Mutants had lower fitness. That’s it. They functioned worse in comparison to the conserved sequence. It’s trivial.
We already know this is the case merely by looking at variants of the sequence in the human population. As I showed you 8 days ago in this post.
You can’t calculate FI if you have not actually tested unexplored regions of sequence space, because then you don’t know the actual ratio of nonfunctional to functional sequences.
Heck, even for the blatant misapplication of FI Gpuccio has managed to dream up, he’d still have to include every known variant of the protein that exists in all populations, not just the single canonical sequence. If some organism lives with a mutant version without dying and is capable of reproducing, then the sequence is strictly viable, and needs to be included in any calculation that attempts to derive the fraction of sequences that meets the minimal threshold for function. But Gpuccio has not done so.
Also, the “precision” of the method Gpuccio is using to try to gauge the number of sequences that meet the minimal threshold for function is probing so vanishingly small an area of sequence space that any number he comes up with basically commits a hasty generalization fallacy. He simply cannot extrapolate one small area of sequence space to the rest of it when there is zero reason to think the functional and nonfunctional sequences are uniformly distributed in that space.