Abstract
Protein biosensors are widely used for the monitoring of metabolite concentration and enzymatic activities inside living cells and in in vitro applications. Neutrophil elastase (NE) is a serine protease of relevance in inflammatory diseases whose activity can lead to pathological conditions if unregulated. This study focuses on the structural characterization of a biosensor for NE activity based on Förster resonance energy transfer (FRET). The cleavage by NE results in dissociation of the FRET fluorescent protein pair and alteration of the fluorescent emission spectrum. We have used small angle x-ray scattering at a high intensity synchrotron source, combined with model-free analysis of the scattering data, to demonstrate the structure of the biosensor and the effect of its exposure to NE on size and shape. These investigations, together with biochemical studies, established the nanostructure-activity relationship that may contribute to the detailed understanding of the FRET-based biosensor and guide the rational design of new biosensor constructs.