Skip to comments.CRYSTALLOGRAPHY
Posted on 02/28/2014 8:12:13 PM PST by neverdem
Since modern crystallography dawned with X-ray diffraction experiments on crystals by Max von Laue in 1912 and William and Lawrence Bragg (a father-and-son team) in 1913, and was recognized by Nobel prizes in physics for von Laue in 1914 and the Braggs in 1915, the discipline has informed almost every branch of the natural sciences. This Nature special issue explores the highlights, evolution and future of the field. And in July 2014, NPG will publish Nature Milestones: Crystallography to further celebrate the International Year of Crystallography
(Excerpt) Read more at nature.com ...
I checked 4 links at the Nature source. All of them opened to their stories, but I know if all will open without a subscription.
FReepmail me if you want on or off my health and science ping list.
I don’t have a subscription but I was able to access an article.
Crystallography is a wonderful technique, but not every molecule can be crystallized.
I earned my PhD by studying a protein that is too unstable to be crystallized. Its structure had to be deduced by other methods. Everyone in the lab got excited when someone managed to crystallize a small part of a similar protein, because it gave us clues as to the real structure of “our” protein...
There have been such advances in crystallography in the past couple decades. Molecules that were thought too difficult to crstalize have been. Membrane proteins, including a number of GPCR and the ribosome.
These feats have been awarded Nobel prizes.
Yet, I feel in the ribosome case, one of the pioneers in ribosome structure and function wasn’t awarded the prize as they gave it to three crystallographers for, to my mind, essentially confirming the structure he’d determined by other means.
I am aware of the advances being made to crystallize membrane proteins, as that particular field was advancing when I was in graduate school in the late 1990s.
The problem with the protein I worked with was that it is incredibly unstable. I used to synthesize it in vitro with 35S labeling; even if I ran a gel immediately after synthesis, the lane was a mess of degradation products. I never quantitated how much was lost to degradation, but I can estimate that less than a quarter—probably less than 10%—of the protein was intact.
That instability makes crystallography impossible.
We were collaborating with a crystallographer to try to crystallize smaller fragments of the protein, for instance, just the basic helix-loop-helix domain—but we just couldn’t synthesize it in quantities sufficient for crystal studies. So when someone published a crystal structure of a related basic helix-loop-helix protein, we were ecstatic.
It’s a lot harder to detemine structure without being able to get crystals. you need to be more clever.
Computer modeling and force fields are very useful tool now. Back when you were doing that they quite nascent and needed expensive computers.
Did you ever figure out why your protein was so unstable?
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?
Anyone ever figure out why it was cut up so readily?
As I said, there is a very good biological reason the protein is unstable. Degradation is the main mechanism of down-activation of this protein once it has been activated; its function is necessary, but absolutely must be turned off once it has fulfilled its function. Its continued activity is lethal. This protein is the aryl hydrocarbon receptor, which was first identified for its role in mediating dioxin related toxicity.
As for how it degraded so rapidly despite every effort to prevent degradation--I just don't know. You would think that boiling in a beta-mercaptoethanol/SDS buffer would deactivate any protease that might be present, but no!
“As for how it degraded so rapidly despite every effort to prevent degradation—I just don’t know.”
That was my question because you are right about how weird that is.
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