Protein Function Structure will determine the function of the protein
Dec 17, 2015
Key ideas and terms
• protein can bind a ligand in the binding site
• For an enzyme, the ligand is a substrate and they bind in what is called the active site
• ligand has to be the correct shape
• ligand has to have the complementary charges and hydrophobicity or hydrophilicity
Lock and Key Hypothesis
• Protein and ligand have complementary shapes.
• Interactions must also be complementary– If enzyme charge is
negative, substrate must be positive
– If pocket is nonpolar, ligand must be nonpolar
• Antibodies
Induced Fit
• Induced Fit: when the protein and ligand bind, the protein may change conformation to allow for tighter binding
• Frequently, both the ligand and the protein change conformation
Examples: O2 binding proteins: myoglobin and hemoglobin
• oxygen is not water soluble yet needs to be transported
• diffusion is not effective
• myoglobin is found primarily in muscle tissue
• Hemoglobin is in the blood
• Both proteins contain heme
Heme Group
the iron must be a 2+ to bind oxygen. The heme group is buried deep within the protein so that the iron is not oxidized to 3+
• there must be flexibility in the protein to allow for oxygen to attach and then let go
• Iron has 6 coordination sites. – Four of them used by porphyrin. Unshared pairs on nitrogen
complex to iron
– Fifth and 6th for oxygen and protein
• Heme is planar
Myoglobin
• has heme group
• eight alpha helical segments
• dense hydrophobic core
– all but two polar groups on outside
– room for only 4 water molecules
• flat heme in pocket
• iron coordinated to poryphorin and H
• As well as the heme
Myoglobin Binding Curve
• Hyperbolic binding curve
• Relatively insensitive to small changes in oxygen concentration
Myoglobin
• The P50 (oxygen partial pressure required for half saturation) for myoglobin is very low
• Myoglobin has a high affinity for oxygen-an important characteristic for a protein that must extract oxygen from the small amounts present in blood.
• At the oxygen concentration existing in the capillaries, the myoglobin in adjacent tissues is nearly saturated.
• When cells are metabolically active, their internal PO2 falls
to levels where myoglobin will lose (deliver) its oxygen.
Hemoglobin
Quaternary structure : 4 subunits
• Each subunits is like myoglobin
• Each subunit has heme group
• 2 alpha chains; 2 beta chains
• Few contacts between alpha and betas, more between alphas and betas
Hemoglobin
• Exists in two states
– R state (high affinity for O2)
– Where would this state be favored? • In the lungs
– T state (low affinity for O2) (deoxyhemoglobin)
– Where would this state be favored? • In the tissue
– Sensitive to pressure changes
– On oxygenation, one pair of subunits shifts with respect to the other by a rotation of 15 degrees.
Oxygen Binding to Heme in Hemoglobin
• Fe is coordinated to a histidine in helix 8 of the Hb molecule
• In deoxy form, porphyrin is puckered and Fe is out of the plane of the heme
• When oxygen binds the Fe (at other coordination site) the Fe is pulled into the plane of the heme
• This pulls on the histidine, which pulls on the helix, changing the shape of the molecule.
Fe2+
His F8
0.6 A
O2
Hemoglobin
• Conformational changes in hemoglobin alter its binding ability
• Binding of oxygen in one subunit causes conformational changes in the next subunit
• This is called cooperative binding• This can happen because it is composed of 4 independent
subunits• produces a different binding curve that is sigmoidal• The modulation of the affinity of a site for a ligand by
ligand binding at another site is called Allostery.
Bohr Effect
• Hemoglobin's affinity for oxygen is decreased in the presence of carbon dioxide and at lower pH.
• Carbon dioxide reacts with water to give bicarbonate, carbonic acid free protons via the reaction:
CO2 + H2O ---> H2CO3 ---> H+ + HCO3-
• Protons bind at various places along the protein and carbon dioxide binds at the alpha-amino group forming carbamate.
• This causes a conformational change in the protein and facilitates the release of oxygen.
Bohr Effect
• Blood with high carbon dioxide levels is also lower in pH (more acidic). (recall the equilibrium)
• Conversely, when the carbon dioxide levels in the blood decrease (i.e. around the lungs), carbon dioxide is released, increasing the oxygen affinity of the protein.
Bohr Effect Summary
• High CO2 in tissues
• Higher H+• Lower pH
• Affinity for O2 decreases
• O2 released to tissues
• T state favored
• Low CO2 in lungs
• Lower H+• Higher pH
• Affinity for O2 increases
• O2 binds hemoglobin
• R state favored
Hemoglobin and CO poisoning
• Other ligands can compete with oxygen for binding to hemoglobin
• The binding of oxygen is affected by molecules such as carbon monoxide (CO) (For example from tobacco smoking, cars and furnaces). CO competes with oxygen at the heme binding site.
• Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amount of CO can dramatically reduces hemoglobin’s ability to transport oxygen.
• Hemoglobin also has competitive binding affinity for Nitrogen Dioxide and Hydrogen sulfide .
Oxygen and Carbon Monoxide
• Oxygen and carbon monoxide same size and shape.
• Carbon monoxide, however has formal charge
• Sticks to Fe better• Blocks oxygen
binding
O OFe2+
Fe2+
C O+
Hemoglobin and 2,3 DPG
• In people acclimated to high altitudes, the concentration of 2,3-diphosphoglycerate (2,3-DPG) in the blood is increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower oxygen tension.
• This phenomenon, where molecule Y affects the binding of molecule X to a transport molecule Z, is called a
heterotropic allosteric effect.
O P O
O
O
C
C
CH2OH
H
OOP O
O
O
Sickle Cell Anemia
• Sickle cell disease is caused by an abnormal adult hemoglobin, called hemoglobin S. People with sickle cell disease make hemoglobin S instead of hemoglobin A.
• Single amino acid substitution – glutamate changed to valine
• To show condition, have to have mutation in both genes (Homozygous)
• http://www.scinfo.org/sicklept.htm
Sickle vs normal hemoglobin
• first 9 amino acids of normal hemoglobin beta chain v h l t p e e k s
• first 9 amino acids of sickle hemoglobin beta chain v h l t p v e k s
Notice the single amino acid change?