Biochemical tools are needed to understand the molecular mechanisms by which metal ions affect abundance and colonization of gut bacteria and resistance to pathogens. The overall goal of this research is to develop and apply new metal ion biosensors for elucidating metal dynamics in complex systems like the gut microbiota. Fluorescent protein reporters are among the most powerful tools for studying live biological systems and can be used to detect and track biological analytes like metals. To determine how metal ions are used by gut bacteria and probe the competition between bacterial species and hosts, novel sensors with oxygen independence and near-infrared (NIR) emission are required. Although fluorescent metal ion sensors are indispensable tools for studying metal homeostasis in living systems, current protein-based platforms cannot be applied to the many gut bacteria that can only grow under oxygen-free conditions and do not lie in the preferred NIR transparency window for in vivo imaging. To achieve these requirements, we are investigating the use of several oxygen-independent and NIR-emitting cofactor-based fluorescent proteins as platforms for new sensors for metals such as zinc and copper. Using fluorescence intensity and fluorescence lifetime-based detection approaches, we report that the flavin-binding fluorescent protein CreiLOV selectively binds copper(II) with a micromolar affinity over other biologically relevant metal ions in cuvettes and in live bacterial cell suspensions. Site-directed mutagenesis of CreiLOV can lead to increased metal-binding affinities, facilitating its use as a metal ion sensor. We will discuss an orange fluorescent cyanobacteriochrome protein that is sensitive to zinc ions.  Finally, we will share our progress developing NIR-emitting fluorescent protein-based sensors for detecting transition metals.

Designing new oxygen-independent metal ion sensors using cofactor-based fluorescent proteins

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.