Active applications

Introduction

In the more active applications of fluorescent proteins5, biochemical parameters such as metabolite concentrations, enzyme activity, or protein–protein interactions can be detected by their effects on the fluorescence properties of the designed indicators. Such indicators can be further divided into molecules with single chromophores versus composites in which the emission intensity is dependent on the energy transfer between two chromophores.

Description6

The availability of a broad selection of colors of fluorescent proteins has enabled researchers to develop methods to probe whether two proteins are within a distance of less than 10 nm of each other. The observation of colocalization for two different proteins-of-interest fused to different colors of fluorescent protein is insufficient to address this question, since the theoretical resolution limit of conventional optical imaging is several hundred nanometers. To obtain information on the proximity of two proteins at better than 10 nm resolution, investigators exploit the phenomenon of Förster (or fluorescence) resonance energy transfer (FRET)20,21.

The greater the extent of the spectral overlap between the donor emission and the acceptor excitation, the more efficient the energy transfer is for a particular FRET pair of fluorescent proteins. Since spectral overlap is constant, and orientations are assumed to be random, the efficiency of FRET is generally a good indicator of distance between the donor and acceptor fluorescent proteins. Accordingly, by expressing the donor fluorescent protein as a fusion with one protein-of-interest and the acceptor fluorescent protein as a fusion with a second protein-of-interest, the distance between the two proteins-of-interest can be inferred from the FRET efficiency measured using live cell fluorescence microscopy.

In contrast to intermolecular FRET, used for the investigation of protein-protein interactions, intramolecular FRET between two fluorescent proteins fused in the same polypeptide chain can be used to investigate small molecule dynamics and enzyme activities in a live cell. These intramolecular FRET constructs are often referred to as "reporters" or "biosensors" of biochemical activities5. One of the most well known examples of such reporters are the "Cameleons" which enable imaging of intracellular calcium ion concentrations22. Although the BFP-EGFP pair was the first set of engineered fluorescent proteins that had the appropriate spectral overlap to allow their use as a FRET pair, it was soon superseded by the still popular CFP-YFP pair that has been used in vast majority of FRET experiments to date22. However, with the advent of the numerous Anthozoa-derived fluorescent proteins, a wide variety of new FRET pair combinations has been explored. One application that has been made possible with these new combinations is the simultaneous imaging of two different FRET pairs in a single cell23.

The general design of FRET-based fluorescent probes is shown in figure:

[From Ref. 5] a) An intermolecular fluorescence resonance energy transfer (FRET)-based probe consists of two different proteins (X and Y) that are labelled with CFP and YFP, respectively, which interact and bring the fluorophores into close proximity, thereby increasing the FRET efficiency. b) An intramolecular FRET-based probe consists of either a cleavable linker or a conformationally responsive region sandwiched between a FRET pair. c) Cameleon is an intramolecular FRET-based probe that is used to measure intracellular Ca2+. d) Intramolecular phosphorylation-sensitive FRET probes have been constructed with specificities for various different kinases.


Variants derived from Aequorea green fluorescent protein have proven to be fairly tolerant of a variety of dramatic structural manipulations, including the genetic insertion of a second protein or protein domain24,25, circular permutation24,26, and even splitting into two polypeptide chains that are competent to fold into a functional fluorescent protein when brought into close proximity. In certain cases, fluorescent protein chimeras that incorporate a genetically inserted second protein, or circularly permuted fluorescent proteins with interacting proteins or peptides fused to the new N- and C-termini, can be used as single fluorescent protein-based (as opposed FRET-based) biosensors. These biosensors are designed such that the binding of the second protein to its ligand, or the ligand-dependent interaction of the attached proteins and/or peptides, results in a change in the protein environment (and thus the fluorescence properties) of the chromophore. Single fluorescent protein-based biosensors have been successfully used for the imaging of localized calcium ion24,27,28 and hydrogen peroxide29 concentrations.

Yet another strategy for the creation of single fluorescent protein-based biosensors is to modify the beta-can itself, such that the fluorescence properties of the chromophore are dependent on external factors. Some representative examples of biosensors of this type include ones for halide ions30 and cellular redox potential31,32.

[From Ref. 5] a) Aequorea fluorescent protein (AFP) can be engineered to be directly sensitive to a small molecule of interest.

[From Ref. 5] b) Insertion of a conformationally responsive domain into AFP can result in a chimaera in which fluorescence is modulated by the conformational change.

[From Ref. 5] c) Alternatively, interacting proteins (or peptides) can be fused to the amino and carboxyl termini of circularly permuted AFP.

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