Myoglobin and hemoglobin are two oxygen (O2)-binding proteins with a very simi- lar primary structure (see Fig. 5.10). However, myoglobin is a globular protein com- posed of a single polypeptide chain that has one O2 binding site. Hemoglobin is a tetramer composed of two different types of subunits (2α and 2β polypeptide chains, referred to as two αβ protomers). Each subunit has a strong sequence homology to myoglobin and contains an O2 binding site. A comparison between myoglobin and hemoglobin illustrates some of the advantages of a multisubunit quaternary structure.
The tetrameric structure of hemoglobin facilitates saturation with O2 in the lungs and release of O2 as it travels through the capillary beds (Fig. 5.11). When the amount of O2 bound to myoglobin or hemoglobin is plotted against the partial pres- sure of oxygen (pO2), a hyperbolic curve is obtained for myoglobin, whereas that for hemoglobin is sigmoidal. These curves show that when the pO2 is high, as in the lungs, both myoglobin and hemoglobin are saturated with O2. However, at the lower levels of pO2 in O2-using tissues, hemoglobin cannot bind O2 as well as myoglobin (i.e., its percent saturation is much lower). Myoglobin, which is present in heart and
Equation 5.1. The association constant, Ka
for a binding site on a protein Consider a reaction in which a ligand (L) binds to a protein (P) to form a ligand–protein com- plex (LP) with a rate constant of k1. LP dissoci- ates with a rate constant of k2:
L ⫹ P
k1
k2
LP then,
Keq⫽ k_ 1
k2
⫽ _ [LP]
[L][P] ⫽ Ka⫽ 1 _ Kd
The equilibrium constant, Keq, is equal to the as- sociation constant (Ka) or 1/Kd, the dissociation constant. Unless otherwise given, the concen- trations of L, P, and LP are expressed as mol/L, and Ka has the units of (mol/L)⫺1.
10 20 30 40 50 60 70 80 90 100 0.5
1.0
Partial pressure of oxygen (mm Hg)
Saturation with oxygen
Tissues Lungs
P50
=2.8
P50=26 Myoglobin
Hemoglobin
FIG. 5.11. O2 saturation curves for myoglo- bin and hemoglobin. Note that the curve for myoglobin is hyperbolic, whereas that for he- moglobin is sigmoidal. The effect of the tet- rameric structure of hemoglobin is to inhibit O2 binding at low O2 concentrations. P50 is the partial pressure of O2 (pO2) at which the protein is half-saturated with O2. P50 for myo- globin is 2.8 torrs, and that for hemoglobin is 26 torrs, where 1 torr is equal to 1 mm Hg.
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skeletal muscle, can bind the O2 released by hemoglobin, which it stores to meet the demands of contraction. As O2 is used in the muscle cell for generation of ATP during contraction, it is released from myoglobin and picked up by cytochrome oxidase, a heme-containing enzyme in the electron transport chain that has an even higher affi nity for O2 than myoglobin.
A. Oxygen Binding and Heme
The tertiary structure of myoglobin consists of eight α-helices connected by short coils, a structure that is known as the globin fold (see Fig. 5.10). This structure is unusual for a globular protein in that it has no β-sheets. The helices create a hydro- phobic O2-binding pocket containing tightly bound heme with an iron (Fe2⫹) atom in its center.
Heme consists of a planar porphyrin ring composed of four pyrrole rings that lie with their nitrogen atoms in the center binding a Fe2⫹ atom (Fig. 5.12). Negatively charged propionate groups on the porphyrin ring interact with arginine and histidine side chains from the hemoglobin, and the hydrophobic methyl and vinyl groups that extend out from the porphyrin ring interact with hydrophobic amino acid side chains from hemoglobin. Altogether, there are about 16 different interactions be- tween myoglobin amino acids and different groups in the porphyrin ring.
Organic ligands that are tightly bound to proteins, such as the heme of myoglo- bin, are called prosthetic groups. A protein with its attached prosthetic group is called a holoprotein; without the prosthetic group, it is called an apolipoprotein.
The tightly bound prosthetic group is an intrinsic part of the protein and does not dissociate until the protein is degraded.
Within the binding pocket of myoglobin and hemoglobin, O2 binds directly to the Fe2⫹ atom on one side of the planar porphyrin ring (Fig. 5.13). The Fe2⫹ atom is able to chelate (bind to) six different ligands; four of the ligand positions are in a plane and taken by the central nitrogens in the planar porphyrin ring. There are two ligand positions perpendicular to this plane. One of these positions is taken by the nitrogen atom on a histidine, called the proximal histidine, which extends down from a myoglobin or hemoglobin helix. The other position is taken by O2.
The proximal histidine of myoglobin and hemoglobin is sterically repelled by the heme porphyrin ring. Thus, when the histidine binds to the Fe2⫹ in the middle of the ring, it pulls the Fe2⫹ above the plane of the ring. When O2 binds on the other side of the ring, it pulls the Fe2⫹ back into the plane of the ring. The pull of O2 bind- ing moves the proximal histidine toward the porphyrin ring, which moves the helix containing the proximal histidine. This conformational change has no effect on the function of myoglobin. However, in hemoglobin, the movement of one helix leads to the movement of other helices in that subunit, including one in a corner of the subunit that is in contact with a different subunit through salt bridges. The loss of these salt bridges then induces conformational changes in all other subunits, and all four subunits may change in a concerted manner from their original conformation to a new conformation.
B. Cooperativity of Oxygen Binding in Hemoglobin
The cooperativity in O2 binding in hemoglobin comes from conformational changes in tertiary structure that take place when O2 binds. The conformational change of hemoglobin is usually described as changing from a T (tense) state with low affi nity for O2 to an R (relaxed) state with a high affi nity for O2. Breaking the salt bridges in the contacts between subunits is an energy-requiring process, and consequently, the binding rate for the fi rst O2 is very low. When the next O2 binds, many of the hemo- globin molecules containing one O2 will already have all four subunits in the R state, and therefore, the rate of binding is much higher. With two O2 molecules bound, an even higher percentage of the hemoglobin molecules will have all four subunits in the R state. This phenomenon, known as positive cooperativity, is responsible for the sigmoidal O2 saturation curve of hemoglobin (see Fig. 5.11).
Myoglobin is readily released from skeletal muscle or cardiac tissue when the cell is damaged. It has a small molecular weight, 17,000 Da, and is not complexed to other proteins in the cell. (Da is the abbreviation for Dalton, which is a unit of mass approximately equal to 1 H atom. Thus, a molec- ular weight of 17,000 Da [equivalent to 17 kDa]
is approximately equal to 17,000 g/mole.) Large injuries to skeletal muscle that result from physi- cal crushing or lack of ATP production result in cellular swelling and the release of myoglobin and other proteins into the blood. Myoglobin passes into the urine (myoglobinuria) and turns the urine red because the heme (which is red) remains covalently attached to the protein. Dur- ing an acute MI, myoglobin is one of the fi rst proteins released into the blood from damaged cardiac tissue; however, the amount released is not high enough to cause myoglobinuria. Labo- ratory measurements of serum myoglobin were used in the past for early diagnosis in patients like Anne J. Because myoglobin is not present in skeletal muscle and the heart as tissue-specifi c isozymes, and the amount released from the heart is much smaller than the amount that can be released from a large skeletal muscle injury, myoglobin measurements are not specifi c for an MI. Due to the lack of specifi city in myoglobin measurements, the cardiac markers of choice for detection of MIs are the heart isozymes of the troponins (I and/or T) and creatine kinase.
V
P M
P
M Heme
M M
N V Fe N N 2+N
FIG. 5.12. Heme. The Fe2⫹ is bound to four nitrogen atoms in the center of the heme porphyrin ring. Methyl (M, CH3), vinyl (V,MCHFCH2 ), and propionate (P, CH2CH3COO⫺) side chains extend out from the four pyrrole rings that comprise the por- phyrin ring.
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CHAPTER 5 ■ STRUCTURE–FUNCTION RELATIONSHIPS IN PROTEINS 69
C. Agents that Affect Oxygen Binding
The major agents that affect O2 binding to hemoglobin are shown in Figure 5.14.
1. 2,3-BISPHOSPHOGLYCERATE
2,3-Bisphosphoglycerate (2,3-BPG) is formed in red blood cells from the glycolytic intermediate 1,3-bisphosphoglycerate (see Chapter 19). 2,3-BPG binds to hemoglo- bin in the central cavity formed by the four subunits, increasing the energy required for the conformational changes that facilitate the binding of O2. Thus, 2,3-BPG low- ers the affi nity of hemoglobin for O2. Therefore, O2 is less readily bound (i.e., more readily released in tissues) when hemoglobin has bound 2,3-BPG. Red blood cells can modulate O2 affi nity for hemoglobin by altering the rate of synthesis or degrada- tion of 2,3-BPG.
2. PROTON BINDING (BOHR EFFECT)
The binding of protons by hemoglobin lowers its affi nity for O2 (Fig. 5.15), contrib- uting to a phenomenon known as the Bohr effect (Fig. 5.16). The pH of the blood decreases as it enters the tissues (and the proton concentration rises) because the CO2 produced by metabolism is converted to carbonic acid by the reaction catalyzed by carbonic anhydrase in red blood cells. Dissociation of carbonic acid produces protons that react with several amino acid residues in hemoglobin, causing confor- mational changes that promote the release of O2.
C N
Fe
O O
HC CH
NH
Proximal histidine
Heme – Fe Helix C
N
Fe
HC CH
NH Deoxyhemoglobin
A B O2 Hemoglobin
O2
FIG. 5.13. A. O2 binding to the Fe2⫹ of heme in hemoglobin. A histidine residue called the proximal histidine binds to the Fe2⫹ on one side of the porphyrin ring and slightly pulls the Fe2⫹ out of the plane of the ring; O2 binds to Fe2⫹ on the other side. B. O2 binding causes a conformational change that pulls the Fe2⫹ back into the plane of the ring. As the proximal histidine moves, it moves the helix that contains it.
Sickle cell anemia is really a disease caused by an abnormal quaternary structure. The painful vaso-occlusive crises experienced by Will S. are caused by the polymerization of sickle cell hemoglobin (HbS) molecules into long fi bers that distort the shape of the red blood cells into sickle cells. The substitution of a hydrophobic valine for a glutamate in the β2-chain of hemoglobin creates a knob on the surface of deoxygenated hemoglobin that fi ts into a hydrophobic binding pocket on the β1-subunit of a different hemoglobin molecule. A third hemo- globin molecule, which binds to the fi rst and second hemoglobin molecules through aligned polar interactions, binds a fourth hemoglobin molecule through its valine knob. Thus, the polymerization continues until long fi bers are formed.
Polymerization of the hemoglobin molecules is highly dependent on the concentration of HbS and is promoted by the conformation of the deoxygenated molecules. At 100% O2 satura- tion, even high concentrations of HbS will not polymerize. A red blood cell spends the longest amount of time at the lower O2 concentrations of the venous capillary bed, where polymeriza- tion is most likely initiated.
HbO2 Hb + O2
Hydrogen ions
2,3-Bisphosphoglycerate Covalent binding of CO2 1
2 3
FIG. 5.14. Agents that affect O2 binding by hemoglobin. Binding of hydrogen ions, 2,3-bisphosphoglycerate, and carbon dioxide to hemoglobin decrease its affi nity for O2.
Tissues Lungs
40 80 120
20 0 40 60 80 100
pO2
% Saturation
7.6 7.2 6.8 pH Hb
FIG. 5.15. Effect of pH on O2 saturation curves. As the pH decreases, the affi nity of he- moglobin for O2 decreases, producing the Bohr effect.
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In the lungs, this process is reversed. O2 binds to hemoglobin (due to the high O2 concentration in the lung), causing a release of protons, which combine with bicarbonate to form carbonic acid. This decrease of protons causes the pH of the blood to rise. Carbonic anhydrase cleaves the carbonic acid to H2O and CO2, and the CO2 is exhaled. Thus, in tissues where the pH of the blood is low because of the CO2 produced by metabolism, O2 is released from hemoglobin. In the lungs, where the pH of the blood is higher because CO2 is being exhaled, O2 binds to hemoglobin.
3. CARBON DIOXIDE
Although most of the CO2 produced by metabolism in the tissues is carried to the lungs as bicarbonate, some of the CO2 is covalently bound to hemoglobin. In the tissues, CO2 forms carbamate adducts with the N-terminal amino groups of deoxy- hemoglobin and stabilizes the deoxy conformation, resulting in more O2 delivery to the tissues. In the lungs where the pO2 is high, O2 binds to hemoglobin and this bound CO2 is released (Fig. 5.17).
VIII. PROTEIN FOLDING
Although the peptide bonds in a protein are rigid, fl exibility around the other bonds in the peptide backbone allow an enormous number of possible conformations for each protein. However, every molecule of the same protein folds into the same stable three-dimensional structure. This shape is known as the native conformation.
A. Primary Structure Determines Folding
The primary structure of a protein determines its three-dimensional conformation.
More specifi cally, the sequence of amino acid side chains dictates the fold pattern of the three-dimensional structure and the assembly of subunits into quaternary struc- ture. Proteins become denatured when they lose their overall structure. However, under certain conditions, denatured proteins can refold into their native conforma- tion, regaining their original function. This indicates that the primary structure es- sentially specifi es the folding pattern. In some cases, proteins are assisted in folding by heat shock proteins (some of which are also called chaperonins), which use the energy provided by ATP hydrolysis to assist in the folding process.
A cis-trans isomerase and a protein disulfi de isomerase also participate in fold- ing. The cis-trans isomerase converts a trans peptide bond preceding a proline into the cis conformation, which is well suited for making hairpin turns. The disulfi de isomerase breaks and reforms disulfi de bonds between the -SH groups of two cys- teine residues in transient structures formed during the folding process. After the protein has folded, cysteine-SH groups in close contact in the tertiary structure can react to form the fi nal disulfi de bonds.
It is important to note that there is very little difference in the energy state of the native conformation and a number of other stable conformations that a protein might assume. This enables the protein to have the fl exibility to change conforma- tion when modifi ers are bound to the protein, which enables a protein’s activity to be regulated (similar to 2,3-bisphosphoglycerate binding to hemoglobin and stabilizing the deoxy form of hemoglobin).
B. Fibrous Proteins
1. COLLAGEN
Collagen, a family of fi brous proteins, is produced by a variety of cell types, but principally by fi broblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells. Type I collagen, or collagen(I), the most abundant protein in mammals, is a fi brous protein that is the major component of connective tissue. It is found in the extracellular matrix (ECM) of loose connective tissue, bone, ten- dons, skin, blood vessels, and the cornea of the eye. Collagen(I) contains about 33% glycine and 21% proline and hydroxyproline. Hydroxyproline is an amino RBC
RBC CO2
H2CO3
H+
O2 H2O Carbonic
anhydrase
Carbonic anhydrase HbO2
HHb Tissues
Tissues A
CO2
H2CO3
H+
O2 H2O
HCO3– HCO3–
HbO2 HHb Exhaled
Lungs B
FIG. 5.16. Effect of H⫹ on O2 binding by hemoglobin (Hb). A. In the tissues, CO2 is re- leased. In the red blood cell, this CO2 forms carbonic acid, which releases protons. The protons bind to Hb, causing it to release O2 to the tissues. B. In the lungs, the reactions are re- versed. O2 binds to protonated Hb, causing the release of protons. The protons bind to bicar- bonate (HCO3⫺), forming carbonic acid which is cleaved to water and CO2, which is exhaled.
RBC, red blood cell.
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CHAPTER 5 ■ STRUCTURE–FUNCTION RELATIONSHIPS IN PROTEINS 71
acid produced by posttranslational modifi cation of peptidyl proline residues (see Fig. 4.9).
Procollagen(I), the precursor of collagen(I), is a triple helix composed of three polypeptide (pro-α) chains that are twisted around each other, forming a ropelike structure. Polymerization of collagen(I) molecules forms collagen fi brils, which provide great tensile strength to connective tissues (see Fig. 5.1; the representation of a fi brous protein). The individual polypeptide chains each contain about 1,000 amino acid residues. The three polypeptide chains of the triple helix are linked by interchain hydrogen bonds. Each turn of the triple helix contains three amino acid residues, such that every third amino acid is in close contact with the other two strands in the center of the structure. Only glycine, which lacks a side chain, can fi t in this position, and indeed, every third amino acid residue of collagen is glycine.
Thus, collagen is a polymer of (Gly-X-Y) repeats, where Y is frequently proline and/
or hydroxyproline and X is any other amino acid found in collagen.
Procollagen(I) is an example of a protein that undergoes extensive posttrans- lational modifi cations. Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues. These reactions occur after the protein has been synthesized and require vitamin C (ascorbic acid) as a cofactor of the enzymes, which are prolyl hydroxylase and lysyl hydroxylase.
Hydroxyproline residues are involved in hydrogen bond formation that helps to sta- bilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose).
The side chains of lysine residues may also be oxidized to form the aldehyde allysine. These aldehyde residues produce covalent cross-links between collagen molecules to further stabilize the triple helix. An allysine residue on one collagen molecule reacts with the amino group of a lysine residue on another molecule, form- ing a covalent Schiff base (a nitrogen-carbon double bond) that is converted to more stable covalent cross-links. Aldol condensation may also occur between two allysine residues, which forms the structure lysinonorleucine.
2. TYPES OF COLLAGEN
At least 28 different types of collagen have been characterized (Table A5.1) . Although each type of collagen is found only in particular locations in the body, more than one type may be present in the extracellular matrix at a given location.
There are various types of collagen (fi bril-forming, network-forming, those that as- sociate with fi bril surfaces, transmembrane proteins, endostatin-forming, and those that form periodic beaded fi laments), but this text will focus on the fi bril-forming collagens (types I, II, III, V, XI, XXIV, and XXVII). These collagen molecules form fi brils that assemble into large insoluble fi bers. The fi brils are strengthened through covalent cross-links between lysine residues on adjacent fi brils. The arrangement of the fi brils gives individual tissues their distinct characteristics. Tendons, which attach muscles to bones, contain collagen fi brils aligned parallel to the long axis of the tendon, thus giving the tendon tremendous tensile strength.
3. SYNTHESIS AND SECRETION OF COLLAGEN
Collagen is synthesized within the endoplasmic reticulum as a precursor known as preprocollagen. The presequence acts as the signal sequence for the protein and is cleaved, forming procollagen within the endoplasmic reticulum. From there, it is transported to the Golgi apparatus (Table 5.1). Three procollagen molecules as- sociate through formation of inter- and intrastrand disulfi de bonds at the carboxy terminus; once these disulfi des are formed, the three molecules can align properly to initiate formation of the triple helix. The triple helix forms from the carboxy end toward the amino end, forming tropocollagen. The tropocollagen contains a triple helical segment between two globular ends, the amino and carboxy terminal exten- sions. The tropocollagen is secreted from the cell, the extensions are removed using
CO2
H+ Hb NH+ 3
Hb N COO– + H
Hemoglobin
Carbamate of hemoglobin +
FIG. 5.17. Binding of CO2 to hemoglobin.
CO2 forms carbamates with the N-terminal amino groups of hemoglobin chains. Approxi- mately 15% of the CO2 in blood is carried to the lungs bound to hemoglobin. The reaction releases protons, which contribute to the Bohr effect. The overall effect is the stabilization of the deoxy form of hemoglobin.
The hydroxyproline residues in col- lagen are required for stabilization of the triple helix via hydrogen bond formation. In the absence of vitamin C (scurvy), the melting temperature of collagen can drop from 42°C to 24°C as a result of the loss of inter- strand hydrogen bond formation from the lack of hydroxyproline residues.
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