proteins / Q000737:chemistry* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016875:tau Proteins