Is there any interference of GFP-tag on protein structure and function?

If the linker between the target protein and the GFP is flexible then it most probably will not interfere with the target protein structure. However if there is no linker or the GFP is fused directly to a structural fold then steric hindrance may distort the structure.

Thanks Nathan for the info., I also received very detailed info. from other colleagues working on protein chemistry.

I just release the information for some other researchers in case if they are interested to know.

It is not true that GFP doesn't affect proteins. Sometimes it does and sometimes it doesn't.

There are many different scenarios;

1) GFP sequence is complementary to regions of the gene. This may cause the mRNA to fold into secondary structures by base complementarity between the gene sequence and GFP sequence. This is not too likely since we do not produce these GFP proteins and therefore our genes aren't likely to contain this sequence either. But this is not true for introns, which can mutate relatively freely so some intronic sequence may be complementary to GFP sequence by chance. If the mRNA or it's precursors (with introns) folds into secondary structures due to GFP sequence then maybe the mRNA will fail to be spliced/processed, fail to be exported from the nucleus to the cytoplasm or interfere with translation. This may cause none/low levels of the protein to be expressed and therefore low signal of fluorescence.

2) Sometimes we are interested in where a protein is, in the cell. So we can tag it with GFP. But proteins which respond to different signals or which have a defined cellular location under different conditions are 'told' to go to these locations by sequences in the amino acids. GFP can interfere with that. If a protein is needed in the endoplasmic reticulum (ER) or needs to be exported; its mRNA can be translated by ribosomes which sit on the ER. Whilst the protein is being synthesized, it is being fed through the ER membrane at the same time. GFP can interfere in this process.

3) GFP can interfere with the function of the protein. Maybe by causing the protein to fold differently. Maybe the active site of the protein is at the same site which GFP is fused. This could block the active site so that other molecules cannot bind or interact with the protein causing it to lose its function. Maybe now it can't interact with other proteins or can't bind to DNA and turn up or down the expression of other genes.

Whenever you tag a protein with GFP it's best to try both N- and C-terminal linkers. And you always want to have some test to show that you have not substantially altered the protein behavior such as an activity assay. I like GST because you can follow the protein even in cell expression and during purification for biochemistry experiments. For in vivo experiments having a self-forming fluorescent probe is ideal for many experiments. It's probably safest to assume that there is some effect and then test to determine if that effect is large enough to alter your experiment significantly. Mostly it is useful and powerful tool. I hope it works well for you.

Thanks John for your feedback and guidance.