Lecture 3 Lecture 3: Myoglobin, Hemoglobin, Immunoglobins Protein Structure of Myoglobin John Kendrew and X-ray diffraction of myoglobin: small, oxygen-binding protein of muscle cells Function: store oxygen and to facilitate oxygen diffusion in rapidly contracting muscle tissue Contains a single polypeptide chain of 153 a.a. and single iron protoporphyrin (heme) group is the same one found in hemoglobin Hb is the oxygen binding protein for red blood cells (erthrocytes) and heme is responsible for deep-red color of byoglobin and hemoglobin Abundant in muscles of diving mammals (whale, seal) wich muscles so rich in protein they are brown; storage and distribution of oxygen by muscle myoglobin allow for long dives Backbone is made up of 8 relatively straight segments of ?helices interrupted by some ? turns and bends The longest helix is 23 a.a. long and shortest 7; all are right handed More than 70% of residues in myoglobin are in these ?helices Most stability derived from hydrophobic interactions Hydrophobic R groups in interior of the myoglobin, hidden from water All but 2 of polar R groups are located on the surface, all are hydrated Molecule is so compact that its interior has room for only 4 molecules of water This denseness is typical of globular proteins All the peptide bonds are in the planar trans configuration ¾ Pro residues are found at bends (praline incompatible for ?helix because of fixed bond angle) 4th Pro residue occurs within a ?helix, creating a kink necessary for tight helix packing Other bends include: Ser, Thr, Asn, all whose bulk and shape make them incompatible with ?helix if they are in close proximity in a.a. sequence The flat heme group rests in crevice in molecule The iron atom at the center of the heme group as two coordination positions perpendicular to the plane of the heme One is bound to the R group of His; other is binding site for O2 molecule Accessibility of the heme group to solvent is restricted. This is important because free heme groups in an oxygenated solution are rapidly oxidized from the ferrous form (active in reversible binding of O2) to Ferric form, (which does not find O2) Chapter 5: Protein Function Ligand: a molecule bound reversibly by a protein Binding site: complementary to the ligand in size, shape, charge, hydrophobic or hydrophilic character Interaction is specific; a protein may have separate binding sites for several different ligands Proteins are flexible: changes in conformation are subtle and reflect molecular vibration/small mvmts of residues throughout protein Induced fit: binding of protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand Conformational change in one subunit often affects the conformation of other subunits 5.1 Reversible Oxygen-binding Proteins O2 is poorly soluble in aqueous solutions and cannot be carried to tissues if it is just dissolved in blood Diffusion through tissues is also ineffective over long distances We needed a protein to transport and store oxygen Reversible binding of oxygen molecules not suited for a.a. side chains; accomplished by transition metals, Iron and Copper, easily bind oxygen Free iron promotes forming highly reactive oxygen species that damages DNA; so iron is found in form that makes it less reactive SO?iron is incorporated into a heme Consists of complex organic ring (protoporphyrin) bound to single iron in ferrous state (Fe2+) Iron has 6 coordination bonds, 4 to nitrogen atoms (that are part of the flat porphyrin ring) and 2 perpendicular to the porphyrin [image] The N atoms (electron-donating) help prevent conversion of the heme iron to the ferric state (3+ state does not bind oxygen) Free heme molecules (not bound to protein) have two open bonds Reacting O2 with two free heme groups can result in irreversible conversion of ferrous 2+ into ferric 3+ This is prevented by burying each heme deep within structure where acess to two open coordinationg bonds is restricted One of 2 bonds is occupied by R chain N of His; other binds O2 When oxygen binds, electrical properties of heme change; so color goes from dark purple of venous blood (O2 depleted) ( bright red of arterial blood (O2 rich) Small molecules like carbon monoxide (CO) and nitric oxide ( NO) coordinate to heme iron with greater affinity than O2 (this is very toxic) Carbon Monoxide Kills? CO has an approx. 250 fold greater affinity for hemoglobin than does oxygen As CO binds to one or two subunits of a hemoglobin tetramer, the affinity for O2 increases substantially in the remaining subunits A hemoglobin tetramer with two bound CO molecules can efficiently bind O2 in the lungs but releases very little in the tissues Moving from CO environment to O2 environment: O2 begins to replace the CO in hemoglobin. The COHb levels drop very slowly, though Myoglobin has single binding site for O2 Myo-b facilities oxygen diffusion in muscle 78% of a.a. residues are found in 8 ? helices (named A through H) Equilibrium expression: EMBED Equation.DSMT4 Equilibrium constant: EMBED Equation.DSMT4 EMBED Equation.DSMT4 is an association constant: measures affinity off the ligand L for the protein, units M-1; a higher EMBED Equation.DSMT4 is a higher affinity of the ligand for the protein The ratio of bound protein : free protein is proportional to the concentration of the free ligand: EMBED Equation.DSMT4 When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligang by the protein does not change the concentration of free (unbound) ligand?so [L] stays contant Binding equilibrium of fraction of ligand-binding sites on the protein that are occupied by ligand: EMBED Equation.DSMT4 Sub EMBED Equation.DSMT4 Value of K determined by plotting ( vs. [L] Any x = y / (y + z) describes a hyperbola The fraction of ligand binding sites occupied approaches saturation asymptotically as [L] increases The [L] at which ½ of available ligand binding sites are occupied (( = 0.5) corresponds to 1/Ka The dissociation constant Kd is the reciprocal of Ka and is in molar concentration It is the equilibrium constant for release of ligand: EMBED Equation.DSMT4 When [L] is equal to Kd, half of the ligand-binding sites are occupied As [L] falls below Kd, progressively less of the protein has ligand bound to it In order for 90% of the available ligand-binding site to be occupied, [L] must be 9 times greater than Kd A low Kd means a higher affinity of ligand for the protein (reciprocal of affinity) Kd is equivalent to the molar concentration of a ligand at which half of the available ligand binding sites are occupied Here, the protein reaches half-saturation The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding, the lower the Kd Same application for oxygen in myoglobin: EMBED Equation.DSMT4 Kd is the [O2] at which half of the available ligand-binding sites are occupied: EMBED Equation.DSMT4 We measure oxygen as a partial pressure: EMBED Equation.DSMT4 Protein Structure Affects how Ligands Bind CO binds to free heme molecules 20,000 better than EMBED Equation.DSMT4 (the P50 for CO binding is 20,000 times lower than EMBED Equation.DSMT4 ); but only 200 times better when heme is in myoglobin This may be because of steric hindrance: when EMBED Equation.DSMT4 binds to free heme, the oxygen molecule is positioned at an angle to the Fe ? O bond. But, when CO binds, the Fe, C and O atoms lie in a straight line In myoglobin, His on the EMBED Equation.DSMT4 -binding side of the heme is too far away to coordiate with the heme iron, but it interacts with the ligand bound to the heme Distal His forms an H bond with EMBED Equation.DSMT4 but not with CO ; this could account the diminished binding of CO to heme CO is forced to adopt a slight angle because the perpendicular arrangement is sterically blocked by the distal His. This weakens the binding of CO Rapid molecular flexing of the a.a. side chains produces transient cavities in structure; EMBED Equation.DSMT4 makes its way in and out by moving through e.g. rotation of R group on distal His HEMOGLOBIN (Hb) Erthrocytes (RBC) are incomplete, vestigial cells, unable to reproduce and survive for 120 days RBC carry hemoglobin, which is dissolved in cytosol at a very high concentration In arterial blood, hemoglobin is 96% saturated with oxygen; in venous blood, hemoglobin is 64% saturated Each 100 mL of blood passing through tissue releases about 1/3 EMBED Equation.DSMT4 it carrers Myoglobin: is insensitive to small changes in the concentration of dissolved oxygen and functions well for oxygen storage; Hemoglobin is better suited to transport oxygen Hemoglobin is structurally similar to myoglobin? Hb is spherical, has 4 heme prosthetic groups, one associated with each polypeptide chain 2 types of globin: two ? chains and two ? chains; fewer than half of the a.a. residues of the ? and ? subunits are identical ?1?1 interface involves more than 30 residues and interaction is very strong that reatment of urea causes tetramer to disassemble into ? ? dimers; dimers remain intact The ?1?2 and ?2?1 interface has 19 residues; hydrophobic interactions predominate at the interfaces; also many Hbonds and ion pairs The strongest subunit interactions occur between unlike subunits. When oxygen binds, the ?1?1 contact changes little, but there is large change in the ?1?2 contact Hb undergoes structural change on binding EMBED Equation.DSMT4 R state and T state Oxygen binds to both, but higher affinity for hemoglobin in R state; EMBED Equation.DSMT4 binding stabilizes the R state When EMBED Equation.DSMT4 is absent, the T state is more stable and is the predominant conformation of deoxyhemoglobin T originally for ?tense? and R for ?relaxed? because T is stabilized by more ion pairs (at the ?1?2 and ?2?1 interface The binding of EMBED Equation.DSMT4 to hemoglobin T state triggers change in conformation to the R state. During transition, individual subunits change a little, but the ?? subunit pairs slide past each other and rotate, narrowing the pocket between the ? subunits In the T state: the porphyrin is slightly puckered: causing the heme iron to protrude on the proximal His side The binding of EMBED Equation.DSMT4 cause the heme to become more planar, shifting the position of the proximal His and the helix (R state) Cooperative Binding Myoglobin binds oxygen with hyperbolic binding curve: ill-suited to this function Because it finds with such high affinity at the lungs, it would not release much in the tissues and v.v. Hb undergoes transition from low-affinity state (T) to high affinity state (R) as more EMBED Equation.DSMT4 bind ( Sigmoid binding curve EMBED Equation.DSMT4 binding to individual subunits of Hb can alter affinity for EMBED Equation.DSMT4 in adjacent subunits The first EMBED Equation.DSMT4 that interacts with deoxyhemoglobin binds weakly because it is in the T state. But after conformational changes communicated to adjacent subunits, additional molecules of EMBED Equation.DSMT4 are easier to bind Thus, T(R transition occurs more readily in second subunit once first is bound The last EMBED Equation.DSMT4 molecule binds to a heme in a subunit already in the R state Allosteric Protein: the binding of a ligand to one site affects the binding properties of another site on the same protein Can be either inhibitors or activators; modulators induce conformational changes interconverting more-active and less-active forms of the protein Homotropic: if ligand and modulator are identical Heterotropic: if ligand and modulator are different EMBED Equation.DSMT4 can be considered as both a ligand and activating homotropic modulator There is 1 binding site for EMBED Equation.DSMT4 on each subunit; the allosteric effects giving rise to cooperativity are mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions The binding sites consist of stable segments close to unstable segments, with unstable segments capable of frequent changes in formation When a ligand binds, the moving parts of the protein?s binding site may be stabilized in a particular conformation Cooperative ligand binding described quantitatively For protein with n binding sites, equilibrium equation: EMBED Equation.DSMT4 Association constant: EMBED Equation.DSMT4 Expression for ?: EMBED Equation.DSMT4 Some more work gives Hill equation: EMBED Equation.DSMT4 Plot of EMBED Equation.DSMT4 vs. log [L] gives Hill plot with slope n (does not reflect number of binding sites, instead degree of interaction between them Slope of Hill plot is nH Hill coefficient: measure of degree of cooperativity If nH = 1, ligand binding is not cooperative, >1 indicates positive cooperativity (hemoglobin?the theoretical upper limit is n?where all the binding sites would bind ligand simultaneously?measured nH is always less than actual # of binding sites); <1 indicates negative cooperativity Models for Cooperative Binding Concerted model: assumes that the subunits of a cooperatively binding protein are functionally identical each subunit can exist in 2 conformations, and that all subunits undergo transition from one conformation to the other simultaneously Sequential model: ligand binding can induce a change of conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacent subunit. There are more potential intermediates states in this model Hemoglobin transports H+ and CO2 CO2 produced by oxidation of organic fuels is hydrated to form bicarbonate: EMBED Equation.DSMT4 Hydration of EMBED Equation.DSMT4 results in an increase in the H+ concentration The binding of oxygen by hemoglobin is influenced by pH and EMBED Equation.DSMT4 concentration Hemoglobin transports about 40% off the total H+ and 15% to 20% of the EMBED Equation.DSMT4 formed in the tissues to the lungs/kidneys The binding of H+ and EMBED Equation.DSMT4 is inversely related to the binding of oxygen At low pH and high EMBED Equation.DSMT4 concentration at tissues, the affinity of hemoglobin for oxygen decreases as H+ and EMBED Equation.DSMT4 are bound, and oxygen is release to tissues At the lungs, as EMBED Equation.DSMT4 is excreted, the pH rises and the affinity hemoglobin to oxygen increases and protein binds more oxygen fofr transport This is Bohr?s Effect; reaction must be rewritten: EMBED Equation.DSMT4 Oxygen-saturation curve of hemoglobin is influenced by the H+ concentration Both oxygen and H+ are bound by hemoglobin but with inverse affinity When [oxygen] is high in the lungs, Hb binds oxygen and releases protons When oxygen is low in the tissues, H+ is bound and oxygen released oxygen and H+ are not bound at same sites oxygen binds to the iron atoms, H+ binds to any a.a. residue Major contributor is His of ? subunits; when protonated, it forms one of the ion pairs to Asp. This helps stabilize deoxyhemoglobin in the T state As [H+] rises, protonation of His promotes release of oxygen by favoring R state ( T state Hb also binds EMBED Equation.DSMT4 inversely related to binding oxygen; it binds to the ?-amino group at the amino-terminal end This reaction produces H+, contributing to Bohr effect; also forms additional salt bridges that help stabilize the T state and promote release of oxygen When [ EMBED Equation.DSMT4 ] is high (in tissues), some EMBED Equation.DSMT4 binds to Hb and the affinity for oxygen decreases, causing it to release Conversely, when Hb reaches lungs, the high [oxygen] promotes binding of oxygen and release of EMBED Equation.DSMT4 Oxygen binding regulated by BPG BPG: 2,3-biphosphoglycerate is present in high conc. in erythrocytes Great reduces affinity of hemoglobin for oxygen (inverse relationship between binding oxygen and BPG Binding process equation: EMBED Equation.DSMT4 BPG binds at site distant from oxygen-binding xite BPG plays big role in adaptation to lower p EMBED Equation.DSMT4 at high altitudes; at high alts, the delivery of EMBED Equation.DSMT4 to tissue is reduced (in normal is 40% off max carried by blood) After a few hours, the BPG concentration rises, decreasing the affinity of Hb for oxygen This has small effect on binding of EMBED Equation.DSMT4 but big effect on release of EMBED Equation.DSMT4 in tissues When person returns to sea level, BPG decreases, effect on releasing decreases Hypoxia: BPG concentrations cause the lowered oxygenation of tissues The binding site of BPG is the cavity between the ? subunits in the T state Cavity is lined with positively charged a.a. that interact with negatively charged BPG Only 1 BPG is found to each hemoglobin (4 for EMBED Equation.DSMT4 ) BPG lowers the affinity for EMBED Equation.DSMT4 by stabilizing the T state The transition to the R state narrows the binding pocket for BPG, minimizing affinity Without BPG, hemoglobin is converted to the R state more easily Sickle Cell Anemia allele is inherited from both parents fewer and abnormal erythrocytes: long, thin, crescent shaped RBC that looks like blade of sickle when hemoglobin is deoxygenated, it becomes insoluble and forms polymers that aggregate into tubular fibers normal hemoglobin (A) remains soluble on deoxygenation the insoluble fibers of deoxygenated hemoglobin (S) are responsible for the deformed sickle shape Results from single a.a. substitution Val instead of Glu at position 6 in two ? chains The R group of Val has no charge where Gly has negative charge Hb S therefore has two less negative charges than A This creases a ?sticky? hydrophobic contact point which causes long, fibrous aggregates Sickled cells are fragile and rupture easily ( anemia (lack of blood) Capillaries become blocked by the long, abnormally shaped cells Heterozygous individuals have small but significant resistance to malaria Antibodies have 2 identical antigen-binding sites Immunoglobulin G (IgG) is major class of anti-body molecule has 4 polypeptide chains: 2 large heavy chain, 2 light chains, linked by noncovalent and disulfide bonds Y-shaped molecule: at ?hinges? can be cleaved with proteases Cleavage with papain frees the basal fragment (Fc) and two branches (Fab- antigen binding fragments) Each branch has single antigen-binding site Immunoglobulin fold: motif in all ? class of proteins. There are 3 domains in each heavy chain and 1 in each light Variable domains create the antigen-binding site (heavy and light chains have one variable domain each) IgG is the major antibody in secondary immune responses, initiated by memory B cells; the most abundant immunoglobulin in the blood When IgG binds to an invading bacterium/virus, it activates macrophages to engulf the invader A class for receptors on macrophages recognizes and binds to the Fc region if IgG residues lining the antigen-binding site are hypervariable: very likely to differ Specificity depends on complement between antigen and its binding site (shape, location or charged/nonpolar, Hbonding groups) Conformational changes in the antibody/antigen occur that allow the compleentary groups to interact fully Typical antigen-antibody interaction is strong (low Kd values means high affinity) Selected antibody can be covalently attached to a resin and used in chromatography column When a mixture of proteins is added to the column, the antibody specifically binds its target protein and retains it on the column while other proteins are washed through (protein purification) Another technique: an antibody is attached to a radioactive label/detectable reagent When antibody binds target protein, label reveals presence of protein in solution or location in gel ELISA: proteins are adsorbed into an inert surface surface is washed with solution of nonspecific protein to block proteins introduced in subsequent steps from adsorbing Surface treated with solution containing primary antibody: antibody is against the protein off interest Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These are linked to an enzyme that catalyzes a reaction that forms colored product Immunoblot assay: proteins are separated by gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane Membrane is treated successively with primary antibody, secondary antibody linked to enzyme and substrate Colored precipitate forms
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