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I'm working on building some FRET reporters. In addition to a cleavage site (of varying composition from 15-18AA), a 1 AA linker, I'm using Venus and Cerulean.

Initially I was worried that 18AA would be too long, but it does in fact give a fair signal. For reference, I believe the Förster radius (Ro) is 54Å.

I am now working on further optimization. Does the removal of the intial methionine cause any problems for Venus if it is the second protein in the FRET pair? The basic layout is:

Cerulean - Link - Cleavage - Link - Venus

And I'm wondering if cutting the Met from Venus might bring me 1 AA closer and therefore a slightly better signal. I have seen it published both ways, and I was wondering if anyone had tried both or just knew theoretically that it wouldn't matter.

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    $\begingroup$ why do you want to remove the Met-1. Removal of 1 amino acid may only reduce the length by ~3Å (twice the peptide bond) and if i am not wrong Forster's radius is ~60Å and therefore the distance of interaction = 2x60Å. A 3Å reduction may not be very significant $\endgroup$ – WYSIWYG Oct 30 '13 at 7:01
  • $\begingroup$ @WYSIWYG I didn't mean to imply that the reduction of the radius was the only advantage. It also gives me a better restriction site in the DNA, and is possibly more flexible in the AA sequence. $\endgroup$ – Atl LED Oct 30 '13 at 13:27

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