Fluorescent Protein Sensors of Post-Translational Modifications

Abstract

The present invention includes a fluorescent compound that can detect an activity, such as an enzymatic activity, and exhibits quenching. The fluorescent compound can include a fluorescent protein, such as an Aequorea-related green fluorescent protein. The fluorescent compound can include a substrate site for an enzymatic activity such as a kinase activity, a phosphatase activity, a protease activity, and a glycosylase activity. The fluorescent compound of the present invention can be used to detect such enzymatic activities in samples, such as biological samples, including cells. The present invention also includes nucleic acids that encode the fluorescent compounds of the present inventions, and cells that include such nucleic acids or fluorescent compounds.

Description

TECHNICAL FIELD[0001]The present invention generally relates to compositions and methods for the detection of activities, such as enzymatic activities, using fluorescent compounds that are modified by the activity such that they exhibit a chance in their sensitivity to quenching.BACKGROUND[0002]Fluorescent compounds have been used in the art to detect a wide variety of biological phenomenon, such as changes in ion concentration, specific binding reactions, subcellular localization, and enzymatic reactions. In the case of detecting changes in ion concentration, specific binding reactions, and subcellular localization, the fluorescent compound is used as a label to detect such specific binding or localization. In some cases, the fluorescence of the fluorescent compound is altered after an enzyme has acted on the fluorescent compound or a molecule binds with the fluorescent compound. For example, the activity of beta-galactosidase on the substrate fluorescein di-beta-D-galactopyranosid...

AI Technical Summary

Benefits of technology

[0019]The present invention recognizes that fluorescent compounds, such as fluorescent proteins, can exist in at least two states and that the fluorescent properties of these two states can be different, preferably after exposure of the fluorescent compound to quenching conditions. Particularly, the stability of the fluorescence in one state can be different from the stability of the fluorescence in the second state, which can be detected by their susceptibility to quenching. The first and second states of the fluorescent compound can be caused by the action of a chemical or enzyme. Thus, the pres...

Problems solved by technology

Exposure of the fluorescent compound to chemicals or enzymes can cause such modifications.

Such moieties can also destabilize the tertiary structure of a fluorescent protein, which can result in destabilized fluorescence under quenching conditions.

Furthermore, enzymatic activities such as proteases can alter the tertiary structure of a fluorescent protein, which can also result in destabilized fluorescence under quenching conditions.

Many variations in the above sequence are allowed, but generally exhibit poorer kinetics.

Specifically, replacement of Glu 5 or Glu 6 by a non-charged Ser or Thr residue can significantly disrupt the fluorescence, folding, or sensitivity of GFP to quenching.

Although this motif produces a codon distribution as equitable as available with standard methods of oligonucleotide synthesis, it results in a bias against peptides containing one-codon residues.

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