CLASS NAME: Fundamentals I (Hour 1)Scribe: Ashley Russell

DATE: 8/11/2010Proof: Meghan Guthman

PROFESSOR: Dr. DeLucasTITLE OF LECTUREPage1 of 7

Chromatographic Function [From 8.10 amino acid ppt, starts slide 49)]:
So I’m gonna real quick finish… so what I did is I took the last few slides and just added them to the beginning of what I should have started with today. So I’ll just kinda finish talking about where I left off which was, we were talking about if you wanted to purify and amino acid, what are ways you can do it; what are some of the chromatographic procedures. I started talking about functional groups that are actually attached. So I don’t know how many of you did take biochemistry in undergraduate school, but you basically have a tube with a column, and you have a media in there and that media has functional groups attached to it. They can be plus charged or minus charged. They can even be, not have a charge, be sort of just hydrophobic. So you can imagine if what’s bound to that column is something with a plus charge, and you shot over it arginine mixed with aspartic acid, the aspartic acid is going to stick more, because it will interact with those plus charges. As opposed to the arginine which is to slide through a little faster and so you can separate those two amino acids depending on the pH, depending on the ionic strength you can change how much you separate them, how fast they come off. So if you have a mixture of several amino acids, you might play with those conditions until you get the optimum separation. It probably won’t separate everything, in fact it wont and so then you can go to the opposite charge column for those that still aren’t separated and try to improve that separation. If you have nonpolar amino acids, then having a media that is basically non polar would cause a very intense interaction between those two and you could bind it. Then how do you get that off? Well, you increase the salt concentration, you are going to make them stick even more and so generally you put something in there that is also nonpolar and it competes with the protein and knocks it off. So, depending on what column you are using, the solvent you flow over this long column. (I say long column, today we do some things in the column, maybe not more than 0.5cm in diameter and we’re purifying sometimes amino acids in some of these medias sometimes a milligram. So on a preperative scale, there are machines that do this on an automative way and you are constantly flushing the column and sending the next batch of amino acids over it to very quickly purify it.
Reversed phase chromatography of amino acids
What I just said is pictorially shown on the next few slides here and it just shows in one case a negative particle, with positive amino acids would bind to it and vice versa, so it’s all pretty self explanatory. When you run amino acids over columns, you detect them in some cases optically, and what comes off of the column, depending on when it comes off, you’ll see a peak come off and that peak can indicate not only what it is, but also how much of it you have. So you can to some extent quantatate the amount of each amino acid you have in a mixture. So, that’s about all, you know, I want to say for something like this. But it just is basically a mixture of amino acids get placed on a column, they bind at different amounts and as they elute off they come off, this one came off first, then this one then this one and so now you have all of these peaks. And then to determine what that is, you either have a, I mean this has already been done in the past, so we know where that comes off under specific conditions, so you know, you know, under specific conditions (a certain column of a certain length), the one that comes off first is gonna be aspartic vs glutamic and so just from where history has told us they come off, you know. But, early on, we had to identify what each of those amino acids were and there are a bunch of techniques that can allow you to determine exactly what the mass is. Today, mass spec can do that and you can tell which one is an aspartate vs a threonine and so on. So if we had to, we could go back an identify what each of those are. This method of solution based chromatography to identify amino acids is kind of old. Today we use mass spectrometry to do this. Time of flight mass spec can do it quickly and its very automated and it tells you what amino acids you have in a mixture. To quantitate or sequence it there is a whole series of other steps and im gonna go through that as I go into the next chapter.
Reverse Phase Chromatography of amino acids [S3]
When I talked about if you just want to separate non-polar acids, there is a column called reverse phase chromatography and its because it is the opposite of what we all did early on which was to separate proteins based on charges and polar groups. So this is basically a hydrophobic type column and it is used a lot to separate drugs. A lot of the drugs we make always have to have a big hydrophobic component because generally they all have to be water soluble but they all have to get through a cell membrane and so a hydrophobic phenol ring, for example, will interact with this column and bind, so you can purify one compound , one potential drug from another using reverse phase chromatography. When this first started being used for proteins (insert minute long rambling about tears. If you want to hear it, start at minute 7 of the audio. General idea: He used reverse phase chromatography to separate out about 65 proteins found in tears. IT was “really an exciting part of his early career. Unfortunately, he found flying in space to be much more exciting and had to leave the tear proteins alone)
Amino Acid Analysis:
There are many ways to analyze amino acids and I just listed them here. And what I did for you, and you don’t have to know this, I just want you to get a feel for the many different ways we can do this. There are advantages and disadvantages for some of these ways and many of them require to identify the amino acids, you have to derivitize it. That takes a little bit of time, but now there are automated procedures to do that. Some are faster than others, but anyways, what I did is in green I put advantages and in red I put some of the disadvantages of each of these techniques. And the newest of these, time of flight mass spec is probably the mainstay today for doing this because not only can you do it very quickly, you can do it on a mixture of proteins, and so it gives you an advantage, actually you can take all of the proteins that are in a cell and start identifying what proteins are in that cell and what are their amino acid content, all from one cell. And as I told you, there are literally hundreds of thousands of proteins in a cell, so things are really getting sophisticated with mass spec these days.
The Sequence of Amino Acids in a Protein:
Um, so, let’s see here. Hold on. So, um, the sequence of an amino acid of the actual contiguous sequence as we make this peptide bond is a unique characteristic for each protein. Each protein has a different sequence. That sequence is encoded by a nucleotide sequence of DNA. So everything, all proteins, the genetic information comes from DNA for eukaryotes, and we always read this sequence by convention from the amino terminus to the carboxy terminus and that’s the way it’s actually made on the ribosome in eukaryotes. So if you look at all the proteins that are out there, and today it’s amazing what informatics does in biology and crystallography. (Insert ramblings at 11:30 about how flying in space opens neat-o doors and you get to go to dinner with important people (CEOs of companies that are trying to sequence the human genome in 1994) to talk about how neat-o flying in space is. Used informatics to search all of the proteins known to be related to bone formation or deformation in different species. Then, looked up all the species that are not known to have osteoporosis. Subtracted the two and had a set of 35 proteins that are specific to species that suffer from bone loss. Some were obvious and others were not.
Amino Acid Composition:
When you see databases like this that talk about what amino acid is most common, which is least common. This is what you typically see for globular proteins, but you can look at something like this and if its not an aqueous protein, one that is in your blood, or in the interstitial fluid, but one that is in a membrane, then this won’t look this way. You’ll have a higher predominance of hydrophobic amino acids, vs the polar or charged amino acids obviously, because a membrane protein has a big portion that has to go through that membrane, so that stalk, or transmembrane portion, is gonna be composed almost completely of hydrophobic amino acids. So, just from looking at things like this you can almost say what kind of protein it might be. If it’s a protein that imparts strength, fibrous protein like collagen, it has some unique amino acids. You will see much higher values for those so databases like this are incredible but what I have learned is that I’m not smart enough, when you really want to look deep into all these databases to understand all of these correlations, and so today in my lab, and we are not the only ones that do this, (but we have really pioneered this with several things) we rely on artificial neural networks to help us. We can put in a lot of data, and that neural net can look at correlations that I would never be able to figure out if I spent years. It’s just like the best chess players are the best because they can look at the board and think 6 or 7 moves ahead. The ones that aren’t so good are the 3. Neural networks not only do that, but they look for hidden correlations in the data and bring about information you never would have thought possible.

**insert Anna Lucy’s question about protein structure:

If you look at most of the amino acids that make up a fibrous protein, they have some polar groups but most of it is very hydrophobic in nature. A globular protein, I think this is what you are asking, (Why is that more soluble?), it depends on the globular protein, but if you look at a protein that has to sit in water, the amino acids that are out here are generally going to be polar. And I say general there are sometimes on the surface, hydrophobic pockets. It is usually just a small area usually, but it is where one protein might interact with another, or where a substrate might come in and bind. So you always seem to have a mixture of hydrophilic polar, but uncharged on the surface of these proteins, but you always seem to have pockets of hydrophobic amino acids on these proteins also. It just depends on not only the makeup of the amino acids but where they sit. These globular proteins as you go in are very hydrophobic (70-90% hydrophobic). This really helps the proteins fold because they want to get away from the water and so as you go out further and further you will see more of the polar amino acids.

Protein Structure Nomenclature [S56]:
A little bit of nomenclature is important, just so you know when people say words like “oligopeptide” what it means. Each amino acid in a protein is called a “residue” and if you have more than 12 residues, instead of just calling it a peptide, we call it a oligopeptide. This is just a number… sometimes people will call one with 15 residues a peptide, but in general, when you write a paper if you have more than 12, you call it an oligopeptide.
If you have several dozen residues, it is a “polypeptide”. Proteins are composed of one or more polypeptide chains. So if it has just one chain, all contiguous but it is all bonded with that peptide bond, you call it a “monomeric protein”. If you have 2 or more chains, you call it a multimeric protein. Now if those 2 chains are identical, you can call it a “homo multimeric protein”. If there are 2 of them, a simpler way to describe it is to call it a homodimer. If they are different it is a “hetero multimeric protein” and if there are 2 it is a “heterodimer”. If there are 3 chains, it is a heterotrimer. It’s pretty obvious, but that is the nomenclature that we all use in my world as we describe proteins. A simpler way, as you write these, instead of having to write that out; if it’s a homodimer we call that 2. If it is a heteromultimer, in this case we are just giving you the example of a tetramer 4 polypeptides, 2 are the same and the other 2 are the same, then that would be 22. So that is the nomenclature we adopt. As we go on and you get lectures from some other people, I am sure that you are going to see that. For example, I think you will see it later on with me and hemoglobin, and probably you are going to get a more detailed lecture about that anyways. So you should know this nomenclature and how to interpret it.
Protein Structure (1)
We talked about amino acids and the different ways you can catagorize them. There are two general ways you can talk about proteins: globular or fibrous. If you want to get more detailed and talk about each segment of how that protein is made and built up, the first part is called the primary structure and that is just the linear sequence of amino acids.
Secondary structure when we talk about that, we are talking about characteristic patterns of how the primary structure folds upon itself and it makes certain themes as it does that. One is an alpha helix and the other one is a beta strand. Then there are things like pleated sheets of beta strands. Those are the secondary structural elements.

Protein Structure (2)
The tertiary structure is when we take those secondary elements and fold it all together. So its how it all twists and turns (an alpha helices here, a beta strand here) to make this tertiary structure.
Then there is quantenary structure is usually 2 or more of these polypeptide units. Two different proteins or two different domains that interact together, different ways they interact. That is what we refer to as quaternary structure, so subunit organization in proteins is what it is called.
Proteins are always moving. There are wonderful modelers’ motion pictures of these things and they are all just shaking like I was on launch. That’s the way these are, they are vibrating like mad. There are parts of proteins that have loops. These loops may open and close and it’s a pretty big movement and when it happens pretty big other changes occur through the protein. These are called conformational changes. You will hear us talk about different conformations of proteins and how they orient themselves differently maybe to accept a substrate or expel it, or maybe to interact with another protein. Proteins have all kinds of different biological functions, so we can also classify them not just based on structural aspects but their biological function.
Proteins- Large and Small (1)
So there are all different types of proteins. Here is an example: Insulin is a very small protein basically. Glutamine synthetase is involved in a synthesis process. Connectin proteins are elastic proteins in muscle and they are very large in molecular weight. Look at the span of molecular weight and the different sizes of proteins. The only thing they are trying to point out in the book is that they all come in different sizes and different conformations.
Proteins- Large and Small (2)
This is just a table that shows you the same thing and the subunit organization is described here
using that nomenclature of alpha, beta, gamma that I described.
Proteins- Large and Small (3)
And that just shows you when we determine the structure of proteins using xray crystallography, we know where every atom is within a hundredth or a thousandth of an angstrom. But one thing is, when you put all those atoms for a protein like this, what you see is just a big blob of balls, and you can’t understand anything
. So sometimes we look at it like this, and this is just sort of a surface representation of where those atoms are. And in some cases where you see an opening there, it might be for a particular enzyme a hydrophobic pocket, for this, where it binds a metal ion. We can color these different ways. In general, not here though, this is just from the book, but if we color parts of a molecule red on the surface it usually means that it is charged.