Figure 1. Design and development of phosphorylation-dependent enhanced Tb³⁺ luminescence tyrosine kinase peptide biosensors.

A) General biosensor design strategy for kinase biosensors capable of phosphorylation induced enhanced Tb³⁺ luminescence, where X is any amino acid, Φ is a hydrophobic antenna containing residue and [−] is an acidic amino acid. B) The detection strategy using the phosphorylation-dependent physical changes in the biosensors that result in enhanced Tb³⁺ luminescence. C) To develop a kinase specific peptide based biosensor, we first obtain all known phosphorylated substrates for a given kinase as the foreground as well as all unphosphorylated tyrosine centered sequences for the substrates and validated proteins that interact with the kinase as the background. Data from positional scanning peptide library screens from the Turk laboratory were also included²³. (1) A positional scoring matrix, where values represent the preference for each amino acid at every position, and a site-selectivity matrix (SSM), representing the degree to which a given position “requires” a given amino acid, are generated from these data. SSM values are centered at one; values greater than one reflect a strong preference for a particular amino acid at that position and values less than one reflect a lack of preference. (2) A library of sequences were generated in silico based on substrate preferences at each positions using the site selectivity score (using the“Generator™” tool). (3) The library is scored against the kinase of interest as well as all other tyrosine kinases and clustered using bidirectional Euclidian distance and filtered to remove any nonspecific or nonsubstrate sequences for the kinases based on the PSM scores (using the “Screener™” tool). Scores are on a scale from 0 to 100, where binary classification (of “Substrate” or “Non-substrate”) was determined based on threshold values through cross-validation. (4) The remaining sequences are scored using a BLOSUM matrix to assess the similarity to the phosphorylation-dependent Tb³⁺-binding α-syn Y125 peptide^24,25 (using the “Aligner™” tool), which enables filtering out of sequences that are predicted to be selective substrates but not to match the Tb³⁺ motif inherent in the target sequence (which in this case was the best-characterized model, the α-syn Y125 peptide, but could be another Tb³⁺-binding sequence of interest). (5) The remaining sequences are validated empirically for kinase specificity and photophysical properties associated with Tb³⁺ luminescence. For each relevant step, the score similarity for each kinase (columns) and sequence similarity to one another across kinases (rows) were clustered using bidirectional Euclidian distance.