Revision:Pre-clinical: Overview of Protein Structure and Function

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The Old Basics

As we should know (be worried otherwise), DNA is a template for RNA and thus, ultimately, proteins. DNA is transcribed into RNA, which is translated into proteins. Each three nucleotides on the sequence encodes one primary amino acid (there are twenty of these, for reference*).

Protein structure may be described in four degrees:

• The primary structure of a protein is the sequence of amino acids that make it connected by peptide (N-C) bonds. Side-chains may be charged, polar or non-polar, affecting the affinity of the protein for water and the conformation of the folded protein. The length of the chain affects its flexibility and consequently its folding.
• The secondary structure of a protein describes a protein's basic three dimensional shape prior to conformational folding: due to electrostatic attraction between amino acids, this typically forms either an α-helix or a β-pleated sheet.
• The tertiary structure is the three dimensional conformation of the protein: the protein folds into the structure that requires the lowest energy to maintain - so factors like charged side-chains will affect this, since, exempli gratia, two positively charged amino acids will repel and therefore require energy to force into proximity, thus the protein would conform to have these away from each other.
• The quaternary structure describes the structure of a complex of multiple polypeptide chains interacting, exempli gratia haemoglobin.

Structure and Function

The entire structure of a protein is ultimately decided by its primary structure. Though molecular chaperones may assist proteins into their lowest energy conformation, they don’t affect the structure. The N and unbonded O atoms present in the peptide bonds form hydrogen bond with other areas of the (poly)peptide. These hydrogen bonds produce α-helices when the bond involves those of an amino acid 4 amino acids away, and β-pleated sheets when the bond is to adjacent polypeptides. In forming the tertiary structure, these helices and sheets are important, and are conformed by further bonding and, importantly, electrostatic forces from side-chains and hydrophobic and hydrophilic areas interacting in their environment. Disulphide bridges also occur but not in the cytosol: the reducing nature of the cytoplasm removes any present, however they can form extracellularly and in the endoplasmic reticulum, where there is an oxidative environment. Disulphide bridges stabilise the existing conformation, giving proteins such as lysozyme in tears durability and longevity.

α-helices are often found as the trans-membrane domain of integral membrane proteins. These will have any hydrophilic side-chains directed to the inside of the helix, allowing the hydrophobic chains to anchor the protein in the lipid bilayer.

As in all things, structure is related to function. Thus, some proteins are globular, especially those that are to be secreted, like enzymes; some are elongated, such as collagen; and some are shaped to form complexes as homomers and heteromers, such as haemoglobin. Proteins perform various functions. To name but a few major categories:

• Binding and transport: ligands and receptors
• Catalysis: enzymes
• Switching: signalling pathways
• Structuring: cytoskeletal proteins

Protein function is related to conformation. It is reliant on changes therein, produced by environmental changes (exempli gratia enzyme activation by change in pH), substrate binding and regulatory peptide binding (exempli gratia CDK and cyclin). Phosphorylation of the protein is often involved. This is especially relevant considering the hydrolysis of ATP produces ADP and one inorganic phosphate, which therefore may be used to change the conformation and thus function of a protein.

The binding sites of proteins are responsible for their functions (especially regarding enzymes). The conformation of the protein determines the atoms present at these binding sites, and so the exact three-dimensional locations where non-covalent forces may act between the protein and the binding target (such as hydrogen bonds) will occur.

As such, proteins may be targeted by drugs for treatment of various diseases. For example, HIV protease is required for replication of HIV; drugs that occupy the active site inhibit the enzyme functioning to replicate HIV.

Regulation of Protein Function

Protein activity and function must be regulated to ensure that the correct functions are being carried out at the correct time, at the correct place, at the correct speed and in the correct amount. This is regulated by four processes:

1. Synthesis: to control whether the protein is present
2. Localisation: to control where the protein is acting
3. Modification: to control the activity of the protein
4. Degradation: to remove the protein where it is no longer required

Much of this is co-ordinated by the regulation of gene expression in response to specific stimuli, such as markers and hormones in immune responses, or factors related to cell division.

Protein synthesis is undertaken by ribosomes, which are present both free in the cytosol (especially in polysomes) and, importantly, are studded along the membrane of the rough endoplasmic reticulum (RER), forming the ‘rough’ component. To simplify, ribosomes receive mRNA and create a peptide by matching and bonding amino acids from complementary tRNA. The product (not necessarily ‘finished’) is transported to the next compartment -- usually the Golgi apparatus -- in a transport vesicle for further modification.

Further to the synthesis of the protein, some modification of proteins occurs in the RER and the Golgi apparatus: most pertinently glycosylation and phosphorylation. Importantly, however, further modification for activation or conformation changes often occur in situ after the protein has been localised. This further modification is also often phosphate-intensive; whether it involves phosphorylation (kinases, remember?) or dephosphorylation (phosphatases, duh) to inhibit or activate the protein, the modification is responsible for conformational change in the protein, which affects its function.

Exempli gratia, the binding of growth factor or cytokines to a receptor tyrosine kinase causes dimerisation and phosphorylation, allowing an adaptor protein from the cytoplasm to bind, to which a Ras protein binds and activates.

Localisation -- transport of the protein to where it is needed -- is controlled by sorting signals defined by the structure of the protein. The conformation is important, as the chemical sequence that is ‘read’ as the sorting signal is not necessarily that of the primary structure, but may involve amino acids from many unrelated points along the chain due to the tertiary structure of the protein. These sorting signals may also be activated or modified by phosphorylation.

Of Clinical Interest

Due to the highly specific nature of protein function and sorting signals, changes to proteins are largely significant (although some minor ones may not have any tangible effect, but these are, funnily enough, not noticed much). Whilst random damage to a protein will result in their degradation by natural failsafes, mutations in the gene that encodes the protein are much more problematic as the expression of said genes may result in the synthesis of incorrect proteins, as well as too much synthesis, incorrect localisation, failure to degrade and aberrant function. Such mutations may be inherited (exempli gratia in Marfan syndrome) or acquired (exempli gratia from exposure to radiation).

An example of this is cystic fibrosis, most commonly caused by a mutation that deletes phenylalanine at position 508 in the gene encoding CFTR, resulting in the protein having an incorrect tertiary structure, its retention in the endoplasmic reticula, and its degradation without reaching the cell membrane where it should function.

Prion diseases (such as CJD) are caused by infectious mis-folding of proteins that induces the same conformation in proteins it comes into contact with. They are universally fatal and largely untreatable. These proteins have unwanted amyloid folds, and aggregate due to their extremely stable nature, eventually accumulating to the point at which they cause cell death.

* Yes, after so many years happily trotting along knowing we have a redundant genetic code since there only 20 naturally occurring amino acids, suddenly Dr. Wallace and some shifty looking molecular biologists told us that there are actually 22 proteinogenic amino acids floating around. So why the difference in figures? The important thing is that only 20 amino acids (the ones we know and love, like that little rascal methionine) are encoded by the universal genetic code. The other two (those blaggards ornithine and taurine) are only made available through the degradation of other compounds or through dietary supply, and are not synthesised de novo by ribosomes. If you’re interested, ornithine is an important substrate in the urea cycle (next year, methinks...), important for regeneration of arginine.