Protein Function I 9

Question 1

Post translational modifications - Phosphorylation

Answer 1

• transfer of PO4^2- from ATP to Ser, Thr, or Tyr
• catalyzed by Kinases, removed by Phosphatases
• regulation of metabolism and signal transduction
• creates an overall negative charge

Serine -> Phosphoserine
Threonine -> phosphothreonine
Tyrosine -> phosphotyrosine

Question 2

Post-translational modifications: glycosylation

Answer 2

• attachment of complex carbohydrate groups to Asn or Ser
• secreted, extracellular or lumenal proteins
• affects protein stability, targeting and recognition
• bacteria can be designed to bind to sugar groups

Question 3

Post-translational modifications: proteolysis

Answer 3

• enzymatic cleavage of polypeptide chain at specific sites
• activation of other pro teases (eg. Thrombin and hemostasis)
• synthesis of peptide hormones
• if a protein has 3 connected chains, a superficial chain may be taken out by cleavage
• Proinsulin -> active form of insulin

Question 4

Post-translational modification: ubiquitin and proteasomes

Answer 4

• cytosolic proteins are degraded in proteosome after ubiquitination
• organellar/extracellular proteins are degraded in lysosomes

Question 5

Myoglobin

Answer 5

• myoglobin is a monomeric oxygen carrier in muscle cells: consists of 8 alpha helices (ancestor of hemoglobin)
• contains porphyrin heme prosthetic group, wedged between helices E and F
• burying heme in a hydrophobic pocket helps to prevent its oxidation to Fe3+
• makes iron stable
• Fe2+ is coordinated by poryphyrin ring to form heme (high affinity for oxygen)
  
  • also coordinates to O2 and His F8

Question 6

Oxygen binding to myoglobin is hyperbolic

Answer 6

• binding of oxygen to myoglobin can be expressed as a dissociation
• the fractional saturation (Y) of myoglobin with oxygen is given by bound Mb/Total Mb (conc)
• K is inversely proportional to the affinity of Mb for O2
• small Kd means high affinity

Question 7

Myoglobin and Hemoglobin are homologous

Answer 7

• hemoglobin is a tetrameric protein (a2b2) that carries oxygen from lungs to tissues in blood
• despite only 18% sequence identity, Mb and Hb subunits have similar sequences
• invariant residues indicate critical function, while sequence difference indicates evolutionary divergence
• conservative mutations may not change the function
• homology can give clues to function
• area around heme is most structurally conserved

Question 8

Oxygen binding to hemoglobin is sigmoidal

Answer 8

• Hb exhibits a sigmoidal O2 dissociation curve
• allows Hb to release much more oxygen than Mb could at lower oxygen pressure levels in tissue
• sigmoidal behaviour is typical of multisubunit allosteric proteins
• allows Hb to be a better oxygen transporter than Mb

Question 9

Oxygen binding in Hb

Answer 9

• causes a structural change
• O2 binding to one subunit deoxy state moves helices E and F relative to eachother: this is transmitted to other subunits
• heme group is planar in relaxed (oxygenated form) otherwise it is displaced from the centre of the ring

Question 10

Hemoglobin exists in 2 states with different O2 affinity

Answer 10

• hemoglobin can exist in 2 states that differ in intersubunit contacts with O2 affinity:
  
  1. The T (taut, tense, deoxy) state, salt bridges between subunits favour a low affinity conformation
  2. The R (relaxed, oxy) state, these ionic contacts are broken and the O2 affinity is increased

-O2 binding to one subunit in the T state moves helices E and F relative to eachother: this is transmitted to other subunits

Question 11

Hemoglobin: allosteric effectors

Answer 11

• H+ (Bohr effect)
  
  • CO2 production in tissues: formation of H2CO3 (dissociates to H+ and HCO3^-
  • decreased pH protonates key histidine side chained at ab interface (stabilizes T)
• CO2
  
  • reacts directly with N-terminal amino groups to form carbamate (stabilizes T)
  • some Co2 is transported to lungs this way
• BPG (regulatory molecule)
  
  • negatively charges regulatory molecule binds at positive charged hole at centre of Hb, only in T state (stabilizes T)
  • BPG is increased at high altitude and in chronic anemia (favouring O2 release)
  • fetus Hb doesnt bind BPG (higher affinity for O2

Question 12

Molecular basis of sickle cell

Answer 12

1. A point mutation in the DNA codes for structurally altered HbS
2. In the deoxygenated state, HbS polymerizes into long, rope-like fibres
3. Intracellular fibres of HbS distort the erythrocytes into sickle cell shape
   
   • cannot carry oxygen

Question 13

List of post-translational modifications

Answer 13

• phosphorylation
• glycosylation
• proteolysis