Fluorescent protein sensors for detection of analytes

A fluorescent indicator comprising:

• a target peptide moiety;
• a binding protein moiety having an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte, and a peptide-binding region for binding the target peptide moiety;
• a linker moiety that covalently couples the binding protein and the target peptide moiety and is a peptide moiety;
• a donor fluorescent protein moiety covalently coupled to the binding protein moiety;
• and
• an acceptor fluorescent protein moiety covalently coupled to the target peptide moiety,
• wherein the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited, and the indicator is a single polypeptide.

A method for determining the concentration of an analyte in a sample comprising:

• contacting the sample with a fluorescent indicator comprising a binding protein moiety having an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte, a donor fluorescent protein moiety covalently coupled to the binding protein moiety, and an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety, wherein the donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region, altering fluorescence resonance energy transfer between the donor moiety and the acceptor moiety when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the degree of fluorescence resonance energy transfer in the sample corresponding to the concentration of the analyte in the sample.