BCHM 3010 Ligand Binding
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This 4 page Class Notes was uploaded by Morgan Dimery on Saturday January 30, 2016. The Class Notes belongs to 3010 at a university taught by Dr. Cheryl Ingram-Smith in Spring 2016. Since its upload, it has received 27 views.
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Date Created: 01/30/16
Ligand Binding: Oxygen Binding to Hemoglobin & Myoglobin • Hemoglobin and myoglobin are part of the same superfamily-‐ they have the same 3D structure but they do not have the same amino acid sequence (primary structure) • Hemoglobin has four subunits and myoglobin only have one-‐ this gives them different properties Ligand binding is extremely specific • A ligand is a molecule (smaller than a protein) that binds to a protein o It can go on the protein and then come back off o Only specific ligands can bind to a certain protein-‐ they have to be able to bind it and then hold it in the correct orientation • Ligand binding is dependent on size, shape, charge, & hydrophobic/hydrophilic character o Hydrophobic regions nicely shield each other • Ligand binding can cause conformational changes-‐ this is known as the induced fit and it causes tighter binding Quantitative Description • The process of ligand binding is described by k a and kd o k = a association rate constant-‐ 2 order (mol/s) st o k = d dissociation rate constant-‐ 1 order (molarity) o Eventually the process will get to equilibrium and they will be equal K =a k a d k • K a is the association constant-‐ describes how quickly proteins and ligands come together and then go apart • We are able to figure out the fraction of sites that are occupied! The stronger the binding between protein and ligand the lower the K dvalue You want there to be a high affinity!! • It is possible to show the binding of proteins to ligands using a graph-‐ it is a good way to compare different proteins • K d gives you an idea of how well a protein binds to its ligand • When [L]=K half of the ligand binding sites are occupied-‐ the protein has d reached half saturation for ligand binding θ= [L] d [L]+K The Globin Superfamily • Oxygen-‐binding proteins • Protein side chains do not have an affinity for oxygen • Transition metals can bind to oxygen, but when they are by themselves they generate free radicals which are not good o Heme: a big ring with an Fe in the middle • The solution to the free radical problem was to put heme into a protein-‐ this prevents free radicals, makes it more stable, and allows for the oxygen molecules to be captured The way that hemoglobin and myoglobin are with oxygen is very different because of their different numbers of subunits!! • Myoglobin-‐ 1 subunit, function is oxygen storage in muscles • Hemoglobin-‐ 4 subunits, function is oxygen transport to tissues • The Fe atom in heme has four nitrogens as well as two additional bonds perpendicular to the plane of the ring o One of these bonds is for His, the other is for an oxygen-‐ myoglobin has one heme molecule, so it has one bond available for oxygen Side note-‐ a graph showing ligand sites being bound will at first increase dramatically but then level off because all of the sites have become filled-‐ it doesn’t matter how much you increase the concentration anymore • When a ligand is a gas, the binding is expressed in partial pressure o P : 50partial pressure of oxygen that is needed for half of the binding sites to be filled-‐ this is different for myoglobin and hemoglobin Carbon Monoxide • CO can actually bind to heme better than oxygen can-‐ this is what makes it so toxic, it displaces the oxygen • Usually it can bind 20,000 times better than oxygen, but having it in a protein makes it only 250 times better • Oxygen binds to heme at a tilt, and CO binds to it straight up-‐ this is why CO binds to heme better/easier • Luckily, the shape that is formed by all the His/myoglobin bonds causes steric hindrance for the CO, but it creates a perfect fit for the oxygen to bind to the heme Myoglobin is only used for oxygen storage, not oxygen transport!! • Myoglobin does not release oxygen, it keeps its site bound o This is because of the partial pressures in the lungs and tissues o It will only give up oxygen if the muscles really need it • Myoglobin only has only subunit, therefore, it does not have any other subunits to be influenced by-‐ no cooperative binding • Hemoglobin has four different subunits that all fold in very similar ways with alpha helices o They interact and change each other’s affinity for oxygen-‐ this is known as cooperative binding • Hemoglobin has a T (tense) state and an R (relaxed) state • The R state binds oxygen very well, but the T state is more stable when there is low oxygen binding (it has a lower affinity for oxygen) • During the R state the heme becomes planar and it can bind to the oxygen much better • Oxygen binding is what triggers the T and R state to go back and forth between one another! • Hemoglobin binds oxygen well in the lungs, and then releases it to the tissues o As more oxygen is bound, hemoglobin goes from a T state to an R state o These different transitions create a sigmoidal curve in a graph for hemoglobin, hemoglobin is never fully in one state-‐ represents cooperativity between subunits • In summary of hemoglobin: it has a low affinity state that doesn’t bind oxygen well. When there is a lot of oxygen present the hemoglobin changes it state from T to R-‐ the R state has a high affinity for oxygen, so the oxygen will bind well • There is both positive cooperativity and negative cooperativity o Positive-‐ the first binding event causes more binding (higher affinity) at other sites (hemoglobin) o Negative-‐ the first binding event causes less binding (lower affinity) at other sites • Cooperativity can be explained with math-‐ the hill coefficient (n) measures the degree of cooperativity o n=1; no cooperativity o n<1; negative cooperativity o n>1; positive cooperativity • When you plot cooperativity on a graph the slope of the line is the hill coefficient o For hemoglobin the line begins with a slope of 1, changes to a slope of 3, and then goes back to having a slope of 1. o This is because at the beginning all of the subunits are the same at low affinity, then they become different from one another, and then they go back to the same thing again at a high affinity • There are also models that represent cooperativity by showing all subunits in the same conformation or the subunits in either conformation (R or T) Cooperativity is essentially the same thing as allosteric • For allosteric proteins binding of the ligand at one site effects the binding at another site-‐ positive or negative • Homotropic is when the normal ligand is what effects the protein (like in hemoglobin) • Heterotropic is when a different ligand effects the protein Hemoglobin is also used for 2O export!! • CO 2 and H ions are produced by metabolism and are in tissues as waste products + • After the hemoglobin drops off the oxygen to the tissues it bring2 CO and H back to the lungs (mostly H ions) • The binding of CO2 and 2 O do not happen at the same time or at the same site-‐ there is no competition for 2he O • Oxygen does not bind well in low pHs-‐ tissues have a lower pH than the lungs do o Oxygen does not bind well in the tissues (it is released), an2 CO /H can bind o The effect that pH has is known as the Bohr Effect 2-‐3-‐bisphosphoglycerate (BPG) regulates oxygen binding to hemoglobin • This is considered a negative heterotopic regulator • It comes from glycolysis as a small negatively changed molecule o It binds to the positively charged hemoglobin and stabilizes the T state, which is the state that doesn’t bind oxygen well o This is very useful in the adaptation to lower oxygen levels at high altitudes • BPG reduces affinity of oxygen so that even when oxygen levels are lower the same amount of oxygen is released to the tissues • There is more BPG at higher altitudes, which helps to deliver more oxygen even though you are actually binding less Sickle Cell Anemia • Hereditary disease • Fewer erythrocytes, abnormal hemoglobin • Glu à Val; negatively charged amino acid to a nonpolar amino acid (uncharged)-‐ now there are more hydrophobic regions that want to clump together o This causes red blood cells to be misshapen and then oxygen can’t bind as well • In Africa, carriers of sickle cell anemia are more resistant to malaria
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