If GFP is fused to a mitochondrial targeting signal peptide, we know it gets expressed and imported into the mitochondrion, where its fluorescence can be visualized. But we also know that proteins that are imported into the mitochondrion in this manner are unfolded and fed through the TOM/TIM complex as a strand, and then refolded on the inside. And thirdly, we know that the GFP chromophore is formed by irreversible spontaneous reaction between residues on the inside of the barrel structure, that these residues are not all adjacent to each other on the unfolded protein strand sequence, and that the chromophore has covalent bonds that permanently crosslink those residues. So how does the GFP chromophore remain intact during mitochondrial import?
I think you already gave the answer.
To be clearer: Have you ever tested if your GFP is in fact IN your mitochondria?
Hi,
The chromophore of GFP is made from Ser65–Tyr66–Gly67. So the amino acid residues are next to each other in unfold primary sequence. All fluorescent proteins (GFP and GFP-like) have conserved region of Tyr-Gly (a part that made up chromophore). This may be the reason why the chromophore can remain intact during import process to mitochondria.
Hi Beverley,
it is a fundamental question on GFP / GFP fusion protein folding. GFP per se fold into its native 3D structure in second order, and chromophore maturation in minute order, and this folding is sequential and an autocatalytic reaction.Thus, after synthesis, GFP fold without chaperones / carrier proteins and randomly diffuse to the most part of the cell. To contrast, GFP fusion protein (in you case GFP with Mt. signal peptide) folding depend on several factors. e.g. solubility and position of the fusion protein placed, and linker sequence etc. In this case, I think, GFP fold first and then participate import process. as you know, GFP is very stable protein and once it fold do not unfold while it transporting. For this reason, the chromophore remain intact during the process. Note that folded GFP can unfold (95C in 8M urea for 5 min), but it quickly fold back to native structure when it dilute into proper buffer. This is beyond your question. My question is do you see the GFP fluorescence and it is located in Mt. ?
I would rather argue that GFP is first imported and then folds inside the mitochondria. Import process in this case occurs faster than the folding either due to co-translational insertion (reported for some mitochondrial proteins) or due to slower folding of the fusion GFP variant. Once imported it can fold and 3D structure of GFP facilitates autocatalytic formation of the chromofore.
As was mentioned chromofore is a tripeptide within the GFP sequence, so it cannot be lost during the import process, it is a part of the protein. However it only becomes a fluorophore once GFP is folded and intramolecular environment favors cyclization of the tripeptide.
Regarding import vs folding speed: if you have a GFP folding faster than import (example: superfolder GFP) it will not be imported into the organelle, but rather will end up stuck at the pore, unable to go inside.
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases