Biologically, we had a very good reason for the instability of the protein.
Chemically, I was never able to show anything definitive. I loaded up my reactions with protease inhibitors, but the protein showed the same degradation pattern on gels. There was always the intact protein band at the top (about 95 kDa), another major band at about 50 kDa, a third major band at about 30 kDa, and between the major bands were several minor bands/smear.
I know that the cDNA had some cryptic Kozak sequences, which could explain the two smaller major bands. But they did not account for the minor bands/smear.
If I were to make a standard control (from liver extracts) for Western blotting, and store it ready to load on a gel in the -20, it would degrade within a couple of weeks. That was after it was mixed with the beta-mercaptoethanol/ SDS loading buffer and boiled for 2 minutes. The raw extracts and in vitro synthesized proteins had to be stored at -80.
Needless to say, for structural studies, this protein was quite frustrating. However, I was able to generate some very nice functional and protein interaction data, sufficient to earn a PhD, and I can’t complain about that.
I have not worked with that protein since graduate school, and I don’t know if there is any structure data other than computer models.
I must say, it is unusual to encounter someone on FR who understands the language of biochemistry. Most of my PhD scientist colleagues at work don’t even understand it. :)
I did my PhD in the late 80’s. I studied structure-function of GPCR.
When I began, the amino acid sequences weren’t known. The nAchR was known, but it’s topography in the membrane was still debatable. When rhodopsin was sequenced, and then the b-adrenergic receptor was found to be homologous, molecular biological methods allowed the protein sequences to be known for many of them via cloning.
Now there’s the sequence of so many genomes.
As for your protein, a protein that gets cleaved up like that in storage is rare and almost nightmarish.
Anyone ever figure out why it was cut up so readily?