Proteins I

Okee-Dokee. Last time we saw how amino acids combine to make peptides. If we increase the number of amino acids in our peptide chain, we start moving into the realm of proteins. Remember, however, that we prefer to leave the name proteins for polypeptide chains that have a biological purpose.

The role of proteins

As you probably know, proteins are in charge of of almost everything that needs to be done in living organisms. Depending on the specific role of the protein, we can clasify then according to:

1) Enzymes. Enzymes are proteins that catalyze chemical reactions that would otherwise take eons to happen under normal biological conditions (we have to remember that we are i) in water, and ii) at 37^oC and normal pressure...). They bind to their reactants with very high specificity, which means that they will only accept either the natural reactant (ligand) or a very close version of it. This allows them to pick and choose the right substrate for a reaction among a soup of possible molecules. Enzymes can either make a bond between two substrates, or break a bond in a substrate to give us two products.

2) Transport. Many proteins also bind tightly to specific molecules, but instead of doing chemistry with them they basically move them from point A to point B. One case is hemoglobin, which binds oxygen as blood goes through the lungs and then releases it were needed (oxidation of fuels). In other cases, point A is outside the cell membrane and point B inside, and the protein will help a polar molecule to get through the membrane (i.e., glucose transport).

3) Storage. Many seeds (wheat, beans, rice, etc.) have proteins that are a source of nutrients (amino acids) for the germinating plants. Obalvumin in eggs and Casein in milk are also examples of nutrient storage proteins.

4) Motion. These ones give cells or organisms the ability to contract, change shape, or move. Examples are Actin and Myosin, which are responsible of muscle contrations, and tubulin, which among other things forms cell falgella.

5) Structural. Several proteins arrange as filaments and give structural support to cells and tissues. The best example is collagen, which is the major component of tendons, cartilage, and leather. Hair, nails, and feathers are made up by the protein keratin. Tubulin forms part of the cytoskelleton, and plays an important role during cell division by guiding the replicated chromosomes towards the ends of what will become the two new cells.

6) Defense. We have two types. Ones are made by the organism against the attack of another organism at a macroscopic level: toxins made by spiders, snakes, etc. are obviusly a defense mechanism. Some plants and fungi make toxic proteins to avoid being eaten by animals or attacked by fungi/bacteria. At the molecular level we have inmunoglobulins, that recognize and remove from solution materials (cells, proteins, viruses) foreign to the organism. At this particular moment, mine are out to lunch, because I have the nastiest flu you can get, and except for some fever, my 'defense proteins' are not doing much...

7) Regulation. Many proteins regulate cellular activity, an example being hormones. Hormones are transmiters of signals and information. Their receptors, usually G-proteins located in the cell membrame, receive this infomation, amplify it, and the result is something being done (or not) by the cell. For example, Insulin regulates sugar metabolism. Other regulatory proteins bind to DNA controling the biosysnthesis of enzymes and RNA molecules involved in cell division.

Size and mass

How about their sizes and compositions? As we said, we left the name proteins for realtivelly big polypeptide chains. We have a wide range of sizes, from 50 to 8,000 residues, wich corresponds to molecular wights in the ~6,000 to ~1,000,000 Daltons (Da), or 6 to 1,000 kilo Daltons (kDa). Most of the naturally occuring proteins have less than 2,000 residues (~220 KDa).

Additionally, proteins can occur as a single polypeptide chain, or unit (monomeric), or as multisubunit proteins, in which several chains or units (called subunits in this case) are held together by non-covalent interactions (salt-bridges as in HIV protease). The units in a multisubunit protein can be identical (oligomeric proteins), or different. Each unit in a polymeric protein are called protomers.

The last thing concerning sizes and masses. If we take the average of the amino acids found in natural proteins, we find that smaller amino acids predominate. The average mass of one residue (amino acid minus water) will therefore be around 110 Da.

Non-amino acids components

In many proteins we will find molecules that are not amino acids, but occur always in that protein, and allows them to function properly. These proteins are conjugated proteins, and the non-amino acid parts are called the prostetic groups. Conjugated proteins can be classified according to the chemical nature of the prosthetic group. Then we will have glycoproteins, in which the non-amino acid part is a sugar (or more likely a polysacharide), lipoproteins (the non-amino acid is a lipid), and metalloproteins (a metal). We will see a large number of small molecules participating as prosthetic groups. 9 times out of 10 the prosthetic group plays a very important role in the biological function of the protein housing it, as it usually has a very different chemical reactivity, and will allow the protein to perform reactions that are imposible to perform with residue side chains alone (Dehydrogenases have NAD or FAD prostetic groups to aid in the oxydation/reduction reactions).

Protein purification

Those of you taking the Biochem Lab are probably doing this hands-on, and others will probably do it at some other point in their studies/careers. We will describe briefly different techniques used in protein purification.

The first thing we have to realize is that our protein is going to form part of some living organism, so unless the protein is being released, we have to kill it. If it is a bug like E. Coli,no problem. Now, if the protein we want is in the mammalian brain, then things get ugly. Be as it may, after killing, lisying, choping, slicing-and-dicing our source of protein, we will have an homogenate or crude extract form the tissue/cell. This will not only contain the protein of interest, but it will also have other (hundreds ~ thousands) of proteins, plus lipids, DNA, RNA, small molecules, and other sticky and slimy things. Most of the time, there will be chunks of tissue in our homogenate, bits and pieces of partially destroyed cells, cell membranes, etc., etc. If our protein is soluble in aqueous solution (buffer), we first centrifugate the homogenate. All insoluble material will be pelleted down, and the soluble stuff will remain in the supernatant. In this step we removed all the solid material. In the next steps (which we won't describe), proteins are separated from soluble small molecules by different procedures. One of the most common coarse separion steps is salting out, in which proteins are precipitated out of solution by varying the salt content of the solution ((NH₄)₂SO₄ is commonly used)

At a certain point, we will have different solutions, each containing some of the proteins from the tissue/cells we used. Now we have to use finner methods to separete each of the proteins in each solution. In particualr, we have to use a method that allows us to separate the protein we are interested in as easy as possible. Some ot the techinques are:

1) Ion-exchange chromatography. As we saw for amino acids, we can separate species that have different charges at different pHs. As you recall, the total charge of the protein will depend on the solution's pH and the pK_as of the  residues present in the protein. In a protein, however, we have to remember that we can have tenths or hundreds of amino ionizable groups, so we will have different affinities for the ion exchange resin depending on the protein mixture we have.

2) Size-exclussion chromatography. The name says it all (basically). This method separates proteins according to their size. The chrromatography columns contains a cross-linked polymer (Sephadex, Sepharose) that has pores of different sizes. Proteins smaller in size than the pores will get into the pores as we pass them through the columns, and they will therefore take a lot longer to go through the whole lenght of the column in comparison to a large proteins that don't fit into the Sephadex pores.

3) Affinity chromatography. This one is one of the best methods, but usually the hardest to be able to use. Say that our protein binds certain small molecule (i.e., glucose). If glucose is present in solution, the protein will bind strongly to it. If we attach a glucose molecule to a solid suport (usually through a linker), then our protein will become inmobilized to this solid support, because it will bind to the ligand molecule attached to the support bead. Therefore, we load our affinity colmn with our soution of proteins, and basically wash it with buffer. After we washed out all the junk in our solution that does not bind to the column, we pass a solution with a high concentration of the ligand. The ligand in solution will compete for the binding site of the protein with the ligand bound to the solid support, which will traduce in the protein being released from the column. This chromatographic method will usually be highly specific for the protein we are looking for, but normally requires a solid support that has a covalently linked ligand. It is therefore pretty expensive/imposible if we have some esoteric protein that binds something really funky.

What about using a support that has a covalently attached prosthetic group? As we saw before, many proteins have a non-amino acid part for proper function. If we are interested in separating a protein that bears a prosthetic group, we can use a column that has mimics of the prosthetic group covalently attached to the solid beads.

Great. Now we purified our protein, and lets assume it was an enzyme. What about its activity? As you probably know, the more we purify something, the more of it we will loose in the different purification steps. If we started, say, with a couple of grams of protein, we may end up with just a few milligrams of purified enzyme. In the very begining, we have a certain enzyme activity (units). As we start purifying the enzyme, we will loose a considerable ammount of it, and the activity will go down. However, if we now consider the activity with respect to the total mass of protein (most of which is not active at the begining), we will see that the specific activity of our protein (units/mg) will increase with each purification step.

Mixture identification and characterization

These are all techniques used to separate, in some cases, large ammounts of protein. What if we just want to see what proteins we have in a mixture, or are trying to determine the purity of a purified protein? The most commonly used technique is electrophoresis, which is the 'thin layer chormatography' of the biochemist.
It is based on the migration rates of charged species in an electric field.

As we said before, a protein will have different charges at different pHs. If we put this protein in a solution and apply an electric potential across the solution, the ions will move toward the electrode of opposite charge. The electrophoretic mobility, m, of the species will be determined by:

were V is the velocity, E is the electric potential, Z is the charge, and f is the frictional coeficient: The more charge or voltage, the faster we move; the more friction (i.e., he bigger the particle or the more viscous the solution), the slower we move. We cannot do it in solution, because the protein would diffuse, so we do it in a cross-linked polymer matrix or gel that holds the buffer and guides the travel of the protein. We normally use polymerized acrylamide gels (Poly-Acrylamide GEls, or PAGE). If we use the protein 'as is', the charge will depend on the nature of the protein and the pH. This is called a native gel, because we are performing electrophoresis in the native state of the protein. It is very tricky, because we may not know the charge of our protein, but it is necessary in some cases in which what is described below cannot be done.

We are normally interested in separating or 'seeing' our protein as a function of molecular weight. Native gels do depend on the mass (actually, on the size and shape), but they also depend on the charge, which may vary inconsistently from protein to protein. There is no relation between charge and mass - it can be anything. What we do is we give every protein the same charge to mass ratio: We coat them with a soapy substance called sodium dodecyl sulfate (NaO₃SO-(CH₂)₁₁-CH₃, or SDS). The greasy alkyl chain will interact with the greasy protein, and the outside of the protein will have a charge that is proportional with its size/mass. Furthermore, SDS, being a soap, will make all the proteins adopt more or less similar conformations, so the charge to mass ratio will be mantained almost constant. Therefore, the separation will depend to a large extent on the mass (molecular weight) of the protein. This is called SDS-PAGE electrophoresis.

Now, we usually carry out our gels using a lane for standards, proteins of known mass, and another lane for our unknowns. We can interpolate the molecular wight of our protein by comparing how much it traveled with respect to the known proteins:

A variation of this is isoelectric refocussing and two dimensional (2D) electrophoresis. We first separate the proteins according to their pI, and then we do the electrophoresis. In order to separate proteins as a function of their pI, we make a column with a gel that has a pH gradient. We load the proteins at the begining of the gel column, and we turn on an electric potential across the gel. Since the proteins have different charges at different pHs they will start moving. However, at certain point they will get into a region of the pH gradient that corresponds with their pI - they don't have a charge anymore, and they stop migrating. At this point, we have all proteins separated by their pI. In the second stage, we take the gel out of the column, and use it to load a normal electrophoresis gel, and we run a normal electrophoresis at constant pH (in a buffer). in this way we have a separation as a function of mass in one dimenssion, and pI in the other - It is very useful to map thousands of proteins in very complex mixtures.

Next time we will start analyzing the different levels of protein structure, starting by primary structure and the determination of primary structure.

Prepared by Guillermo Moyna, 1999.