(a) Proteomics       

• The proteome is the entire set of proteins expressed by a genome
• The proteome is larger than the number of genes, particularly in eukaryotes, because more than one protein can be produced from a single gene as a result of alternative RNA splicing
• Not all genes are expressed as proteins in a particular cell type
• Genes that do not code for proteins are called non-coding RNA genes and include those that are transcribed to produce tRNA, rRNA, and RNA molecules that control the expression of other genes.
• The set of proteins expressed by a given cell type can vary over time and under different conditions
• Some factors affecting the set of proteins expressed by a given cell type are the metabolic activity of the cell, cellular stress, the response to signalling molecules, and diseased versus healthy cells.

(b) The synthesis and transport of proteins

(i) Intracellular membranes

• Eukaryotic cells have a system of internal membranes, which increases the total area of membrane
• Because of their size, eukaryotes have a relatively small surface area to volume ratio. The plasma membrane of eukaryotic cells is therefore too small an area to carry out all the vital functions carried out by membranes.
• The endoplasmic reticulum (ER) forms a network of membrane tubules continuous with the nuclear membrane
• The Golgi apparatus is a series of flattened membrane discs
• Lysosomes are membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates
• Vesicles transport materials between membrane compartments

 (ii) Synthesis of membrane components   

• The cytosol is the fluid part of the cytoplasm
• Lipids and proteins are synthesised in the ER
• Rough ER (RER) has ribosomes on its cytosolic face while smooth ER (SER) lacks ribosomes.
• Lipids are synthesised in the smooth endoplasmic reticulum (SER) and inserted into its membrane
• The synthesis of all proteins begins in cytosolic ribosomes
• The synthesis of cytosolic proteins is completed there, and these proteins remain in the cytosol
• A signal sequence is a short stretch of amino acids at one end of the polypeptide that determines the eventual location of a protein in a cell.
• Transmembrane proteins carry a signal sequence, which halts translation and directs the ribosome synthesising the protein to dock with the ER, forming RER
• Translation continues after docking, and the protein is inserted into the membrane of the ER

 (iii) Movement of proteins between membranes

• Once the proteins are in the ER, they are transported by vesicles that bud off from the ER and fuse with the Golgi apparatus
• As proteins move through the Golgi apparatus they undergo post-translational modification
• Molecules move through the Golgi discs in vesicles that bud off from one disc and fuse to the next one in the stack. Enzymes catalyse the addition of various sugars in multiple steps to form the carbohydrates.
• The addition of carbohydrate groups is the major modification
• Vesicles that leave the Golgi apparatus take proteins to the plasma membrane and lysosomes
• Vesicles move along microtubules to other membranes and fuse with them within the cell

 (iv) The secretory pathway

• Secreted proteins are translated in ribosomes on the RER and enter its lumen
• The proteins move through the Golgi apparatus and are then packaged into secretory vesicles
• These vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell
• Peptide hormones and digestive enzymes are examples of secreted proteins.
• Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins
• Proteolytic cleavage is another type of post-translational modification.
• Digestive enzymes are one example of secreted proteins that require proteolytic cleavage to become active.

(c) Protein structure, ligand binding and conformational change

(i) Amino acid sequence determines protein structure   

Amino Acids

• Proteins are polymers of amino acid monomers
• Amino acids are linked by peptide bonds to form polypeptides
• Recognise the chemical structure of a peptide bond from a diagram.
• Amino acids have the same basic structure, differing only in the R group present
• Amino acids are classified according to their R groups:
  • basic (positively charged – look for NH_2/3);
  • acidic (negatively charged – look for – COOH);
  • polar (hydrophilic – look for OH);
  • hydrophobic (look for hydrocarbon chains)
• R groups of amino acids vary in size, shape, charge, hydrogen bonding capacity and chemical reactivity.

Protein structure

• The wide range of functions carried out by proteins results from the diversity of R groups
• The primary structure is the sequence in which the amino acids are synthesised into the polypeptide
• Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure — alpha helices, parallel or anti-parallel beta-pleated sheets, or turns
• The polypeptide folds into a tertiary structure
• This conformation is stabilised by interactions between R groups: hydrophobic interactions; ionic bonds; London dispersion forces; hydrogen bonds; disulfide bridges
• Disulfide bridges are covalent bonds between R groups containing sulfur.
• Quaternary structure exists in proteins with two or more connected polypeptide subunits
• Quaternary structure describes the spatial arrangement of the subunits.
• A prosthetic group is a non-protein unit tightly bound to a protein and necessary for its function
• The ability of haemoglobin to bind oxygen is dependent upon the non-protein haem group.

Factors affecting R groups

• Interactions of the R groups can be influenced by temperature and pH
• Increasing temperature disrupts the interactions that hold the protein in shape; the protein begins to unfold, eventually becoming denatured.
• The charges on acidic and basic R groups are affected by pH.
• As pH increases or decreases from the optimum, the normal ionic interactions between charged groups are lost, which gradually changes the conformation of the protein until it becomes denatured.

(ii) Ligand binding changes the conformation of a protein          

Ligands

• A ligand is a substance that can bind to a protein
• R groups not involved in protein folding can allow binding to ligands
• Binding sites will have complementary shape and chemistry to the ligand
• As a ligand binds to a protein-binding site the conformation of the protein changes
• This change in conformation causes a functional change in the protein

Allosteric molecules

• Allosteric interactions occur between spatially distinct sites
• Many allosteric proteins consist of multiple subunits (have quaternary structure)
• Allosteric proteins with multiple subunits show co-operativity in binding, in which changes in binding at one subunit alter the affinity of the remaining subunits
• The binding of a substrate molecule to one active site of an allosteric enzyme increases the affinity of the other active sites for binding of subsequent substrate molecules.
• This is of biological importance because the activity of allosteric enzymes can vary greatly with small changes in substrate concentration.
• Allosteric enzymes contain a second type of site, called an allosteric site
• Modulators regulate the activity of the enzyme when they bind to the allosteric site
• Following binding of a modulator, the conformation of the enzyme changes and this alters the affinity of the active site for the substrate
• Positive modulators increase the enzyme’s affinity for the substrate, whereas negative modulators reduce the enzyme’s affinity.
• The binding and release of oxygen in haemoglobin shows co-operativity

The influence and physiological importance of temperature and pH on the binding of oxygen

• Changes in binding of oxygen at one subunit alter the affinity of the remaining subunits for oxygen.
• A decrease in pH or an increase in temperature lowers the affinity of haemoglobin for oxygen, so the binding of oxygen is reduced.
• Reduced pH and increased temperature in actively respiring tissue will reduce the binding of oxygen to haemoglobin promoting increased oxygen delivery to tissue.

(iii) Reversible binding of phosphate and the control of conformation 

• The addition or removal of phosphate can cause reversible conformational change in proteins
• This is a common form of post-translational modification
• Protein kinases catalyse the transfer of a phosphate group to other proteins
• The terminal phosphate of ATP is transferred to specific R groups
• Protein phosphatases catalyse the reverse reaction
• Phosphorylation brings about conformational changes, which can affect a protein’s activity
• The activity of many cellular proteins, such as enzymes and receptors, is regulated in this way
• Some proteins are activated by phosphorylation while others are inhibited
• Adding a phosphate group adds negative charges.
• Ionic interactions in the unphosphorylated protein can be disrupted and new ones created.