Protein bonds and structure
The most basic unit of proteins are amino acids, there are 20 amino acids, that form different combinations of binding to give us a protein.
The process begins with the primary structure, proteins are polymers of smaller monomers known as amino acids, there are 20 different amino acids all containing a basic amino group and an acidic carboxyl group. This allows them to take on the form of a zwitterion where the carboxyl group loses an H+ and the amino group gains an H+. This function also enables amino acids to form longer chains with other amino acids i.e. a polypeptide chain, this is carried out through Ribosomes during protein synthesis. The polypeptide chains are joined together via peptide bonds and are coded by the anticodon section of a tRNA molecule that works using an anticodon – codon complementary binding, as each codon codes for one amino acid.
Amino acids are able to show variability due to the R group, which is home to a variety of side chains. It also allows amino acids to differ from each other via physical and chemical properties.
The secondary structure discovered by Linus Pauling and Robert Corey via the X-ray diffraction pattern of Keratin is what allows the polypeptide chain to differentiate further.
The peptide bonds within the polypeptide chain resonate, this means the bond length isn’t quite a double bond nor a single bond, the bond length lies in between the both, therefore in order to generate a secondary structure there must be no rotation around the planar peptide bond, which meant other regions had to be flexible. These are called the phi and psi bonds, the phi bond is the bond between the amino group and central carbon, whilst the psi bond is the bond between the carboxyl group and the central carbon, the rotation of these groups is what allows the maximum number of bonds to be formed, which was the third finding that there will be hydrogen bonds formed between the nitrogen of one amino acid to the hydrogen of the other amino acid. This derived either a helical structure or a beta-pleated sheet.
If we look at alpha helices in detail we notice that these are amphipathic, this means that the helix has “2-faces”, a hydrophobic side and a hydrophilic side. This is generated when an amino acid is either hydrophobic or hydrophilic and is positioned every three to four residues along the polypeptide backbone. This is where the rule comes from that alpha helices contain 3.6 residues per 360° rotation. This enables the helix to undergo many functions such as being a membrane protein and allowing substances to pass through.
The next secondary structure mentioned was beta sheets or beta strand. A beta strand is in an extended conformation which is why it was named strand. In a beta strand, the distance between adjacent amino acids is 3.4 Angstroms, this is further than in alpha helices which lie at 1.5Angstroms. Just liked alpha helices these are also held by stable hydrogen bonds. Beta strands will form beta sheets, which will either be antiparallel or parallel. The parallel strands consist of fewer hydrogen bonds and are therefore are more unstable that antiparallel beta sheets which contain more hydrogen bonds, therefore are more common in proteins, they can also be referred to as beta pleated sheets, due to the conformation they take up looking like pleated fabric. Lastly, beta sheets can also convert to beta turns, which is where a single beta strand will turn at a point creating a kink, therefore giving a turn structure, this is why most proteins are globular in shape, it is due to the constant turns and loops of the beta strands.
The tertiary structure is then when the secondary structures couple up to form more functional proteins. The overall globular, three-dimensional shape of a protein is referred to as the 3° structure. It’s in the proteins nature to want to bend and twist further to attain maximum stability as a low energy state. Within the tertiary structure of proteins, there are an array of bonds. The hydrogen bonds are still present between the Nitrogen to Hydrogen, but as well as this there are hydrophobic interactions, these are what primarily drive the protein folding process, known as hydrophobic collapse. To put this in context, a polypeptide chain is made within the cytoplasm where it is subject to fluid environments as soon as it’s produced therefore it is already conforming and changing into a tertiary structure, but it’s not in an active state. For example, there may be polar side chains and nonpolar side chains on the polypeptide chains, due to which once released in the fluid-filled cytoplasm, the hydrophobic regions will turn inwards and collapse down, whilst the polar component (hydrophilic) will remain pointed outwards, therefore causing hydrophobic collapse.
There are also a few Van der Waal interactions within the structure, caused via weak electrostatic forces of attraction, due to the difference in charge. Due to the acidic and basic nature of amino acids, there will also be ionic interactions like salt bridges. These will form between an acidic amino acid side chain and a basic amino acid side chain. The disadvantage with these bonds is that water can come along and interfere and try to make bonds instead of an amino acid with another amino acid, therefore are better if they’re formed within the protein. Lastly, a tertiary structure will also form disulphide bonds, these consist of covalent linkages and are really important for extracellular proteins, as these needs to travel from one location to another through mediums such as the blood. We will look at this in more detail later on in the document.
Lastly, proteins also uptake a quaternary structure, this I where they contain protein subunits. These can either be homodimer – same subunits or heterodimer – different subunits. The subunits interact which each other and this is what deems the quaternary structure. A common quaternary structure protein is haemoglobin which is made up of two alpha globin and two beta globin, each which contain an iron-containing Haem group, therefore maximises oxygen transportation.
Whilst some of the bonds holding proteins are remarkably strong i.e. hydrogen bonds and disulphide bridges, there are able to get broken within laboratory environments. For example, altering the temperature of the environment the protein is in, any temperature above the optimum will cause the protein to denature causing inactivation and denaturation. The same goes for pH levels, too high or too low can cause denaturing of the protein i.e. due to the inability of charge matching between the substrate and enzyme active site. We can also alter the ionic strengths, where the salt ionic balance changes, this is seen with milk when it curdles due to the acid build up. We can also use some denaturing agents like organic solvents and chaotropic solvents such as urea, and guanidinium hydrochloride.
Proteolytic enzymes are also able to act on the protein, where enzymes such as proteases are able to ‘chop up’ the enzyme.
The peptide bond within proteins is also able to get hydrolysed by boiling it in 6M concentration of Acid.
The Anfinsen Experiment
In this next section, we link the idea of urea and how it plays a role in not only breaking proteins but how they also reform again.
The Anfinsen experiment was able to show that the information for protein folding resides in the primary amino acid sequence. Refer to image 1.0.
The Anfinsen experiment involved the use of denatured and purified RNaseA, this consisted of four disulphide bonds, this was in the presence of 8M of urea and excess ß-mercaptoethanol (BME), the result of this on the RNaseA was that it fully unfolded inactive polypeptide. The cause of this unfolding was due to Urea, the urea is what unfolds the proteins through disrupting the polar interactions within the protein without altering the covalent structure. The reverse procedure was also carried out, where Anfinsen removed the urea and BME by dialysis and then bubbles oxygen gas, he did this to show how the structure of the protein folded up correctly again with more than ninety percent enzymatic activity. However, Anfinsen was able to see that if he first removed only the BME, and then bubbled oxygen gas through (re-oxidising it), and then removed the urea, the resulting protein was only 1% active. This is because the 8 cysteine residues that formed the four disulphide bonds, formed random disulphide bonds during the refolding process, therefore the scrambled protein was stuck in this misfolded state 99% of the time.
Finally, to prove that it is, in fact, the primary amino acid sequence of RNaseA that determines the three-dimensional structure, the addition of trace amounts of ß-ME, which breaks the bonds and allows them to reform to give 99% enzymatic activity.
The overall experiment shows that the folded and active form of a protein has the lowest free energy. All the information needed by a protein to fold to this structure is encoded in the primary structure.
Synthesis of insulin and the link with the Anfinsen Experiment
The synthesis of Insulin begins with pre-proinsulin which is the polypeptide chain, it contains no disulphide bridges, this gets transported to the rough endoplasmic reticulum where it gets transferred into proinsulin via the cleavage of the signal sequence on the amino terminus. Next, the proinsulin makes its way into the Golgi where it forms into mature insulin, where sulphide bridges get formed.
Insulin gets synthesized as preproinsulin in pancreatic ß-cells. The pre-proinsulin is made up of an amino chain, a carboxyl chain, a connecting peptide (C chain) in the middle and a signal peptide, this is what directs the polypeptide chain into the rough endoplasmic reticulum. The signal gets cleaves as the polypeptide chain gets translocated into the lumen of the rough endoplasmic reticulum, where it now forms proinsulin.
Proinsulin needs to turn into insulin which is a process of maturation, as this is the active insulin
Proinsulin gets matured into active insulin via cellular endopeptidases. These cleave at two positions, therefore, releasing the C-peptide fragment, therefore leaving two chains, the A carboxyl chain and the B amino chain, these are held together by two disulphide bonds.
This results in the mature insulin being produced which gets packaged inside mature granules where it waits for signals to get released, and also to get exocytosis via vagal nerve stimulation from the cell into the circulation.
Therefore the ‘mature’ insulin is also called insulin; this insulin is not compatible with the Anfinsen experiment. This is due to the fact that the structure of insulin are two strands held by disulphide bridges, and the addition of ß-ME, would cause those bonds to break, therefore leaving two separate polypeptide strands that are not forced to go back together, the chances of these two chains re-joining is a simple chance mechanism, it’s not like how it works in polypeptide chains where there are intermolecular interactions that determine the folding. Therefore, mature insulin is unable to work in the Anfinsen experiment.