Monitoring GTPCH‐1 Interaction with GFRP Using Time‐Resolved Fluorescence Resonance Energy Transfer

Abstract

GTP Cyclohydrolase I (GTPCH‐1) is the rate‐limiting enzyme for tetrahydrobiopterin (BH4) synthesis and we have recently shown that it is inhibited by GTP Cyclohydrolase Feedback Regulatory Protein (GFRP) in the endothelium. The complex formation of GTPCH‐1 and GFRP is modulated by several factors including phosphorylation of the former and mechanical forces acting upon the endothelial cell. We have designed and optimized a time‐resolved fluorescence resonance energy transfer (TR‐FRET) assay to monitor the interaction of GTPCH‐1 with GFRP in the presence of GTP and BH4 as cofactors. This assay uses GTPCH‐1 labeled with a europium chelate as FRET donor and GFRP labeled with APC as acceptor and requires low concentrations of proteins to achieve an optimal FRET signal. This assay is highly sensitive and stable and has achieved a robust performance in 384‐well and 1536‐well formats with a signal‐to‐background ratio greater than 12 and a Z’ factor greater than 0.75. In the absence of GTP and BH4 this assay only generates a background signal, confirming that these are needed for protein interaction and demonstrating specificity of the assay. Because it features a one‐step “mix‐and‐read” format, this assay could be particularly suited for use in high throughput screening for GTPCH‐1 modulators. Supported by HL39006 and a VA Merit Grant.