BIOC 460, spring 2008LEC 8 and 9, Protein Function: ligandbinding and allosteric regulation ofhemoglobin 1Lectures 8 and 9Protein Function: Ligand Binding andAllosteric RegulationReading: 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 = myoglobinJmol structure of myoglobin:http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.htmlJmol 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.html• Oxygen Binding to Myoglobin and Hemoglobin• Allosteric Regulation of Hemoglobin FunctionKey Concepts• Ligand binding fundamentally important in biochemicalphenomena.• Heme (Fe protoporphyrin IX) in myoglobin and hemoglobin binds O2reversibly, without oxidation of the heme Fe+2 which is required for O2binding.• Myoglobin and hemoglobin's structures and ligand binding propertieshave evolved differently for the different functions of the two proteins,and the structure-function relationships are very well understood.– Mb is monomeric, 1 O2 binding site per molecule, hyperbolicbinding curve (no cooperativity).– Hb is tetrameric, 4 O2 binding sites per molecule, sigmoid bindingcurve indicative of cooperative ligand binding (structuralcommunication between different binding sites by conformationalchanges).– Hb is thus an allosteric protein.BIOC 460, spring 2008LEC 8 and 9, Protein Function: ligandbinding and allosteric regulation ofhemoglobin 2Key Concepts, continued• Hb is an allosteric protein.– R state ("oxy" conformation, high O2 binding affinity) stabilized byO2 binding (O2 is a homotropic effector)– T state ("deoxy" conformation, low O2 binding affinity) stabilized bybinding of protons (H+), CO2, and/or 2,3-bisphosphoglycerate (2,3-BPG) (all heterotropic effectors, allosteric inhibitors)– Allosteric regulation of O2 binding to Hb is important to enhance theability of Hb to RELEASE O2 in the tissues.• 2,3-BPG is needed in human erythrocytes (red blood cells) to reduceO2 binding affinity enough to get effective release of O2 in tissues.– 2,3-BPG binds in central cavity of Hb (stoichiometry1 BPG/Hb tetramer).• Fetal Hb (HbF) has different quaternary structure from adult HbA (α2γ2vs. (α2β2)– Sequence difference between γ and β reduces HbF's affinity for2,3-BPG, thus increasing its affinity for O2 under physiologicalconditions.Learning Objectives• Terminology: ligand, fractional saturation, prosthetic group, cooperativity,protomer, binding site, allosteric (allosteric site, allosteric effector, allostericregulation)• Briefly describe the tertiary structure of myoglobin and the hemoglobinsubunits (the "globin fold"), explain how the helices are designated, and theroles of the proximal and distal His residues in heme and oxygen binding.• Write a general protein-ligand binding/dissociation reaction in both theassociation and dissociation directions. What is the mathematicalrelationship between the association and dissociation equilibriumconstants?• Describe how and where in the structure of myoglobin and hemoglobin O2binds, including roles of protein functional groups and heme, and theoxidation state of the heme Fe required for O2 binding.• Sketch the O2 binding curve [Y (fractional saturation) vs. pO2] forNONcooperative ligand binding to a protein, such as that for O2 binding tomyoglobin. On same plot, sketch a binding curve that shows cooperativity(cooperative ligand binding), such as that for O2 binding to hemoglobin,and explain (again) what is meant by cooperativity. On both curves,indicate the value of P50, the pO2 at which fractional saturation of proteinwith O2 is 0.5. In what part of the cooperative binding curve (what part ofthe [ligand] concentration range) is protein predominantly in conformationwith low ligand binding affinity, and in what part of the ligand concentrationrange is the predominant form the high binding affinity conformation?BIOC 460, spring 2008LEC 8 and 9, Protein Function: ligandbinding and allosteric regulation ofhemoglobin 3Learning Objectives, continued• Explain how hemoglobin works physiologically (in vivo), i.e., howcooperativity in O2 binding to hemoglobin facilitates “loading” of O2 in thelungs and “unloading” of O2 in the tissues. Include the role of the R state(oxy conformation) and the T state (deoxy conformation) of hemoglobin.• Briefly describe the structural change that occurs when O2 binds to theheme of a subunit of hemoglobin, includinga) what in the heme structure triggers the protein structural change whenO2 binds,b) how that first protein structural change is communicated to othersubunits to change the quaternary structure and the O2 binding affinityof the other subunits, andc) effect of the quaternary structural change on size of the central cavity.• Explain the effect of 2,3-bisphosphoglycerate on the affinity ofmammalian hemoglobin for oxygen, and describe where on thehemoglobin molecule 2,3-BPG binds, how many molecules of 2,3-BPGbind to one hemoglobin tetramer, and predominantly by what type ofnoncovalent interactions the 2,3-BPG is bound. Does 2,3-BPG bind tothe R state or the T state of hemoglobin?Learning Objectives, continued• Explain why maternal red blood cells release O2 and fetal red blood cellsbind O2 in the placenta in terms ofa) the difference in protein primary structure and quaternary structure(subunit composition) between HbA (adult, maternal) and HbF (fetalhemoglobin),b) the effect of the structure of HbF on its 2,3-BPG binding affinitycompared to HbA,andc) the resultant difference in O2 binding properties of the 2 hemoglobinsand its physiological significance.• Discuss the Bohr effect (H+ binding and CO2 binding), in terms ofa) the effect of increasing concentrations of either of these ligands on theO2 binding curve for hemoglobin,b) the physiological significance of this phenomenon.• For a mutant in which the T-R equilibrium is shifted toward the R state,what type of change would you expect in P50? Does that mean O2 affinityof mutant is higher or lower than normal?• For a mutant in which the T-R equilibrium is shifted toward the T state,what type of change would you expect in P50? Does that mean O2 affinityof mutant is
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