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  • at Oregon State University.

  • Kevin Ahern: So we're moving along nicely with the schedule.

  • And though we don't have to stay on it exactly,

  • we've been on it pretty good.

  • I've been pleased with the interactions

  • and also pleased with the questions I'm getting,

  • both in class and out of class.

  • So you guys seem to be engaging in this material

  • and that's a very good indicator of success.

  • So if you have questions, please feel free.

  • Come see me. Come see the TA's.

  • And we're here to help in any way that we can.

  • I only have one tiny little thing to say today

  • regarding the last of protein structure.

  • It's actually sort of an anecdote more than anything else.

  • And then I want to talk about techniques

  • for characterizing and/or purifying proteins.

  • One of the things that biochemists spend

  • a tremendous amount of time doing is just that: isolating,

  • characterizing, understanding proteins, enzymes, etc.

  • And so what you've learned so far about structure of proteins,

  • you will discover will be useful as tools

  • for learning how to isolate them.

  • And so I'll spend some time talking about that today

  • and also on Monday.

  • The anecdotal thing I wanted to mention to you

  • is the very last item on the protein structure page,

  • and it's actually this right here.

  • I've mentioned hydroxyproline to you already

  • and I want to reiterate something here.

  • Now, if you recall, I said that there are 20 amino acids

  • that we find commonly in proteins,

  • but we find modified amino acids in proteins.

  • And the point that I want to emphasize is that those

  • modified amino acids that we see happen post-translationally,

  • meaning that the modifications occur

  • after the amino acid is built into the protein.

  • So, in the case of hydroxyproline, for example, I gave you,

  • I showed you or described to you how Vitamin C

  • was involved in that reaction that modified the proline.

  • That happened after the proline

  • had been built into the protein.

  • The same is true of all the other things there are here.

  • Carboxyglutamate is an important modification,

  • as we will see,

  • that occurs as an important consideration in blood clotting.

  • Carbohydrate-asparagine adduct,

  • where we see, in this case,

  • addition of a carbohydrate to an asparagine residue,

  • this is really imporant in the production synthesis

  • of glycoproteins that we'll talk a little bit about later.

  • Phosphoserine, Phosphorylation is something

  • that you're going to hear a lot about later in the term

  • because phosphorylation is a means of controlling

  • or signaling through proteins.

  • And it's a very, very important mechanism for us to understand.

  • It's specifically phosphorylation

  • that I want to address briefly at the moment.

  • And that is that phosphorylation of amino acids

  • has to occur on side chains and side chains

  • that have hydroxyl groups.

  • So the three amino acid side chains that have hydroxyl groups,

  • of course, are the tyrosine, serine and threonine.

  • These are the three amino acids that get phosphorylated

  • or can be phosphorylated.

  • And we'll see a bit of a pattern

  • to how that phosphorylation occurs.

  • Not surprisingly, you might think,

  • well, why do these have such big effects?

  • You saw a big effect with hydroxyproline

  • because it was a part of that important structural

  • consideration for making a strong collagen.

  • In the case of phosphorylation,

  • what we're doing is we're converting,

  • excuse me, we're converting a side chain

  • from being hydrophilic to actually being ionic.

  • And so, in essence, what we've done is we've changed it from,

  • say, a partial charge to a fully negative charge.

  • In this case, we see two minus groups there.

  • Now, based on what I told you so far about protein structure,

  • you might imagine that changing the charge

  • of a specific location of a protein

  • might have structural considerations for that protein.

  • Imagine that previously we had a negative charge,

  • let's say a glutamic acid residue,

  • that was close to this proline

  • before we put the phosphate on there.

  • When we put the phosphate on there,

  • here's this negative charge before

  • that didn't really have that much interaction with the OH,

  • but now there's two minus charges over here.

  • What's going to happen?

  • Well, of course they're going to repel,

  • and when they repel,

  • that's going to change the configuration.

  • It's going to change the shape of that protein slightly.

  • And, as we will see,

  • and I've mentioned previously,

  • changes in the shape of proteins

  • can have some dramatic effects on the action of those proteins,

  • and we're going to talk more about those

  • as we get further along.

  • So those are some things that are other modifications

  • that can happen to proteins.

  • But I want you to be aware

  • that virtually any time you see a modified amino acid

  • in a protein it is because it has happened

  • after the amino acid has been put into the protein.

  • Okay, so that's the last of what I want to say

  • about general considerations of protein structure.

  • Now I'd like to turn our attention to characterizing proteins.

  • The first part of the characterization

  • I'll talk about is actually purification.

  • And purification isn't a spiritual purification,

  • but it's actually a physical purification.

  • I'll tell you a brief story.

  • When I was working in my very first lab after I had graduated,

  • I worked in a laboratory where we did HPLC,

  • and we had to have very pure solvents.

  • And I was very impressed by this notion

  • of purification that happens in there,

  • the need for purity in all biochemical materials.

  • And so I was very, very impressed

  • with these solvents that we used,

  • and we got them from this company that had purified stuff.

  • So I remember writing a letter to the companyótongue in cheek,

  • of courseósaying that, you know,

  • we found that not only were their solvents very pure,

  • but we had to do a spiritual purification

  • of these solvents before we used them, as well.

  • Of course, I wrote this as if it were completely serious,

  • sent it to the president of the company,

  • and, to my delight, I got this letter back

  • from the president of the company

  • congratulating me on describing for him

  • a new way of purifying his solvents that he could use for HPLC.

  • It was a good exchange.

  • So purification really had a big impact on me

  • as a very young biochemist.

  • Purification is important.

  • When we want to characterize,

  • let's say, a protein or an enzyme,

  • we need to have it isolated away from everything else.

  • When we try to understand an enzymatic reaction,

  • for example, we say, "Okay, well, ìI'm interested in this enzyme.

  • ìI'm interested in the reaction

  • that this enzyme catalyzes."

  • If I only have the soup of the cell, that is,

  • the cytoplasm of the cell that contains this,

  • I not only have that one enzyme that I'm interested in,

  • but I have several thousand other enzymes in there.

  • So it's important for me to understand what

  • this enzyme does that I be able to purify this

  • enzyme away from all those other proteins.

  • And so understanding how to purify one

  • protein apart from others is a very,

  • very important consideration in biochemistry.

  • Well, there are several techniques that

  • we use in order to do this,

  • and I'm going to go through and sort of describe

  • a few of the basic ones to you and then show you some

  • of the applications of these technologies.

  • You can't walk into a biochemistry

  • lab without finding a centrifuge.

  • It's almost impossible to do that and

  • that's because the use of centrifugal force as a means

  • of separating molecules on the basis of their size is a very,

  • very valuable tool.

  • Not surprisingly,

  • different things can be spun down.

  • We talk about "spinning them down."

  • That is, will they precipitate out of solution or

  • will they move to the bottom of the tube?