An Introduction to Proteins

An Introduction to Proteins in 30 minutes 






In the biotechnology industry, the majority of research is directed towards characterizing the structure and function of proteins. New methods are continually being developed to accomplish this.  


Light scattering technologies are relatively new to the field of protein characterization but the potential for characterizing them using methods that do not consume the sample is great.  


This technical note provides an introduction to the field of proteins, their composition and structure, and how size, molecular weight and ζ- potential measurements can be applied. 






Amino Acids

Amino acids are small molecules with a common structure. They have a central carbon attached to an amino and a carboxyl group, a hydrogen atom, and a fourth functional group (R).  This functional group is variable and it is this that changes in each of the 20 or so amino acids the body uses to build proteins.




This basic structure of amino acids is shown in figure 1 in both its charged and uncharged states. A couple of examples of amino acids are shown in figure 2.  





The different functional groups on the amino acids confer them with different properties. For example, the amine group on the lysine will gain or lose its charge depending on the local environment.  


The sulphur groups on cysteine residues can bond together covalently to form di-sulphide bridges when two of these amino acids are brought close together.  

Amino acids join to one another via bonds known as peptide bonds between the carboxyl carbon and the amino nitrogen. When the bond forms, a water molecule is released (figure 3).  



Amino acids can join together using these peptide bonds in chains of almost any sequence, which are subsequently known as polypeptides. 


A short sequence of up to a dozen or twenty amino acids is generally known simply as a peptide. When a polypeptide is of an appropriate sequence, size and structure, it is
functionally a protein. 



Protein Structure 


The function of a protein is determined by its structure rather than its sequence of amino acids. However, the sequence of the amino acids is a key factor in determining the final structure of the protein.  


A protein with no fixed structure is said to be in a random coil formation. This has very little regular structure and no activity. Functional proteins have a very tightly regulated structure held together by hydrogen bonds and Van der Waals forces between nearby amino acids, di-sulphide bridges between cysteine residues, and hydrophobic interactions.  


The structure of a protein has four levels of complexity.

Primary structure simply describes the sequence of the amino acids in the polypeptide chain. 


Secondary structure describes the large regular sub-structures that form as the protein folds.  

There are two major sub-structures that form as secondary structure. These are the α- helix and the β-sheet. Certain amino acids in sequence are known as helix formers (including e.g. methionine, alanine and leucine). They form tight coils known as α-helices, which can be joined by loops (short amino acid sequences with loose structure). 


Hydrogen bonds between the doublebonded oxygen of one amino acid and the amino hydrogen four amino acids along the helix hold the structure together. The structure of an α-helix is shown in figure 4.  




α-helices form in many proteins and are frequently found in membrane spanning proteins – proteins that sit in the cell membrane. These are used to
transmit signals or ions or molecules across the membrane either into or out of the cell. These proteins may have multiple trans-membrane domains.  


As an example of this, Gproteins transmit external signals into the cell. These have a regular structure including 7 membrane spanning helices. A representation of this is shown in figure 5.   




β-sheets are formed when straight chains of amino acids in the polypeptide run past each other in opposite directions (anti-parallel) as the protein chain folds.  


Hydrogen bonds form between the carboxyl oxygen and the amino hydrogen of opposing amino acids giving the structure rigidity. These chains are joined at either end by loops or turns. 




This flat linear structure is called a β- pleated sheet or β-sheet. Figure 6A shows the hydrogen bonding between amino acids and figure 6B shows ‘green fluorescent protein’ in which β- sheets, shown in yellow, form a barrel structure. 


Tertiary structure is the final structure that forms from the secondary structures. It is the final 3-dimensional structure of the protein. It is held in place by hydrogen bonds and Van der
Waals forces, hydrophobic interactions, and disulphide bridges. 


Some proteins only function when two or more polypeptide chains come together to form dimers/trimers etc., collectively known as oligomers. These can be made from identical
sub-units (component proteins) and be called homomers, or different subunits and be called heteromers.  


The arrangement made by the formation of oligomers is known as quaternary structure. For example, the final structure of hemoglobin is a homotetramer made up of four subunits each itself heterotetrameric consisting of 2 pairs of sub-units α and β (figure 7). 






Post-translational modifications 


The process of manufacturing a protein within a biological cell is called translation. It is at this stage that amino acids are joined together in sequence and the protein folds. This process is performed by ribosomes, themselves partially protein, which translate the sequence of a strand of RNA (similar to DNA) into a sequence of amino acids for a protein.  


Subsequent to this process a number modifications can take place that affect the activity of the protein. These can include glycosylation, where chains of sugars are bound to the surface of the protein. This is frequently used to target proteins to particular locations within the cell e.g. the cell membrane.  


Phosphorylation, the addition of a phosphate group can be used to modify the activity of a protein. Multiple glycosylation and phosphorylation sites may be present

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