Development and Characterization of Conformation-Preferring Antibodies Targeting Phosphorylated Threonine 19 in...

Activity-dependent synaptic plasticity is governed by posttranslational mechanisms that regulate the stability and molecular organization of postsynaptic protein complexes. Proline-directed phosphorylation of the N-terminus of PSD-95 promotes synaptic weakening during NMDAR-dependent LTD, yet this type of phosphorylation also alters the cis-trans isomerization of the adjacent peptidyl-prolyl bond. Despite its predicted importance, these conformational changes have not been directly measured using existing molecular tools. Here we describe the development of novel conformation-preferential antibodies that distinguish structural states of PSD-95 when threonine 19 (T19), a site implicated in NMDAR-LTD, is phosphorylated. These antibodies were validated biochemically and in cellular assays, where signal increased following GSK3β-mediated phosphorylation and was lost upon dephosphorylation. These reagents represent the first conformation-preferential antibody-based tools capable of reporting phosphorylation-dependent conformational states of PSD-95 at T19. This strategy validates and expands prior framework for developing conformational-sensitive antibodies, an approach that can be applied to other synaptic proteins.