Protein Function Flashcards
What is quaternary structure?
- Two or more polypeptide chains/subunits
* Subunits divided into functional regions/domains
What makes up haemoglobin?
- 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
What is the quaternary structure and function of IgG?
- Four subunits (2 heavy, 2 light chains)
- Bind antigen ligand
- Variable regions at end undergo conformational change
What happens in one subunit experiences conformational change within a protein?
• Conformational change of one subunit has flow on effect through whole complex (e.g. hb and O2 binding)
Are enzymes usually multimeric?
Yes
What is special about the structure of pyruvate dehydrogenase?
(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
What types of structure do insoluble fibrous proteins have? What’s an examples?
Quartenary
• Eg. Collagen, fibrils in connective tissue, amyloid fibrils
What gives collagen strength?
• Collagen strong because repeating trinucleotide forms helical structure, 3 helices wrap around each other
What is the structure of myoglobin?
- 16.7 kDa, 153 AA
- Muscle protein, red, 70% α helix
- 8 α helices (7-23 AA)
- Diving animals, seals
What is the function of myoglobin?
- Store oxygen in muscles
* Release oxygen, muscle contraction and energy
What allows seals to dive for a long time?
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
How does O2 interact with Heme?
• O2 binds reversibly to heme group Fe2+ (allow O2 to be released when needed)
What is the heme prosthetic group?
o Planar, porphyrin ring
o Fe binds to histidine residue and O2
What are pO2 and θ and how do they interact?
• 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
What is involved in the binding equilibrium?
• 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]
What is Kd and Ka?
• 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)
What’s the equation for θ?
θ = ([L])/(Kd+[L])
How does Kd relate to LP binding?
- 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
Which concentration units are used for Kd and Ka?
d: conc, a: conc ^-1
What is biotin?
- 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
What is cooperative binding?
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
What happens during the binding of O2 to Hb?
• 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
What are the T and R states of Hb?
- Low affinity Hb = T state (tense)
* High affinity Hb = R state (relaxed)
How does H+ influence O2 binding to Hb? Why is this good for the body?
- 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)
What is the mechanism for the effect of H+ on O2 and Hb binding?
- 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
How does Co2 influence O2 and Hb binding? What is the Bohr Effect?
- 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
How does 2,3, BPG influence O2 and Hb binding?
- 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
How does altitude impact oxygen delivery?
- 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.
What is special about foetal Hb?
- γ not β subunits
* lower affinity for BPG than maternal Hb, higher affinity for O2
What is the hill plot?
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
What are the features of Hb’s nH?
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
What does nH > 1 mean?
nH > 1: positive cooperativity. Bind one site, increase binding at other sites
What does nH = 1 mean?
nH = 1: not cooperative binding, sites are independent
What does nH < 1 mean?
nH < 1: negative cooperativity. Bind one site, decrease binding at other sites
How come carbon monoxide is more dangerous than anaemia?
- 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
What happens in sickle cell anaemia?
- Mutant β gene, allele for S Hb, both parents
* Deoxygenated S Hb forms polymers which aggregate to insoluble tubular fibres
What is the chemical reasoning behind sickle cell anaemia?
• 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
What are the consequences of having Hb s?
o Less RBCs o Abnormal RBCs o Painful, life threatening o Fragile RBCs, rupture, less O2 in blood o Block capillaries
