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BCMB20002 Biochemistry > Protein Function > Flashcards

Protein Function Flashcards

1
Q

What is quaternary structure?

A
  • Two or more polypeptide chains/subunits

* Subunits divided into functional regions/domains

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2
Q

What makes up haemoglobin?

A
  • 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
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3
Q

What is the quaternary structure and function of IgG?

A
  • Four subunits (2 heavy, 2 light chains)
  • Bind antigen ligand
  • Variable regions at end undergo conformational change
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4
Q

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

A

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

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5
Q

Are enzymes usually multimeric?

A

Yes

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6
Q

What is special about the structure of pyruvate dehydrogenase?

A

(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

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7
Q

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

A

Quartenary

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

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8
Q

What gives collagen strength?

A

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

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9
Q

What is the structure of myoglobin?

A
  • 16.7 kDa, 153 AA
  • Muscle protein, red, 70% α helix
  • 8 α helices (7-23 AA)
  • Diving animals, seals
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10
Q

What is the function of myoglobin?

A
  • Store oxygen in muscles

* Release oxygen, muscle contraction and energy

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11
Q

What allows seals to dive for a long time?

A

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

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12
Q

How does O2 interact with Heme?

A

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

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13
Q

What is the heme prosthetic group?

A

o Planar, porphyrin ring

o Fe binds to histidine residue and O2

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14
Q

What are pO2 and θ and how do they interact?

A

• 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

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15
Q

What is involved in the binding equilibrium?

A

• 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]

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16
Q

What is Kd and Ka?

A

• 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)

17
Q

What’s the equation for θ?

A

θ = ([L])/(Kd+[L])

18
Q

How does Kd relate to LP binding?

A
  • 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
19
Q

Which concentration units are used for Kd and Ka?

A

d: conc, a: conc ^-1

20
Q

What is biotin?

A
  • 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
21
Q

What is cooperative binding?

A

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

22
Q

What happens during the binding of O2 to Hb?

A

• 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

23
Q

What are the T and R states of Hb?

A
  • Low affinity Hb = T state (tense)

* High affinity Hb = R state (relaxed)

24
Q

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

A
  • 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)
25
Q

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

A
  • 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
26
Q

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

A
  • 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

27
Q

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

A
  • 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
28
Q

How does altitude impact oxygen delivery?

A
  • 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.
29
Q

What is special about foetal Hb?

A
  • γ not β subunits

* lower affinity for BPG than maternal Hb, higher affinity for O2

30
Q

What is the hill plot?

A 
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
31
Q

What are the features of Hb’s nH?

A

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

32
Q

What does nH > 1 mean?

A

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

33
Q

What does nH = 1 mean?

A

nH = 1: not cooperative binding, sites are independent

34
Q

What does nH < 1 mean?

A

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

35
Q

How come carbon monoxide is more dangerous than anaemia?

A
  • 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
36
Q

What happens in sickle cell anaemia?

A
  • Mutant β gene, allele for S Hb, both parents

* Deoxygenated S Hb forms polymers which aggregate to insoluble tubular fibres

37
Q

What is the chemical reasoning behind sickle cell anaemia?

A

• 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

38
Q

What are the consequences of having Hb s?

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

BCMB20002 Biochemistry (7 decks)

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