Lecture 13A - Protein Function

Question 1

Myoglobin (Mb)

Answer 1

• O2 carrying protein is muscle
• Composed of only one polypeptide chain
• Protein is 80% a-helical with 153 amino acids
• O2-binding cofactor is heme
• Protein is globin

Question 2

Heme Cofactor

Answer 2

• Termed a porphyrin (tetrapyrrole)
• Binds Fe2+ in the center which is the site of O2 binding
• Heme binds between E and F helices
• HisF8 (“proximal”) coordinates the Fe2+
• HisE7 (“distal”) restricts the site of the O2 binding site
• Globin modifies O2 binding properties of heme, O2 forced to bind at angle to reduce possibility of oxidation of Fe2+, results in reversible O2 binding

Question 3

Other Molecules Binding to Heme Groups

Answer 3

• CO2 binds tighter than O2 (danger)
• NO may be small molecule regulator
• H2S and CN- are lethal gases

Question 4

O2 Binding Curve for Mb

Answer 4

• X-axis value is the partial pressure of O2 (pO2)
• Y-axis value is “degree of saturation” (YO2)
• Mb curve is hyperbolic

Question 5

Degree of saturation (YO2)

Answer 5

• Also known as fractional saturation
• Number of binding sites occupied / total number of binding sites
• When YO2 = 0.5, pO2=1/Ka
• Define P50 = pO2 at which YO2 = 0.5
• For myoglobin, YO2 = pO2/(pO2 + P50)
• P50 for myoglobin is 3 Torr

Question 6

Hemoglobin (Hb)

Answer 6

• 4 subunits, quaternary structure
• 2 a subunits, 2 b subunits, 4 O2 binding sites
• ab dimer interacts with another ab dimer
• important contacts are a1b2 and a2b1

Question 7

O2 Binding Curve for Hb

Answer 7

• Curve is sigmoidal
• P50 is 30 Torr for the first O2 bound and 0.3 Torr for the last O2 bound
• Hb binds O2 cooperatively, and binds n molecules of O2

Question 8

Hill Plot

Answer 8

• log(PO2) on the x-axis
• log(YO2/1-YO2) on the y-axis
• slope = # O2 molecules that bind at one time
• YO2 = 0.5 = P50 and (YO2/1-YO2) = 1
• Hill constant is 3 for normal hemoglobin, indicating O2 binding is very cooperative
• Hill constant is 1 at low YO2 because each subunit competes with the other independently
• Hill cosntant is 1 at high YO2 because the remaining empty sites are on different molecules and bind independently from eachother

Question 9

Hemoglobin Conformations - Tense State (T state)

Answer 9

• Hb conformation in the deoxy-state
• Low affinity state binds O2 poorly at all 4 binding sites
• Ability of hemoglobin to bind O2 is dependant on equilibrium

Question 10

Hemoglobin Conformations - Relaxed State (R state)

Answer 10

• Hb conformation in the oxy-state
• High affinity state binds O2 well at all 4 binding sites
• Ability of hemoglobin to bind O2 is dependant on equilibrium

Question 11

Structural Changes Upon Binding of O2 - Heme Geometry

Answer 11

• Fe2+ moves 0.6A into the heme plane
• In T state the Fe2+ os 0.6A out of the heme plane on proximal side

Question 12

Structural Changes Upon Binding of O2 - Movement of the F-helix

Answer 12

Moves about 1A because the position of HisF8 is altered on O2 binding and drags the F helix with it

Question 13

Structural Changes Upon Binding of O2 - Changes In Interactions at the a1b2 Interface

Answer 13

• Subunits move relative to eachother
• Example is the His97 Area

Question 14

Structural Changes Upon Binding of O2 - Changes at the C-termini of the a and b chains

Answer 14

• T state is stabilized by a network of salt bridges that must break down to form the R state
• Structural changes break the salt bridges, which is driven by the FeO2 bond’s energy of formation

Question 15

Positive Cooperativity

Answer 15

• Binding of one O2 to one Hb binding site affects the binding of other O2 to the other binding sites to make them easier
• Binding of first one or two O2 to low affinity T-state is difficult
• After binding 1 or two O2, low affinity T-state converts to high affinity R-state
• Subsequent O2 bindings are to the high affinity R-state
• Result is sigmoidal O2 binding curve

Question 16

Alternation between T and R states

Answer 16

• A good thing
• Hb binds O2 in the lungs where pO2 is high
• Hb releases O2 in the body where pO2 is low
• Triggering mechanism is the movement of the F helix as it is dragged by HisF8 (proximal His that binds to Fe2+) upon oxygen binding to the iron

Question 17

Allosteric Proteins

Answer 17

• Cooperativity of O2 binding to Hb is a model for the behavior of other multisubunit proteins (and enzymes) that bind small molecules
• These allosteric effects can be positive (as
  seen for Hb) or negative
• 2 models proposed

Question 18

Symmetry Model (MWC)

Answer 18

• Symmetric arrangement of oligomers (subunits) and each can exist in two different conformational states (R and T)
• A ligand can bind to a subunit in either conformational state
• The molecular symmetry of the protein is conserved during the conformational change
• All subunits therefore must exist as either R or T, not a mixture of subunits

Question 19

Sequential Model (Koshland)

Answer 19

• Ligand binding induces a conformational change in the subunit that the ligand binds to, and cooperative interactions arise through the influence of those conformational changes on
  neighbouring subunits
• The conformational changes occur sequentially as more ligand-binding sites are occupied.