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Abstract Molecular recognition of proteins is key to their biological functions and processes such as protein–protein interactions (PPIs). The large binding interface involved and an often relatively flat binding surface make the development of selective protein-binding materials extremely challenging. A general method is reported in this work to construct protein-binding polymeric nanoparticles from cross-linked surfactant micelles. Preparation involves first dynamic covalent chemistry that encodes signature surface lysines on a protein template. A double molecular imprinting procedure fixes the binding groups on the nanoparticle for these lysine groups, meanwhile creating a binding interface complementary to the protein in size, shape, and distribution of acidic groups on the surface. These water-soluble nanoparticles possess excellent specificities for target proteins and sufficient affinities to inhibit natural PPIs such as those between cytochrome c (Cytc) and cytochrome c oxidase (CcO). With the ability to enter cells through a combination of energy-dependent and -independent pathways, they intervene apoptosis by inhibiting the PPI between Cytc and the apoptotic protease activating factor-1 (APAF1). Generality of the preparation and the excellent molecular recognition of the materials have the potential to make them powerful tools to probe protein functions in vitro and in cellulo. 

Pyk2 is a multi-domain non-receptor tyrosine kinase that serves dual roles as a signaling enzyme and scaffold. Pyk2 activation involves a multi-stage cascade of conformational rearrangements and protein interactions initiated by autophosphorylation of a linker site. Linker phosphorylation recruits Src kinase, and Src-mediated phosphorylation of the Pyk2 activation loop confers full activation. The regulation and accessibility of the initial Pyk2 autophosphorylation site remains unclear. We employed peptide-binding molecularly imprinted nanoparticles (MINPs) to probe the regulatory conformations controlling Pyk2 activation. MINPs differentiating local structure and phosphorylation state revealed that the Pyk2 autophosphorylation site is protected in the autoinhibited state. Activity profiling of Pyk2 variants implicated FERM and linker residues responsible for constraining the autophosphorylation site. MINPs targeting each Src docking site disrupt the higher-order kinase interactions critical for activation complex maturation. Ultimately, MINPs targeting key regulatory motifs establish a useful toolkit for probing successive activational stages in the higher-order Pyk2 signaling complex.