Protein Function

Question 1

What is quaternary structure?

Answer 1

• Two or more polypeptide chains/subunits

* Subunits divided into functional regions/domains

Question 2

What makes up haemoglobin?

Answer 2

• Tetramer of four subunits
• Dimer of Two α, Two β protomers
• Protomer: structural unit of protein with quarte nary structure
• Each chain has single globin domain

Question 3

What is the quaternary structure and function of IgG?

Answer 3

• Four subunits (2 heavy, 2 light chains)
• Bind antigen ligand
• Variable regions at end undergo conformational change

Question 4

What happens in one subunit experiences conformational change within a protein?

Answer 4

• Conformational change of one subunit has flow on effect through whole complex (e.g. hb and O2 binding)

Question 5

Are enzymes usually multimeric?

Answer 5

Yes

Question 6

What is special about the structure of pyruvate dehydrogenase?

Answer 6

(PDH)
• Structure allows direct channelling of substrates to active sites
• Made of 3 different enzymes (3 different genes contribute)
o E1 pyruvate dehydrogenase
o E2 dihyrolipoyl transacetylase
o E3 dihyrolipoyl dehydrogenase

Question 7

What types of structure do insoluble fibrous proteins have? What’s an examples?

Answer 7

Quartenary

• Eg. Collagen, fibrils in connective tissue, amyloid fibrils

Question 8

What gives collagen strength?

Answer 8

• Collagen strong because repeating trinucleotide forms helical structure, 3 helices wrap around each other

Question 9

What is the structure of myoglobin?

Answer 9

• 16.7 kDa, 153 AA
• Muscle protein, red, 70% α helix
• 8 α helices (7-23 AA)
• Diving animals, seals

Question 10

What is the function of myoglobin?

Answer 10

• Store oxygen in muscles

* Release oxygen, muscle contraction and energy

Question 11

What allows seals to dive for a long time?

Answer 11

slow heart rate, breathing, shunt blood to heart/brain/muscles. Lots of mb, positively charged surface to prevent clumping as mb proteins repel each other

Question 12

How does O2 interact with Heme?

Answer 12

• O2 binds reversibly to heme group Fe2+ (allow O2 to be released when needed)

Question 13

What is the heme prosthetic group?

Answer 13

o Planar, porphyrin ring

o Fe binds to histidine residue and O2

Question 14

What are pO2 and θ and how do they interact?

Answer 14

• θ is fractional saturation for binding sites of P/Mb
o fraction of available sites bound to ligand
o [PL]/([P]+[PL])
• pO2 = partial pressure of oxygen

Question 15

What is involved in the binding equilibrium?

Answer 15

• Protein ligand interaction ( P + L ↔ PL ), Mb + O2 ↔ MbO2

• NOT related to Ka for acid-base pKa stuff
• P + L ↔ PL
• Rate constants (f forward, b backward)
o Kf
o Kb
• At equilibrium when rate of forward reaction = rate of backward
o Kf [P][L] = Kb [PL]

Question 16

What is Kd and Ka?

Answer 16

• Association constant for L to P binding is Ka
o Ka = Kf/Kb = [PL]/([P][PL])
o Ka is equilibrium constant for P + L ↔ PL
o Concentration is inverse (conc-1, mM-1 etc)
• Disassociation constant is Kd (inverse of Ka)
o Kd = 1/Ka = [P][L]/[PL]
o Kd is equilibrium constant for release of L from binding site on PL ↔ P + L
o Concentration is normal (conc, mM etc)

Question 17

What’s the equation for θ?

Answer 17

θ = ([L])/(Kd+[L])

Question 18

How does Kd relate to LP binding?

Answer 18

• Kd shows strength of L to P binding (large = low affinity, PL wants to revert to P + L)
• θ = 0.5, corresponding [L] = Kd (half the binding sites are occupied)
• Kd is concentration of L required for half of all available sites on protein to be bound to L

Question 19

Which concentration units are used for Kd and Ka?

Answer 19

d: conc, a: conc ^-1

Question 20

What is biotin?

Answer 20

• Vitamin, can’t be synthesised
• Needed for reactions with Co2 additions
• Binds avidin (protein in egg whites)
• Kd super small (10-15 M), binding basically irreversible
• Too many raw eggs = biotin deficiency

Question 21

What is cooperative binding?

Answer 21

o Work together, make it easier
o One O2 binds, makes it easier for the next ones due to the subunit changes
o Four subunits

Question 22

What happens during the binding of O2 to Hb?

Answer 22

• Hb binds O2 in lungs (pO2 13kPa) and releases O2 in tissue (pO2 4kPa)
• Low affinity Hb = T state (tense)
• High affinity Hb = R state (relaxed)
• More O2 binds, go from T to R state
• First O2 binds globin subunit weakly, T state made unstable, T to R transition made easier
o Shift in subunit pairs
o His residues of B subunits rotate towards centre, no longer involved in ion pairs
• More O2 binds, more Hb molecule in R state, more cooperative ligand binding
• Fourth O2 binds Hb in R state

Question 23

What are the T and R states of Hb?

Answer 23

• Low affinity Hb = T state (tense)

* High affinity Hb = R state (relaxed)

Question 24

How does H+ influence O2 binding to Hb? Why is this good for the body?

Answer 24

• Lower pH, decreased Hb affinity for O2 (H+ stabilise Hb in T state)
• Allows O2 to be offloaded from Hb in tissues (pH is lower because metabolism products)

Question 25

What is the mechanism for the effect of H+ on O2 and Hb binding?

Answer 25

* Protons/H+ might bind N-termini of α subunits, His residue of β subunit * Stabilises T state, θ vs. pO2 curve shifts right * Small pH change = massive affinity change

Question 26

How does Co2 influence O2 and Hb binding? What is the Bohr Effect?

Answer 26

* More Co2 in tissues (catabolism) favours O2 release * CO2 binds more strongly to amino terminal NH2 globin (NOT heme) in T structure than R structure * Binding stabilises T structure, lowers affinity for Hb of O2 * Globin-NH2 + CO2 ↔ Globin-NH-COO- + H+ • Decreased affinity of Hb for O2 at lower pH and increased CO2 = Bohr Effect

Question 27

How does 2,3, BPG influence O2 and Hb binding?

Answer 27

* BPG from intermediate in glycolysis (in RBCs, more at high altitude) * Interacts with Hb, site far from O2 binding site, has effect on O2 binding * Binds Hb, decreases affinity for O2 (like H+ and CO2) * HbBPG + O2 ↔ HbO2 + BPG * Hb single binding site for BPG in centre (larger in T state) * BPG occupies cavity, stabilises T state

Question 28

How does altitude impact oxygen delivery?

Answer 28

* High altitude, decreased pO2 in lungs, decreased O2 delivery. Increased BPG restores O2 delivery to tissue (more BPG binding and decreasing Hb affinity for O2) * No BPG: O2 binds and holds on. Essential for transfer.

Question 29

What is special about foetal Hb?

Answer 29

* γ not β subunits | * lower affinity for BPG than maternal Hb, higher affinity for O2

Question 30

What is the hill plot?

Answer 30

``` Ka = [PLn]/([P][L]n) [PLn] = Ka[P][L]n θ = 〖[L]〗^n/(Kd+〖[L]〗^n ) log (θ/1- θ) = nlog[L] - logKd log(θ/1- θ) vs log[L] = Hill plot Slope should be n (number of binding sites) Slope < number of binding sites Slope measure interaction between binding sites (degree of cooperatively) = nH ```

Question 31

What are the features of Hb’s nH?

Answer 31

Theoretical upper limit for nH is n (n = 4 for Hb) Plot deviates from straight line at high/low concentration of ligand Low [O2] means can only bind low affinity site High [O2] means most sites in high affinity state

Question 32

What does nH > 1 mean?

Answer 32

nH > 1: positive cooperativity. Bind one site, increase binding at other sites

Question 33

What does nH = 1 mean?

Answer 33

nH = 1: not cooperative binding, sites are independent

Question 34

What does nH < 1 mean?

Answer 34

nH < 1: negative cooperativity. Bind one site, decrease binding at other sites

Question 35

How come carbon monoxide is more dangerous than anaemia?

Answer 35

* Anaemic people can survive with half function Hb, but if 50% bound by CO death * CO binds strongly, HbCO accumulates * CO increases affinity of remaining Hb subunits for O2 * Hb molecule binds 2 CO molecules, bind O2 in lungs, cannot release in tissues

Question 36

What happens in sickle cell anaemia?

Answer 36

* Mutant β gene, allele for S Hb, both parents | * Deoxygenated S Hb forms polymers which aggregate to insoluble tubular fibres

Question 37

What is the chemical reasoning behind sickle cell anaemia?

Answer 37

• Glu replaced with Val o Hb S less negative charges than normal Hb (Glu +1, Val neutral) o Lose Glu from each β chain o Different solubility, aggregate I sticky patch

Question 38

What are the consequences of having Hb s?

Answer 38

``` o Less RBCs o Abnormal RBCs o Painful, life threatening o Fragile RBCs, rupture, less O2 in blood o Block capillaries ```