What's cookin', good lookin'? (In your protein...)

With this post we're going to finish up talking about some stuff that I said I would talk about and then we're going to talk about other things later.

I talked in the last post about proteolytic enzymes and how they can chop up a protein. There's different ones and do they it in different ways. For the most part, they're going to target very specific amino acids and cut them either from the N or C terminal. There's endopeptidases and exopeptidases. Endo will cut inside the protein and exo will chop things off the ends. R1 and R2 comes from which side of the protein the stuff favors cleaving off. 

Trypsin and Chymotrypsin are both serine proteases and endopeptidases. They're both digestive enzymes and R1 proteins, so they'll be cleaving off from the N terminal.

Trypsin cleaves after the basic amino acids, but not H and chymotrypsin cleaves off the things with aromatic rings, except H, and it also cleaves after leucine. 

The way I like to remember chymotrypsin is WYFL, because it sounds like a Wiffle Ball.

The next couple are pretty self-explanatory..

There's a whole bunch of controversy in my head (and maybe in the real world) as to whether certain things will still cleave if there's two proline's in a row...According to our professor for the lecture, the answer is actually yes, with some updated news that he gave us...

Lest we forget that this is an R2 protease...cleaving from the C-terminal on...

One of the students in our Lenny-biochem-group came up with this one. I think it's sweet and a great way to remember this particular R2 protease. 

This is also a "cleaver" but it's non-enzymatic. It cleaves from the carboxyl side of Methionine 

Here's a really badly drawn mechanism. 

Some characteristics of the peptide bond that I may or may not have talked about before. There's some double-bond character in the bonds that join to amino acids.

You can have cis or trans conformation, obviously trans is the more appealing one, unless you have a proline next door, in which case cis is allowed.

Those are the 6 atoms that are in a plane

Some proteins, like insulin, have multiple chains connecting them. Insulin has a bunch of disulfide bonds - one that is intra-chain and two that are inter-chain. 

Here's a nice story about insulin. It starts out as preproinsulin - a random coil that has two sequences that later get cleaved off - leader sequence at the N terminal and connecting sequence that lies between the parts that will end up being the two strands of the mature protein. 

The leader sequence is kind of important...it has hydrophobic residues that help get the baby insulin through the cell membranes, since it's not active where it's synthesized. 

After membrane transport, the leader sequence is cleaved off, resulting in proinsulin, and the disulfide bonds within and between chains form. Then there's another cleavage, this time of the connecting sequence, and the mature insulin is formed.

Here's an intro to some stuff I said I was going to talk about, for separating protein pieces apart. In electrophoresis there's an anode and cathone, and charges migrate for separation. Proteins with certain charge densities will end up where you can then elute them from.

Ion exchange chromatography is another way to separate based on charge, using pH differences. 

Getting into a bit more detail about separating proteins and getting the stuff you want.

Affinity chromatography is made to be really super specific for the protein you're trying to isolate. It can be compared to a lock and key, with the protein you're trying to get ending up sticking to the matrix, which you can then elute it out of.

The book gives an example with immobilized metals, which stick well to proteins with sequences of 6-His residues in them in a row. 

Got a bit out of order here, but size exclusion lets you separate proteins based on size. There's pore-like tunnels in the column, where the smaller proteins sort of get "stuck" temporarily, letting the larger proteins elute faster. 

And this is ion exchange chromatography that was briefly mentioned earlier...

A lot of these got really out of order when I was sending them to myself...Regardless...

If you have proteins with like, multiple chains or whatever, and you want to break up the disulfide bonds in those chains, you have two options: The first is oxidation with performic acid. This one is irreversible.

A reversible way to break disulfide bonds is by reduction with betamercaptoethanol. If you want to do it irreversibly, you can block the cysteines you get with iodoacetate. 

The last thing I'll talk about here is this awful mechanism. Edman degradation basically lets you cleave off amino acids from the N terminal (until carboxypeptidase A, off the c-terminal) one by one with phenylisothicyanate and strong acid/water.

First, the phenylisothiocyanate labels the amino acid that's at the end terminal and then you cleave it off with strong acid/water

The Sequenator is basically an automated way of doing this over and over again, but you can't really use it for more than 40 amino acid residues because you'll have zero yield.

This post was disorganized and lame.