Chapter 2: Protein Structures

Joining Amino acids to the protein Primary Structure

We have already learned that the subunits of a protein are amino acids or to be precise amino acid residues. We know that an amino acid consists of a central carbon atom (C_α) and an amino group (NH₂), a hydrogen atom (H), a carboxy group (COOH) and a side chain (R) which are bound to the C_α. A peptide bond is formed via covalent binding of the Carbon atom of the Carboxy group (COOH) of one amino acid to the nitrogen atom (NH₂) of the amino group of another amino acid by dehydration: Figure 2.1: Peptide bond linking two amino acids

A polypeptide chain is a chain of amino acid residues linked together by peptide bonds. The backbone of the polypeptide is given by the repeated sequence of three atoms of each residue in the chain: the amide N, the alpha Carbon C_α and the Carbonyl C. Rotations in the chain take place about the bonds in the backbone, whereat the peptide bond usually is unflexible (see Figure 2). The existence of an amino group (N-Terminal) at one end of the chain and a carboxy group (C-Terminal) at the other end designs a direction to the chain. Conventionally the beginning of a polypetide is its N-Terminal.

Figure 2.2: Torsion (or dihedral) angles of the backbone

NB: See your prescribed textbook for characteristics of the peptide bond (Page 14).

Joining these amino acids together using a peptide bond gives rise to the linear polypeptide chain called the Primary Struture.

A). The primary structure of proteins
Drawing the amino acids
In chemistry, if you were to draw the structure of a general 2-amino acid, you would probably draw it like this:
However, for drawing the structures of proteins, we usually twist it so that the "R" group sticks out at the side. It is much easier to see what is happening if you do that.
That means that the two simplest amino acids, glycine and alanine, would be shown as:

Peptides and polypeptides
Glycine and alanine can combine together with the elimination of a molecule of water to produce a dipeptide. It is possible for this to happen in one of two different ways - so you might get two different dipeptides.
Either:
Or:
In each case, the linkage shown in blue in the structure of the dipeptide is known as a peptide bond. In chemistry, this would also be known as an amide linkage, but since we are now in the realms of biochemistry and biology, we'll use “the peptide bond”.
If you joined three amino acids together, you would get a tripeptide. If you joined lots and lots together (as in a protein chain), you get a polypeptide.
A protein chain will have somewhere in the range of 50 to 2000 amino acid residues. You have to use this term because strictly speaking a peptide chain isn't made up of amino acids. When the amino acids combine together, a water molecule is lost. The peptide chain is made up from what is left after the water is lost - in other words, is made up of amino acid residues.
By convention, when you are drawing peptide chains, the -NH2 group which hasn't been converted into a peptide link is written at the left-hand end. The unchanged -COOH group is written at the right-hand end.
The end of the peptide chain with the -NH2 group is known as the N-terminal, and the end with the -COOH group is the C-terminal.
A protein chain (with the N-terminal on the left) will therefore look like this:

The "R" groups come from the 20 amino acids which occur in proteins. The peptide chain is known as the backbone, and the "R" groups are known as side chains.
You can determine the sequence of the primary structure by using several methods but please how to use enzymatic degradation to do so. We will use Trypsin and Cyanogen Bromide as examples in class.

B). The secondary structure of proteins
Within the long protein chains there are regions in which the chains are organised into regular structures known as alpha-helices (alpha-helixes) and beta-pleated sheets. These are the secondary structures in proteins.
These secondary structures are held together by hydrogen bonds. These form as shown in the diagram between one of the lone pairs on an oxygen atom and the hydrogen attached to a nitrogen atom:

The alpha-helix
In an alpha-helix, the protein chain is coiled like a loosely-coiled spring. The "alpha" means that if you look down the length of the spring, the coiling is happening in a clockwise direction as it goes away from you.
Note: If your visual imagination is as hopeless as mine, the only way to really understand this is to get a bit of wire and coil it into a spring shape. The lead on your computer mouse is fine for doing this!

The next diagram shows how the alpha-helix is held together by hydrogen bonds. This is a very simplified diagram, missing out lots of atoms. We'll talk it through in some detail after you have had a look at it.
What's wrong with the diagram? Two things:
First of all, only the atoms on the parts of the coils facing you are shown. If you try to show all the atoms, the whole thing gets so complicated that it is virtually impossible to understand what is going on.
Secondly, There have no attempt whatsoever made to get the bond angles right. I have deliberately drawn all of the bonds in the backbone of the chain as if they lie along the spiral. In truth they stick out all over the place. Again, if you draw it properly it is virtually impossible to see the spiral.
So, what do you need to notice?
Notice that all the "R" groups are sticking out sideways from the main helix.
Notice the regular arrangement of the hydrogen bonds. All the N-H groups are pointing upwards, and all the C=O groups pointing downwards. Each of them is involved in a hydrogen bond.
And finally, although you can't see it from this incomplete diagram, each complete turn of the spiral has 3.6 (approximately) amino acid residues in it.
If you had a whole number of amino acid residues per turn, each group would have an identical group underneath it on the turn below. Hydrogen bonding can't happen under those circumstances.
Each turn has 3 complete amino acid residues and two atoms from the next one. That means that each turn is offset from the ones above and below, such that the N-H and C=O groups are brought into line with each other.

Beta-pleated sheets
In a beta-pleated sheet, the chains are folded so that they lie alongside each other. The next diagram shows what is known as an "anti-parallel" sheet. All that means is that next-door chains are heading in opposite directions. Given the way this particular folding happens, that would seem to be inevitable.
It isn't, in fact, inevitable! It is possible to have some much more complicated folding so that next-door chains are actually heading in the same direction. We are getting well beyond the demands of our syllabus now. But look at figure 2.8 (2nd edition) or figure 2.9 (3rd edition) in your prescribed text book.
The folded chains are again held together by hydrogen bonds involving exactly the same groups as in the alpha-helix.

C). The tertiary structure of proteins
What is tertiary structure?
The tertiary structure of a protein is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape. This is often simplified into models like the following one for the enzyme dihydrofolate reductase. Enzymes are, of course, based on proteins.
Note: This diagram was obtained from the www.rcsb.org/pdb/ which is RCSB Protein Data Bank. If you want to find more information about dihydrofolate reductase visit this site.
There is nothing particularly special about this enzyme in terms of structure. I chose it because it contained only a single protein chain and had examples of both types of secondary structure in it.
The model shows the alpha-helices in the secondary structure as coils of "ribbon". The beta-pleated sheets are shown as flat bits of ribbon ending in an arrow head. The bits of the protein chain which are just random coils and loops are shown as bits of "string".
The colour coding in the model helps you to track your way around the structure - going through the spectrum from dark blue to end up at red.
You will also notice that this particular model has two other molecules locked into it (shown as ordinary molecular models). These are the two molecules whose reaction this enzyme catalyses.

What holds a protein into its tertiary structure?
The tertiary structure of a protein is held together by interactions between the the side chains - the "R" groups. There are several ways this can happen.

1. Ionic interactions
Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group.
You can get a transfer of a hydrogen ion from the -COOH to the -NH2 group to form zwitterions just as in simple amino acids.
You could obviously get an ionic bond between the negative and the positive group if the chains folded in such a way that they were close to each other.

2. Hydrogen bonds
Notice that we are now talking about hydrogen bonds between side groups - not between groups actually in the backbone of the chain.
Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. This is a classic situation where hydrogen bonding can occur.
For example, the amino acid serine contains an -OH group in the side chain. You could have a hydrogen bond set up between two serine residues in different parts of a folded chain.
You could easily imagine similar hydrogen bonding involving -OH groups, or -COOH groups, or -CONH2 groups, or -NH2 groups in various combinations - although you would have to be careful to remember that a -COOH group and an -NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.

3. van der Waals dispersion forces
Several amino acids have quite large hydrocarbon groups in their side chains. A few examples are shown below. Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.
The dispersion forces set up would be enough to hold the folded structure together.

4. Sulphur bridges
Sulphur bridges which form between two cysteine residues.
If two cysteine side chains end up next to each other because of folding in the peptide chain, they can react to form a sulphur bridge. This is another covalent link and so some people count it as a part of the primary structure of the protein.
Because of the way sulphur bridges affect the way the protein folds, we will count this as a part of the tertiary structure.

C). The Quaternary structure of proteins
Quaternary Structure is the combination of two or more chains, to form a complete unit. The interactions between the chains are not different from those in tertiary structure, but are distinquished only by being interchain rather than intrachain.
Some proteins are composed of identical subunits (chains). A simple example is the dimer of HIV Protease.
Some proteins are composed of non-identical subunits (chains). A simple example is insulin which is made up of two chains, the alpha chain and the beta chain, linked by two disulfide bridges.

In our syllabus we will use Hemoglobin as an example of a protein with a quaternary structure.
To checkout the protein formations from primary struture to quartenary structure please click here.

The most important thing here is that as a pharmacist you should know the three dimensional structures of these protein so that you can design your drugs and medicines accordingly.

TEST YOURSELF
2.1 Choose whether the descriptions below match the
(A) Primary (B) Secondary, C)Tertiary or D) Quaternary Structure
(i) The structure formed by disulphide bridges between different parts of a polypeptide chain.
(ii) The highest level structure found in a protein with two or more peptide chains.
(iii) Folding due to electrostatic interactions between R-groups of the amino acids that make up the polypeptide chain.
(iv) The highest level structure found in a protein with a single peptide chain.
(v) The structure formed by covalent bonds between adjacent amino acid residues in a polypeptide.
(vi) The combination of two or more polypeptide chains.
(vii) The structure due to, amongst other things, van der Waals’ interactions between uncharged R groups.
(viii) The sequence of amino acid residues in a polypeptide chain.
(ix) The structure due to, amongst other things, hydrogen bonding between polar R groups.
(x) The pattern in which the polypeptide backbone folds and is stabilized by Hydrogen bonding between the N-H of one peptide and the C=O of another.
(xi) The alpha-helix
(xii) Always synthesized in the cell from the N-terminal end to the C-terminal end.
(xiii) The structure due to, amongst other things, ionic bonds between oppositely charged R groups.
(xiv) Β-pleated sheet

2.2 A polypeptide is cleaved into peptides by treatment with trypsin and cyanogens bromide, and the peptides are purified and sequenced. The sequences of the peptides are shown below.
Trypsin peptide
Cyanogen bromide peptides
T-1 GASMALIK
C-1 EGAAYHDFEPIDPRGASM
T-2 EGAAYHDFEPIDPR
C-2 TKDCVHSD
T-3 DCVHSD
C-3 ALIKYLIACGPM
T-4 YLIACGPMTK

Determine the primary sequence of the original polypeptide.

2.3
Structure of AspartameAspartame is an artificial sweetener found in Diet Coke, Coke Zero and most sugar-free soft drinks. Although it has roughly the same number of calories per gram as table sugar (sucrose), it is around 200 times sweeter. It was discovered by accident by James Schlatter, a chemist of G D Searle Co. in 1965, when he was testing an anti-ulcer drug. Its use in carbonated drinks was finally approved in 1983, following a decade-long battle against the objections of Dr. John Olney (a neuroscience researcher), James Turner (a consumer attorney) and investigations into the research practices of G D Searle. The objections were based on the amino acids Schlatter used to make Aspartame. Looking at its structure, which amino acids did James Schlatter use to make Aspartame?


2.4 Glutathione is a dietary supplement used as an antioxidant to help protect the body from many diseases and conditions. It is also used to treat infertility (difficulty getting pregnant), cancer, cataracts, and human immunodeficiency virus (HIV). Glutathione is used to detoxify various chemicals from the body.

Carefully observe the structure of glutathione and determine the three amino acids used to synthesize this useful drug. Give the name of this tripeptide.