Molecular sensing fluorescent proteins and FRET pairs

[rwooduk]

Hope this is in the right place, apologies if not.

We have studied green flourescent protein GFP and others (RFP, CFP etc) as a means of analysing an organism / sample, you need two that have unique fluorophores. We have also studied the use of FRET pairs such as florecein-florescien or florecein-rhodamine for studying interatomic distance / molecular structure.

My question is are flourescent proteins and FRET pairs related or are they for completely different analyses?

Think I'm getting the two mixed up, or are they related? i.e. do FP's contain the chemical in Fret pairs?

Hope this question makes some kind of sense I am really not a biologist!

 

[Ygggdrasil]

In biology, we often make use of fluorescence to visualize molecules. Fluorescent molecules will absorb light of a certain wavelength, and emit light of a slightly longer wavelength. The two most common types of fluorescent probes used in biology are small organic dyes (such as fluorescein or rhodamine) and fluorescent proteins (such as GFP, YFP, CFP, etc.).

Förster Resonance Energy Transfer (FRET, sometimes referred to as fluorescence resonance energy transfer) is a phenomenon that can occur with any pair of fluorescent molecules. When FRET occurs, one fluorophore can transfer its energy to a fluorophore that emits at a longer wavelength. This process can occur for any fluorophore, so you can have FRET occur between organic dyes (such as the fluorescein-rhodamine pair you mentioned), between fluorescent proteins (CFP-YFP is a popular pair of proteins to use for this purpose), or between an organic dye and a fluorescent protein.

 

[rwooduk]

In biology, we often make use of fluorescence to visualize molecules. Fluorescent molecules will absorb light of a certain wavelength, and emit light of a slightly longer wavelength. The two most common types of fluorescent probes used in biology are small organic dyes (such as fluorescein or rhodamine) and fluorescent proteins (such as GFP, YFP, CFP, etc.).

Förster Resonance Energy Transfer (FRET, sometimes referred to as fluorescence resonance energy transfer) is a phenomenon that can occur with any pair of fluorescent molecules. When FRET occurs, one fluorophore can transfer its energy to a fluorophore that emits at a longer wavelength. This process can occur for any fluorophore, so you can have FRET occur between organic dyes (such as the fluorescein-rhodamine pair you mentioned), between fluorescent proteins (CFP-YFP is a popular pair of proteins to use for this purpose), or between an organic dye and a fluorescent protein.

The problem I'm having is that if you use florecein-florescien then how would you be able to distinguish between the two in FRET? if that makes sense.

aside from that, that's very helpful thank you!

 

[Ygggdrasil]

Measuring the FRET efficiency between two identical fluorophores (homo-FRET) is difficult because the donor and acceptor fluorescence are not spectrally distinct, but it is possible. Measuring homo-FRET relies on looking at the anisotropy of the emitted light; that is, the polarization of the emitted light compared to the polarization of the excitation light.

When you excite a sample with polarized light, only those fluorophores whose transition dipoles align with the light will be excited. If these fluorophores are rigidly attached to large (greater than tens or hundreds of kilodaltons) molecules, then you would not expect these molecules to rotate much during the lifetime of the excited state (~ a few nanoseconds), so the emitted fluorescence will have the same polarization as the excitation light. FRET, however, will tend to randomize the polarization of the emitted light because FRET can transfer energy to fluorophores whose transition dipoles were not aligned with the original excitation light. Thus, fluorescence anisotropy measurements allow you to measure the FRET efficiency between fluorophores with identical or near-identical emission spectra.

 

[rwooduk]

Measuring the FRET efficiency between two identical fluorophores (homo-FRET) is difficult because the donor and acceptor fluorescence are not spectrally distinct, but it is possible. Measuring homo-FRET relies on looking at the anisotropy of the emitted light; that is, the polarization of the emitted light compared to the polarization of the excitation light.

When you excite a sample with polarized light, only those fluorophores whose transition dipoles align with the light will be excited. If these fluorophores are rigidly attached to large (greater than tens or hundreds of kilodaltons) molecules, then you would not expect these molecules to rotate much during the lifetime of the excited state (~ a few nanoseconds), so the emitted fluorescence will have the same polarization as the excitation light. FRET, however, will tend to randomize the polarization of the excited light because FRET can transfer energy to fluorophores whose transition dipoles were not aligned with the original excitation light. Thus, fluorescence anisotropy measurement can allow you to measure the FRET efficiency between fluorophores with identical or near-identical emission spectra.

wow, that's a very detailed explanation and clears things up immensely, thank you very much!