sciencesoup: The Anfinsen Experiment The previous article went...

sciencesoup:

The Anfinsen Experiment

The previous article went kind of wild with all the different structures and levels of structure within proteins, but there’s essentially one main thing that you need to take away from it all: amino acid sequence is what determines the overall structure of proteins. This idea is described neatly in an experiment by Christian B. Anfinsen.

Anfinsen chose an enzymatic protein called Ribonuclease A to work with, for two reasons: Firstly, because it’s a small, simple protein with only four particular bonds that determine its overall structure—disulfide bonds that run like little bridges between the sulphur atoms of two Cysteine amino acids. Secondly, because its function is to catalyse the hydrolysis of RNA—i.e., break down bonds by adding water. At the start, the enzyme’s activity was 100%, meaning it was busy doing its job breaking down RNA.

But then Anfinsen added a chemical compound called b-mercaptoethanol, which broke down the enzyme’s disulphide bonds and made it unfold into a linear coil. He also added 8 Mole urea, which prevented the protein from reforming the bonds. Essentially, he denatured it.

When the enzyme was fully denatured, he measured its activity again: 0%. This was hardly a surprise—the structure of a protein is fundamental to its function, and so if your protein is entirely unfolded, it’s not going to be doing much catalysing.

Anfinsen then removed the b-mercaptoethanol and urea. He left the enzyme in a tube with oxygen, and eventually, he tested the activity again—finding that it had almost completely regained its activity. Effectively, the disulphide bonds had re-oxidised and joined back up together. The enzyme had refolded itself into its proper shape and could once again catalyse the hydrolysis of RNA. Its activity hadn’t quite fully returned, but it was close.

(Ribonuclease A structure. Source: Wikimedia Commons)

The most important thing is that the protein was able to refold itself—it didn’t need any special help to regain its activity. If it hadn’t refolded itself, we would have to assume that its structure is determined by information that was present when it was synthesised—but Anfinsen showed that the protein itself dictates its own structure.

Since the protein was completely degraded, this could only mean one thing: the information for how to fold into a three-dimensional structure is determined by the most basic component of the protein, the amino acid sequence.

via Tumblr http://ift.tt/1xPkETv July 09, 2014 at 03:42AM