Protein phosphorylation

• Post-translational modification (PTM)

  Covalent attachment of other molecules to the protein after synthesis
• What are the uses of post-translational modifications

  • Used to regulate the activity of many proteins
  • Reversible modifications allow for continuous regulation
• Describe Phosphorylation

  Occurs on Hydroxyl groups of Serine, Thryroninie and Tyrosine.
• What are the effects of phosphorylation

  A conformational change due to a change in electrostatic interactions within the protein

  Allows for interactions with other protein due to the change in electrostatic interactions
• what do protein kinases do

  Phosphorylate a protein using ATP
• what is the effect of phoshorylation

  The phosphate group has 2 negative charges which change the electrostatic interactions
• Which form of glycogen phosphorylase is active

  The phosphorylated version
• Phosphorylation of glycogen phosphorylase

  Phosphorylation of the serine is a form of allosteric regulation.

  Causes conformational change from T state (inactive) to R state (active).

  The regulatory loops move away from the catalytic sites.
• Phosphorylation of glycogen synthase

  Increases negative charge, causing conformational change and inactivation.
• Phosphorylation-dependent regulation

  Phosphorylation alone does not affect enzyme activity, but it promotes the binding of other, regulatory proteins
• Regulatory proteins that bind phosphorylated proteins

  • SH2 domains (bind phosphotyrosine)
  • 14-3-3 proteins (bind phosphoserine)
• Phosphorylation-dependent regulation of plant nitrate reductase

  Nitrate reductase produces toxic nitrite which is only used in the light.

  The 14-3-3 protein binds to the phosphorylated serine reside in nitrate reductase.

  
  

  Dark = Nitrate reductase is phosphorylated by nitrate reductase kinase and binds to 14-3-3 protein. It is inactivated.

  
  

  Light = Nitrate reductase in unbound from 14-3-3 protein and dephosphorylated by protein phosphatase to form nitrate reductase. It is activated.
• Proteolytic activation

  Conversion of inactive precursors (zymogens/proenzymes) into active enzymes by specific cleavage of peptide bonds
• Proteolytic activation of chymotrypsinogen

  Chymotrypsinogen is secreted by the pancreas for transport to the duodenum.

  Trypsin removes two dipeptides producing 3 peptides which are held by interchain disulphide bonds.

  
  

  The disulphide bonds maintain the tertiary structure.

  Cleavage forms the hydrophobic and oxyanion cavities.
• Blood clotting

  • Cascade of zymogen activations
  • Allows amplification of initial weak signals
• Blood clotting cascades

  • Intrinsic response (to collagen, kallikrein and kininogen from blood vessels)
  • Extrinsic response (to Tissue factor 3 from damaged tissues)
• Fibrin formation

  Fibrinogen is converted to fibrinogen using thrombin which removes A and B fibrinopeptides.
• What are the methods of post-translational modifications used

  Phosphorylation, acetylation and methylation.
• What is a serine kinase

  A protein kinase that phosphorylates the hydroxyl group on serine
• What do protein phosphatases do

  Dephosphorylate a protein using ADP
• which version of glycogen phosphorylase is inactive

  The dephosphorylated version
• What version of glycogen synthase is active

  The dephosphorylated version
• Which version of glycogen synthase is inactive

  The phosphorylated version
• What does glycogen phosphorylase do

  Catalyses glucose production from glycogen
• What does glycogen synthase do

  Catalyses the production of glycogen from glucose
• How is glycogen phosphorylase regulated

  Using adrenaline
• How is glycogen synthase regulated

  Using insulin
• Describe the structure of glycogen phosphorylase

  It is a dimer of two subunits
• Describe the structure of glycogen synthase

  Has many phosphorylation sites at the N and C terminals
• What is the structure of fibrinogen

  It is a triple stranded alpha helix with globular domains at the end.

  
  

  It consists of 2 A alpha, B beta and gamma.
• Describe the formation of fibrin

  Thrombin cleaves 4 peptide bonds. This removes an A peptide of 18 residues from the two A alpha chains and 20 residues from the two B beta chains.

  These peptides are called fibrinopeptides and results in the formation of a fibrin monomer.
• What are protofibrils

  Polymerised Fibrin monomers
• What is the subunit structure of fibrinogen

  2 lots of A alpha, B beta, Gamma.
• What is the subunit structure of fibrin

  2 lots of Alpha, Beta and Gamma.
• Describe the polymerisation of fibrin monomers

  This forms protofibrils.

  Cleavage exposes amino acid sequences which interact with the beta and gamma subunits to form crosslinking protofibrils.