BIOC 460 Summer 2010Ligand binding and allosteric regulation of hemoglobin 1*Ligand Binding and Allosteric Regulation*R di B T k&St 6thdCh t 7 183199• Oxygen Binding to Myoglobin and Hemoglobin• Allosteric Regulation of Hemoglobin FunctionReading: Berg, Tymoczko & Stryer, 6th ed., Chapter 7, pp. 183-199problems in textbook: chapter 7, pp. 203-204, #3,4,5,6,8abbreviations used in this set of notes: Hb = hemoglobin, Mb = myoglobinThis material delves deeply into the structure function relationship of the two proteins. In order to understand the nature of this relationship it is highly advised that you look at the molecular graphics routines listed below and throughout the lecture notes! Jmol structure of myoglobin:http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.htmlJmol structure ofhemoglobin1Jmol structure of hemoglobinhttp://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hemoglobin/newhb.htmlJmol structure of hemoglobin with 2,3-bisphosphoglycerate (2,3-BPG) boundhttp://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hbbpg/newbpg.htmlCool animation website: http://www.moleculesinmotion.com/#animations then click on the “animations” link. Another site for RÙT transition:http://www.molecularstation.com/molecular-biology-images/505-protein-pictures/49-hemoglobin-deoxy-oxygen-transition-states.html Key Concepts• Ligand binding fundamentally important in biochemical phenomena.• Heme (Fe protoporphyrin IX) in myoglobin and hemoglobin binds O2reversibly, without oxidation of the heme Fe+2which is required for O2bindingbinding.• Myoglobin and hemoglobin's structures and ligand binding properties have evolved differently for the different functions of the two proteins, and the structure-function relationships are very well understood.–Mb: monomeric, 1 O2binding site per molecule, hyperbolic binding curve (no cooperativity). Used for O2storage in muscle.–Hb: tetrameric, 4 O2binding sites per molecule,sigmoidbinding2Hb: tetrameric, 4 O2binding sites per molecule, sigmoid binding curve indicative of cooperative ligand binding (structural communication between different binding sites by conformational changes). Used for O2transport in red blood cell.– Hb is thus an allosteric protein.BIOC 460 Summer 2010Ligand binding and allosteric regulation of hemoglobin 2Key Concepts, continued• Hb as an allosteric protein.– R state ("oxy" conformation, high O2binding affinity) stabilized by O2binding (O2is a homotropic effector)– T state ("deoxy" conformation, low O2binding affinity) stabilized by binding ofprotons (H+)CO2and/or23-bisphosphoglycerate(2 3-binding of protons (H), CO2, and/or 2,3bisphosphoglycerate (2,3BPG) (all heterotropic effectors, allosteric inhibitors of O2binding)– Allosteric regulation of O2binding to Hb is important to enhance the ability of Hb to RELEASE O2in the tissues.• 2,3-BPG is needed in human erythrocytes (red blood cells) to reduce O2binding affinity enough to get effective release of O2in tissues.– 2,3-BPG binds in central cavity of Hb in the T state (stoichiometry 1 BPG/Hb tetramer)31 BPG/Hb tetramer).• Fetal Hb (HbF) has different polypeptide and quaternary structure from adult HbA (Hb F is 〈2©2; HbA is 〈2®2)– Amino acid differences between © and ® reduces HbF's affinity for 2,3-BPG, thus increasing its affinity for O2under physiological conditions.Ligand Binding• Terminology:– LIGAND: a molecule or ion (usually small) that's bound by another molecule (usually large, e.g., a protein)– COOPERATIVE ligand binding ("cooperativity"): only observed in proteins containing > 1 binding site AND binding of a ligand (O2) t 1 bi di it ff t bi di ffi iti f th bi di it fto 1 binding site affects binding affinities of other binding sites of the same protein molecule.Example: O2binding to Hb• The essence of protein function/action is BINDING (recognition of andinteraction with other molecules).– BINDING: result of specific, usually NONCOVALENT interactions between molecular surfaces4• SHAPE complementarity (lots of van der Waals interactions)• CHEMICAL complementarity (hydrogen bonds, salt linkages)• HYDROPHOBIC EFFECT (hydrophobic ligand minimizes exposure to water by binding in hydrophobic site in protein)BIOC 460 Summer 2010Ligand binding and allosteric regulation of hemoglobin 3General case: ligand binding to a protein• Ironically, in order to understand ligand BINDING, you usually discuss ligand DISSOCIATION!PL P + L• equilibrium dissociation constant Kdfor reaction:5• Concentrations of free protein (empty binding sites) = [P], free ligand [L], and PL complex [P•L] in this expression are the equilibrium concentrations.• Note: Kdissociation= 1/ Kassociation; Units of Kd?*FRACTIONAL SATURATION*• Fractional saturation = Y = fraction of total binding sites on protein ([P]total) occupied by ligand • Y = [occupied binding sites] / [total binding sites] = For:Now:ANDIt can be shown:6• Plot of fractional saturation (Y) vs. [L] is hyperbolic, with Y approaching a limiting value.Note: Kdis a concentration!!!!!!!!!!!!!!!BIOC 460 Summer 2010Ligand binding and allosteric regulation of hemoglobin 4The SHAPE of this curve suggests you are saturating P with L!Units of Y? Minimum and maximum values of Y?Fractional Saturation:•When Y = 0.5, [L] = Kd•Kd= concentration offree ligand needed to HALF-SATURATEthe binding sites7Suppose you have 2 proteins that both bind the same ligand, one with Kd= 10–5M and the other with Kd= 10–7M. Which one has the higher binding affinity for the ligand? •Kdis a dissociation constant•Kdis the free ligand concentration needed to HALF saturate the protein binding site.binding site.• The smaller the value of Kdthe tighter the binding (or smaller the amount of dissociation)8amount of dissociation)BIOC 460 Summer 2010Ligand binding and allosteric regulation of hemoglobin 5We are now going to study how two proteins with almost exactly the same tertiary structureand same ligand binding site (prosthetic group) behave so fundamentally different that one protein is ideally suited for O2storage in muscle tissue and the other 2gprotein is evolutionarily optimized to bind and transport O2in a cooperative manner from the lungs to muscle.St t F ti9Structure FunctionMyoglobin (Mb)http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html• first high-resolution protein X-Ray crystal structure ever
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