Globular Proteins

Globular Proteins UNIT I: Protein Structure and Function

Overview • This chapter examines relationship b/w structure and function for the clinically important globular hemeproteins, and mainly Hb.

II. Globular hemeproteins • Hemeproteins: group of specialized proteins, contain heme as a tightly bound prosthetic group • Role of heme group is dictated by environ. created by 3D structure of protein e.g., • Heme of a cytochrome functions as an electron carrier • Heme of catalase is part of active site of the enzyme  catalyzes breakdown of H2O2 • In Hb and myoglobin, the 2 most abundant heme proteins, heme serves to reversibly bind oxygen

A. Structure of heme • A complex of protoporphyrin IX and ferrous iron (Fe2+) • Iron is held in center of heme molecule by bonds to 4 nitrogens of porphyrin ring • Heme Fe2+ can form 2 additional bonds, one on each side of the planar porphyrin ring e.g., in myoglobin and Hb, one of these positions is coordinated to side chain of His residue of globin molecule, the other is available to bind oxygen

Figure 3.1. • Hemeprotein (cytochrome c). • Structure of heme.

B. Structure and function of Myoglobin • Myoglobin, a hemeprotein present in heart & skeletal muscle • Functions as a reservoir for oxygen & as oxygen carrier that increases rate of transport of oxygen within muscle cell • Consists of a single polyp structurally similar to individual subunit polyp of Hb molecule, making myoglobin useful model for interpreting some complex properties of Hb

α-helical content: • Myoglobin a compact molecule, ~ 80% of its polyp folded into 8 stretches of α-helix • These α-helical regions are terminated either by Pro (its 5-membered ring cannot be accommodated in α–helix), or by β-bends and loops stabilized by H-bonds and ionic bonds • Location of polar and non-polar aa residues: • Interior of myoglobin molecule is composed of almost entirely non-polar aa’s. Packed together forming a structure stabilized by hydrophobic interactions • Charged aa’s located almost exclusively on surface, forming H-bonds with each other and with water

Figure 3.2. • Model of myoglobin showing helices A to H.

Binding of heme group: • Heme group sits in a crevice lined with non-polar aa’s. Notable exceptions are 2 His residues. • One, proximal His, binds directly to iron of heme, 2nddistal His, does not directly interact with heme, but helps stabilize binding of oxygen to ferrous iron • The protein, or globin, portion of myoglobin creates a microenviron. for heme that permits reversible binding of one oxygen molecule (oxygenation). • Simultaneous loss of electrons by ferrous iron (oxidation) occurs only rarely.

Schematic diagram of the oxygen-binding site of myoglobin.

C. Structure and function of hemoglobin • Hb is found exclusively in RBC’s, its main function is transport of oxygen from lungs to capillaries of tissues. • HbA, major Hb in adults, is composed of 4 polyps. 2 α- and 2 β-chains –held together by non-covalent interactions. • Each subunit has stretches of α-helical structure, and a heme binding pocket. • Tetrameric Hb is more complex structurally and functionally than myoglobin e.g., • Hb can transport CO2 from tissues to lungs, and • carry 4 O2 from lungs to cells of the body, further • Oxygen-binding properties of Hb are regulated by interaction with allosteric effectors

Figure 3.3 A. Structure of hemoglobin showing the polypeptide backbone. B. Simplified drawing showing the helices.

1. Quaternary structure of Hb • Hb tetramer can be envisioned as composed of 2 identical dimers (αβ)1 and (αβ)2. • The 2 polyp chains in each dimer held tightly together primarily by hydrophobic interactions (in this case hydrophobic aa residues are localized not only in interior of molecule but also in a region on surface of each subunit. Interchain hydrophobic interactions form strong associations b/w α- and β-subunits in dimers) • Ionic and H-bonds also occur b/w members of the dimer • The two dimers, in contrast, are able to move wrt each other, being held primarily by polar bonds. The weaker interactions b/w these mobile dimers result in the 2 dimers occupying different relative positions in deoxy-Hb as compared with oxy-Hb

Figure 3.4. Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin.

T-form: • deoxy form of Hb “T”, or taut (tense) form. • The 2 αβ dimers interact through a network of ionic and H-bonds  constrain movement of polyp chains • Low oxygen-affinity form of Hb • R-form: • Binding of oxygen to Hb causes rupture of some of the ionic and H-bonds b/w αβ dimers • This leads to a structure called “R” or relaxed form, here polyp chains have more freedom of movement • R-form is high oxygen-affinity form of Hb

D. Binding of oxygen to myoglobin and Hb • Myoglobin can bind 1 O2 molecule, it contains only 1 heme group • Hb can bind 4 O2 molecules, one at each of its 4 heme groups • Degree of saturation (Y) of these oxygen-binding sites on all myoglobin or Hb molecules can vary b/w zero (all sites are empty) and 100% (all sites are full)

1. Oxygen dissociation curve • A plot of Y measured at different pO2 • Curves for myoglobin & Hb show important differences. Myoglobin has a higher oxygen affinity than Hb. Partial pressure of oxygen needed to achieve half-saturation of binding sites (P50) is ~ 1mm Hg for myoglobin & 26 mm Hg for Hb • Note: the higher the oxygen affinity (i.e., the more tightly oxygen binds), the lower P50

Figure 3.5 Oxygen dissociation curves for myoglobin and hemoglobin.

a. Myoglobin • The oxygen dissociation curve for myoglobin has a hyperbolic shape. This reflects myoglobin reversibly binds a single molecule of oxygen • Thus, oxygenated (MbO2) and deoxygenated (Mb) exist in a simple equilibrium: Mb + O2 ↔ MbO2 • Mb is designed to bind oxygen released by Hb at the low pO2 found in muscles. Mb releases oxygen within muscle cell in response to oxygen demand

b. Hb • The oxygen dissociation curve for Hb is sigmoidal in shape. This reflects that subunits cooperate in binding oxygen. • Cooperative binding of O2 by the 4 subunits of Hb means binding of O2 to one heme group increases the oxygen affinity of remaining heme groups in the same Hb molecule, this effect is heme-heme interaction • Although binding of 1st O2 is difficult, subsequent binding of O2 occurs with high affinity, shown by steep upward curve in the region 20-30 mm Hg

Figure 3.6 Hemoglobin binds oxygen with increasing affinity.

E. Allosteric effects • Ability of Hb to reversibly bind oxygen is affected by pO2(through heme-heme interaction), pH of environ. pCO2and availability of 2,3-bisphosphoglycerate. • Collectively called allosteric (“other site”) effectors, as their interaction at one site on Hb molecule affects binding of oxygen to heme groups at other locations on the molecule • Binding of oxygen to myoglobin is not influenced by allosteric effectors of Hb 1. Heme-heme interactions: sigmoidal oxygen-binding curve reflects specific structural changes that are initiated at one heme and transmitted to other heme groups in Hb tetramer. Net effect  affinity of Hb for last oxygen ~ 300x greater than affinity for 1st oxygen

Loading and unloading oxygen: cooperative binding of oxygen allows Hb deliver more oxygen to tissues in response to relatively small changes in pO2. Figure 3.5 indicates pO2 in alveoli of lung and capillaries of tissues - e.g., in lung conc. oxygen is high and Hb becomes saturated (loaded) with oxygen. - in peripheral tissues, oxy-Hb releases (unloads) much of its oxygen for use in oxidative metabolism b. Significance of sigmoidal O2-dissociation curve: Steep slope of O2-dissociation curve over the range of oxygen conc. b/w lungs and tissues permits Hb to carry and deliver oxygen efficiently from sites of high to sites of low pO2 A molecule with hyperbolic O2-dissociation curve, e.g. myoglobin could not achieve the same thing. Instead, it would have max affinity for oxygen throughout this oxygen pressure  would deliver no oxygen to tissues

Figure 3.7. Transport of oxygen and CO2 by hemoglobin.

2. Bohr effect: • Release of oxygen from Hb is enhanced when pH is lowered or when Hb is in pressure of an increased pCO2. Both result in decreased oxygen affinity  shift to the right in O2-dissociation curve. • This change in oxygen binding is called Bohr effect. • Conversely, raising pH or lowering conc. of CO2  greater affinity for oxygen, and a shift to the left in O2-dissociation curve.

Figure 3.8. Effect of pH on the oxygen affinity of hemoglobin.

a. Source of the protons that lower the pH: • Conc. of both CO2 and H+ in capillaries of metabolically active tissues is higher than that observed in capillaries of lung, where CO2 is released into expired air • Note: organic acids e.g., lactic acid, are produced during anaerobic metabolism in rapidly contracting muscle • In tissues, CO2 is converted by carbonic anhydrase to carbonic acid, CO2 + H2O ↔ H2CO3 which spontaneously loses a proton becoming bicarbonate, the major blood buffer H2CO3 ↔ H+ + HCO3- • The proton produced contributes to lowering pH. This differential pH gradient (lungs having higher, tissues lower pH) favors unloading oxygen in peripheral tissues, and loading of oxygen in lung. • Thus, oxygen affinity of Hb responds to small shifts in pH b/w lungs and oxygen-consuming tissues, making Hb a more efficient transporter of oxygen.

b. Mechanism of the Bohr effect: • Deoxy form of Hb has a greater affinity for protons than does oxy-Hb. This fact is caused by ionizable groups, e.g., N-terminal α-amino groups, & specific His side chains that have higher pKa’s in deoxy-Hb than in oxy-Hb. • An increase in conc. of protons causes these groups to become protonated (charged) and able to form ionic bonds (a.k.a salt bridges), which stabilize deoxy form of Hb, producing a decrease in oxygen affinity • Bohr effect schematically: HbO2 (oxy-Hb) + H+ ↔ HbH (deoxy-Hb) + O2 where an increase in protons (or a lower pO2) shifts equilibrium to right, whereas an increase in pO2 (or decrease in protons) shifts equilibrium to left

Figure 3.9. Synthesis of 2,3-BPG. [Note: is a phosphoryl group.] 3. Effect of 2,3 bisphosphoglycerate on oxygen affinity • 2,3 BPG an important regulator of binding of oxygen to Hb • It is the most abundant organic phosphate in RBC, where its conc. ~ that of Hb. • 2,3 BPG is synthesized from an intermediate of glycolytic pathway

Binding of 2,3 BPG to deoxy-Hb 2,3 BPG decreases oxygen affinity of Hb by binding to deoxy-Hb but not to oxy-Hb. This preferential binding stabilizes the “taut” conformation of deoxy-Hb. HbO2 + 2,3-BPG ↔ Hb-2,3-BPG + O2 • Binding site of 2,3 BPG One molecule of 2,3 BPG binds to a pocket, formed by the two β-globin chains, in center of deoxy-Hb tetramer. This pocket contains several positively charged aa’s that form ionic bonds with the negatively charged phosphate groups of 2,3 BPG A mutation of one of these residues can result in Hb variants with abnormally high oxygen affinity 2,3 BPG is expelled on oxygenation of Hb. Figure 3.10. Binding of 2,3-BPG by deoxyhemoglobin.

c. Shift of oxygen dissociation curve • Hb from which 2,3 BPG removed, has a high affinity for oxygen. In RBC, presence of 2,3 BPG significantly reduces affinity of Hb for oxygen, shifting oxygen-dissociation curve to the right. This reduced affinity enables Hb to release oxygen efficiently at partial pressures found in tissues Figure 3.11. Effect of 2,3-BPG on the oxygen affinity of hemoglobin.

d. Response of 2,3-BPG levels to chronic hypoxia or anemia • Conc. of 2,3 BPG in RBC increases in response to chronic hypoxia, e.g., that observed in obstructive pulmonary emphysema, or at high altitudes, where Hb may have difficulty receiving sufficient oxygen. Intracellular 2,3 BPG also elevated in chronic anemia in which fewer than normal RBCs are available to supply body’s oxygen needs. Elevated 2,3 BPG levels lower oxygen affinity of HB, permitting greater unloading of oxygen in capillaries of tissues

e. Role of 2,3 BPG in transfused blood: • 2,3 BPG is essential for normal oxygen transport function of Hb. • e.g., storing blood in acid-citrate-dextrose  decrease of 2,3 BPG in RBCs. Such blood displays an abnormally high oxygen affinity, and fails to unload its bound oxygen properly in the tissues. • Hb deficient in 2,3 BPG thus acts as an oxygen “trap” rather than as an oxygen transport system.

Transfused RBCs are able to restore depleted supplies of 2,3 BPG in 24-48 h. However, severely ill patients may be seriously compromised if transfused with large quantities of such 2,3 BPG –”stripped” blood. • Decrease in 2,3 BPG can be prevented by adding substrates e.g., inosine (hypoxanthine-ribose) to storage medium. • Inosine, uncharged molecule can enter RBC, its ribose moiety is released, phosphorylated, and enters hexose mosophosphate pathway, eventually converted to 2,3 BPG

4. Binding of CO2: • Most CO2 produced in metabolism is hydrated and transported as bicarbonate ion. • However, some CO2 is carried as carbamate bound to uncharged α-amino groups of Hb (carbamino-Hb): Hb-NH2 + CO2 ↔ Hb-NH-COO- + H+ • Binding of CO2 stabilizes T (taut) or deoxy form of Hb, resulting in decrease in its affinity for oxygen. In lungs, CO2 dissociates from Hb, released in breath.

5. Binding of CO: • CO binds tightly (but reversibly) to Hb iron carbon monoxyhemoglobin (HbCO). • When CO binds to one or more of the 4 heme sites, Hb shifts to relaxed conformation, causing remaining sites bind oxygen with high affinity. • This shifts oxygen saturation curve to the left, and changes normal sigmoidal shape toward a hyperbola. • As a result affected Hb is unable to release oxygen to tissues • Affinity of Hb to CO is 220x greater than for oxygen.

Figure 3.12. Effect of carbon monoxide on the oxygen affinity of hemoglobin. CO-Hb = carbon monoxyhemoglobin.

Even minute quantities of CO in environ. can produce toxic conc’s of HbCO in blood. • CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at cellular level. • CO poisoning is treated with 100% oxygen therapy, which facilitates dissociation of CO from Hb.

F. Minor hemoglobins • HbA is one member of a functionally & structurally related family of proteins, the hemoglobins • Each of these oxygen-carrying proteins is a tetramer, composed of 2 α-like and 2 ß-like polyps • Certain Hbs e.g., HbF, are normally synthesized only during fetal development, others e.g., HbA2, are synthesized in the adult although at low levels compared to HbA • HbA can also become modified by covalent addition of a hexose e.g., addition of glucose  glucosylated Hb derivative HbA1c.

Figure 3.13 Normal adult human hemoglobins. [Note: The a-chains in these hemoglobins are identical.]

1. Fetal hemoglobin (HbF): • HbF is a tetramer of two α, plus 2 γ chains (α2γ2). γ chains are members of ß-globin gene family. • HbF synthesis during development: • In 1st few wks after conception, embryonic Hb (Hb Gower 1), composed of 2 zeta and 2 epsilon chains (ζ2ε2), synthesized by embryonic yolk sac • Within few wks, fetal liver begins to synthesize HbF in developing bone marrow • HbF is major Hb found in fetus and newborn, accounting for ~60% of total Hb in RBCs during last months of fetal life • HbA synthesis starts in BM at about 8th month of pregnancy and gradually replaces HbF

Figure 3.14 Developmental changes in hemoglobin.

b. Binding of 2,3 BPG to HbF: • Under physiologic conditions, HbF has a higher affinity for oxygen than does HbA, as a result HbF’s binding only weakly to 2,3 BPG • The γ-globin chains lack some of the positively charged aa’s responsible for binding 2,3 BPG in ß-globin chains • Because 2,3 BPG serves to reduce affinity of HbA for oxygen, weaker interaction b/w 2,3 BPG and HbF  higher oxygen affinity for HbF relative to HbA. • If both HbF and HbA stripped of their 2,3 BPG they then have similar affinity for oxygen • The higher oxygen affinity of HbF facilitates transfer of oxygen from maternal circulation across placenta to RBCs of fetus.

HbA2: • Is a minor component of normal adult Hb, appearing ~ 12 wks after birth  ~ 2% of total Hb. composed of α2δ2. HbA1c: • Under physiologic conditions, HbA slowly & non-enzymatically glycosylated • Extent of glycosylation dependent on plasma conc. of a particular hexose • Most abundant form of glycosylated Hb is HbA1c: has glucose residues attached predominantly to NH2 groups of the N-terminal Val of ß-globin chains • Increased amounts found in RBCs of diabetes mellitus, as their HbA has contact with higher glucose concs during the 120 d lifetime of these cells

Figure 3.15 Nonenzymic addition of glucose to hemoglobin.

III. Organization of the globin genes To understand diseases resulting from genetic alterations in structure or synthesis of Hbs. A. α-Gene family • Genes for α- and β-globin-like subunits of Hb chains occur in 2 separate gene clusters (families) located on two different chr’s • α-gene cluster on chr 16 contains 2 genes for α-globin chains. It also contains zeta (ζ) gene, expressed early in development as a component of embryonic Hb, & a number of globin-like genes that are not expressed (pseudogenes)

B. β-Gene family • A single β-globin gene located on chr 11, plus additional 4 β-globin-like genes: epsilon (ε) gene, two gamma (γ) genes (Gγ & Aγ that are expressed in fetal, HbF), and the δ gene that codes for globin chain found in minor adult HbA2.

Figure 3.16 Organization of the globin gene families.

C. Steps in globin chain synthesis • Begins in nucleus of RBC precursors, where DNA sequences encoding gene is transcribed. • RNA produced by transcription is a precursor of mRNA • Before translation, two non-coding stretches of RNA (introns) must be removed, and remaining 3 fragments (exons) reattached in a linear manner. • Resulting mature mRNA enters cytosol, its genetic info. translated  globin chain

Figure 3.17 Synthesis of globin chains.

Globular Proteins