The BFP and GFP are connected by a flexible linker and FRET is more higher.
The efficiency of energy transfer is strongly dependent on distance, but it is also dependent on the relative orientations of the donor and acceptor dipoles. In your construct, the flexible linker may allow the donor and acceptor to get closer together than whatever you are comparing it to, and/or the linker may allow for a more favorable relative orientation.
Adam B Shapiro Thank for your answer. Can you give me some references about relative orientation.
The orientation factor, kappa-squared, is part of the Forster theory of resonance energy transfer. Since it generally can't be measured experimentally, it is usually assumed to take the value 2/3 based on a random orientation between the donor and acceptor fluorophores. Here is a reference.
https://iopscience.iop.org/article/10.1088/2050-6120/ab8f87/pdf
Adam B Shapiro Thank you for providing me with references. You are right, κ^2 usually assumed to 2/3 but FRET pair of fluorescence portein may be inaccurate.
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