Recently while working on a alanine mutagenesis scanning, I noticed a single amino acid change (Ala to Phe) of one of the residue in a particular extracellular loop of a multispan transmembrane protein caused multiple folds of increased in total protein expression as observed on WB, and 2-3 folds of increased surface expression as detected in flow cyto (compared to wild type). However, functional analysis showed it attenuates the functionality up to 20 folds as compared to wild type protein. Typically, increased protein (for wild type) expression should increase the function of the protein, we found that increased expression of this mutant follows the same trend (but even 10 times more plasmid used for transfection, its function only increases slightly, still about 10 folds lower activity than wild type). Meaning, the lack of activity is not because of the expression since the mutant is more expressed, it's just that more expresion are probably required for the mutant to have comparable activity to the wild type.
I am scratching my head hard lately. What do you think could be the possible reason?
Any opinions are welcome.