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Charles Saladino Introductory Concepts We could begin this lecture by immediately describing the structure of various proteins. However, in order to understand the relationship between the structure and the function of proteins, such as hemoglobin (Hb), it is necessary that we comprehend the critical concept of allosterism, derived from the Greek allos (other) + stereos (solid or space). Although this term is a key feature of many enzymes, it is certainly not limited to these biomolecules, but rather, is critical to the function of many other types of proteins. Before we start with the classic model of hemoglobin to illustrate allosterism, we need to define the term as involving the behavior of multisubunit (thus having quaternary structure) proteins that bind small molecules. This binding of a small molecule, called a ligand, to the protein will change the affinity of that protein for other ligand molecules and/or the activity level of the protein itself. In other words, allosteric effects involve the binding of a ligand at one site, which in turn affects the binding of a ligand at another site (or subunit) in the same protein, probably altering the protein’s activity. To accomplish this, there must be conformational shifts involving interactions between the subunits of the protein. (The conformational shifts involved in some proteins are not well understood, whereas those of others, including hemoglobin, are very well described, as we shall see.) The following are the main features of the symmetry model of allosterism, first formulated in 1965. However, I have included the more recent modifications to this original model: 1. The protein must be an oligomer (meaning having multiple subunits) of symmetrically-related subunits (or approximately symmetrically related - as we will discuss in the case of the α- and β-subunits of Hb). 2. The oligomer (multi-subunit protein) can exist in two conformational states, Taught (T) and Relaxed (R), which exist in equilibrium with each other. 3. A ligand binds to a subunit that is configured to accept it. This binding results in a change in the conformation of that subunit, which, in turn, initiates a change in the conformation of a second subunit to alter that second subunit’s affinity for the ligand (and the activity level of the protein– to be discussed under the topic of enzymes). 4. In other words, when the first ligand binds to the first subunit, the rest of the subunits of the protein change conformation in a sequential manner (not simultaneously as previously thought), so as to accept additional ligands sequentially and generally change the activity of the whole protein. 5. As a result of the sequential change in the conformation of each subunit, the entire protein will go from the Taught State to the Relaxed State or vise versa, depending upon whether you are starting with the protein in the R State or the T
State of configuration. Classically, the T form of the protein is less active, whereas the R form is more active. As we shall see, this phenomenon of allosterism allows for efficient and multiple ways of regulating the ability of a protein to bind a ligand and to become either more active (R form) or less active (T form). Those ligands that bind to the protein or to an enzyme to enhance its function are known as positive allosteric effectors, whereas those that bind to cause a decreased level of functional are referred to as negative allosteric effectors. Structure of Hemoglobin (Hb) When the metabolic pathways associated with aerobic respiration began to appear, so did the ability of cells to produce great sums of additional energy, compared to the relatively small amount of ATP that could be generated from anaerobic respiration. With higher levels of energy production, greater and greater molecular-genetic variability could be supported, and a huge leap in specialized cellular activity could be realized. However, a system had to be available to deliver that insoluble oxygen to the tissues for aerobic respiration. The molecular and cellular environment within the erythrocyte provided just what was needed for delivering oxygen from the lungs to the tissues, within the context of the circulatory system. Within this intra-erythrocytic environment, wherein there are neither nuclei nor mitochondria, are found the well described hemoglobin molecules in great quantity plus an effective bicarbonate buffer system, which we shall discuss shortly in detail. The erythrocyte is the only cell that contains hemoglobin. The primary function of hemoglobin is to transfer oxygen from the lungs to the tissues. It is a tetrameric, heme protein that is comprised of four subunits (hence tetrameric), two identical α-subunits and two identical β-subunits. Thus, Hb has quarternary structure (remember, quarternary refers to a protein having two or more subunits). Both the α- and the β-subunits are structurally related to one another and to the single subunit molecule, myoglobin (which we discuss later in this lecture), even though only about 18% of the amino acids are identical in the twp types of subunits. Each α-subunit is comprised of 141 amino acids, whereas each β-subunit is composed of 146. About 75% of the complete structure of hemoglobin comprised of these four subunits is in the form of the α-helix. Within the center of each of the four subunits is a heme molecule, which is a protoporphyrin ring structure with an iron atom in the center. Familiarize yourself with the heme structure, shown on page 355 of your text, for the second exam. Below is a ribbon diagram of the hemoglobin molecule showing the four subunits, each in a different color and with each containing a heme molecule. As you observe the diagram, consider why each subunit fulfills the criteria of having tertiary structure (review those criteria from last week) and how the bonding of those subunits forms a quaternary protein structure.
The iron atom at the center of the heme is coordinately bonded with four nitrogen atoms and is the actual binding site of oxygen to the hemoglobin molecule see page 355 of your text). Thus, one hemoglobin molecule binds four oxygen molecules. However, the issue is exactly when and how the oxygen molecules bind to the hemoglobin in the lungs and when and how they are released to the tissues. Obviously, the oxygen gradients that exist in the lungs vs. the tissues allow the diffusion of oxygen between the red blood cells and the tissues and the lungs and these erythrocytes. However, the mechanism by which oxygen binds to and is released from a Hb molecule involves the complexities of allosterism and a specific aspect of it called positive cooperativity. Let’s explain that. Now try and picture one alpha subunit and one beta subunit joining to form a dimer with the help of numerous hydrophobic interactions. (This is shown in Devlin. on page 361) However, Hb is obviously composed of two dimers. One dimer (an αand a β-subunit) will then interact with the other dimer (the other αand β-subunit), however, by a small number of relatively weak bonding interactions. This is so that one dimer can rotate about 15 degrees in relation to the other dimer when oxygen binds to the first subunit. So let’s put all this together and define the allosteric change. We begin with the Hb in the Taut state configuration. The first oxygen binds to a hemoglobin molecule while the red cell is in the lungs. This causes an alloseteric, conformational change in the second subunit, which now accepts the second oxygen. This, in turn, repeats as it sequences to the third and then to the fourth subunit, during which time the 15 degree rotation has occurred between the two dimers. The Hb is now in the relaxed state (R), and it is fully saturated with four oxygen molecules, each bound to an Fe atom in the center of each heme. Obviously, there have been allosteric changes within each Hb as oxygen molecules bind. This change in the conformation of one subunit enhancing the binding of oxygen to the next subunit (and so forth) is defined as “positive cooperativity.” The red cells now travel to the tissues where oxygen is released. When the red cells reach the tissues, the pH is somewhat lower (more protons present), due to metabolic activity and due to carbonic acid (see below). These protons displace the oxygen from the Hb by their binding to the Hb in such a manner as to change the position of the heme relative to the α or β subunit. In other words, the protons cause displacement of the oxygen molecules as those protons bond to the heme. This removal of oxygen from the Hb by protons is known as the Bohr Effect. You might have thought that carbon dioxide would take the place of oxygen as it diffuses into the erythrocyte. Actually, only about 20% of the CO2 is carried back to the lungs
bound to N-terminus of the hemoglobin chain in each of the four Hb subunits. Rather, the bulk is formed into the bicarbonate buffer system, as well shall now describe. Bicarbonate Buffer System Without this system operating in our red blood cells, we would be in the world of the inanimate. We need it to survive. Let’s look at it. You should remember from earlier courses that CO2 + H2O --------> H2CO3 (1). So carbonic acid is formed from water and carbon dioxide. Please remember that this reaction is reversible and is catalyzed in both directions by the enzyme carbonic anhydrase (a bidirectional enzyme). Now: H2CO3 then dissociates ---------> H+ + HCO3- (2) If you remember, a buffer is composed of a weak acid (the carbonic acid) and its conjugate base (the bicarbonate ion – HCO3-). I will summarize what happens at the tissues first: 1. The CO2 from the tissues primarily combines with water to give carbonic acid (catalyzed by carbonic anhydrase), as in equation #1. The carbonic acid dissociates as in equation #2. 2. The Bohr Effect occurs where the protons from the tissues moving into the red cells and from the dissociation of carbonic acid (equation 2) remove the oxygen from the Hb. We now have HbH+. Remember, this is the Bohr Effect, delivering oxygen to the tissues. 3. When the red cells get to the lungs, the reverse occurs. Oxygen in high concentration displaces the protons from the Hb (or the 20% of the carbon dioxide that was bound). The protons then combine with bicarbonate ion to form carbonic acid (H+ + HCO3- ------>H2CO3). 4. The carbonic acid is catalyzed by carbonic anhydrase to reverse equation # 1, forming CO2. This is how 80% of the CO2 is carried in the red cells and then released from the erythrocytes when they reach the lung capillaries. Notice the rather remarkable interplay between the bicarbonate buffer system and the Hb. Remember, the Hb can only accept its four oxygen molecules because of the allosteric, positive cooperativity between the subunits. So here is a great example of the structure/function concept I spoke of in the Introductory Lecture. Myoglobin “Myo” comes from the Greek for muscle. Myoglobin is a protein designed to store oxygen in muscle, which requires a ready reserve of that oxygen when it transitions into significant activity, wherein oxygen depletion could easily occur. Diffusion of oxygen from the red cells in the capillaries would be too slow, as we see in concerted muscle activity where oxygen is depleted, lactic acid builds up, and sometimes severe muscle
cramping occurs. Thus, oxygen released from the red cells to the muscle is first bound to and stored in the myoglobin molecules. When the muscle becomes active, oxygen is released from the myoglobin. The structure of myoglobin is, we might say, 25% as complex as hemoglobin. It is a water-soluble, globular heme protein comprised of only one subunit, thus having tertiary structure only. The myoglobin structure is very similar to a single Hb subunit, containing one heme only and being a single peptide comprised of 153 amino acids, with 70% of the chain folded into eight α-helices. This arrangement of amino acid folding is reminiscent of the subunits of Hb. Again, the binding of a single oxygen molecule occurs at the single heme iron. However, there is no positive cooperativity, because there is only one subunit. There is also no interplay with the bicarbonate buffer, as there is with Hb. Only oxygen tension levels (partial pressure) determines whether or not oxygen is bound to myoglobin, which in turn is related to the level of muscle activity. Some Comparisons: Myoglobin vs. Hemoglobin The structural differences between these two proteins are striking, but both do function in order to bind oxygen – Hb to carry it through the vascular system and deliver it, myoglobin to store that oxygen in muscle. Also, these two heme proteins were the first to have their entire three-dimensional structures delineated. As discussed above, both are heme proteins, because they contain heme molecules. These heme molecules are known as prosthetic groups by definition, because they represent a tightly-bound molecule essential to the function of the protein. Importantly, however, is the fact that the heme groups are contained in the interior of the Hb subunits and the myoglobin structure. This is no accident. The interior of the Hb subunits or myoglobin structure consist almost entirely of non-polar amino acids (leucine, valine, etc), whereas the charged amino acid residues (aspartate, glutamate etc) are absent from the inside. The only polar residues are histidines, which play critical roles in binding Fe and thus oxygen. The outside of the Hb subunits (or myoglobin structure), in contrast, consist of both polar and non-polar amino acids. A space-filled model would show very little room inside the structure. This excludes the aqueous environment from the inside where water-insoluble oxygen binds. The following diagram illustrates the position of the heme in relation to the histidines:
Additionally, we note that in order for oxygen to bind iron, the iron must be in the ferrous state (Fe++), not the ferric state (Fe+++). The hydrophobic interior where the heme is located is also conducive to this chemical necessity. Because myoglobin only binds one oxygen molecule, whereas Hb binds four, the partial pressure of oxygen required to saturate each protein would obviously be significantly greater for Hb than for myoglobin. Specifically, if one looks at the saturation curves below, the P50 (partial pressure of oxygen for 50% saturation) of myoglobin molecules is about 2.8 mm Hg vs. that of Hb at approximately 26.0 mm Hg. If you think about it, this makes perfect sense. 0100090000037400000002001c0000000000040000000301 0800050000000b0200000000050000000c02e205c505040000002e 0118001c000000fb029cff000000000000900100000000044000125 4696d6573204e657720526f6d616e0000000000000000000000000 000000000040000002d0100000400000002010100050000000902 000000020d000000320a5a00ffff0100040000000000c305dc0520b 32d001c000000fb021000070000000000bc0200000000010202225 3797374656d00000000000000000000180000000100000068fc210 0e4040000040000002d010100030000000000 2,3-Bisphosphoglycerate (2,3-BPG) and Hemoglobin This compound is derived from the glycolytic pathway, which we have not yet studied. However, keep this on the back burner of your mind when glycolysis (the oxidation of glucose) becomes our topic of discussion. For now, however, you should know that 2,3BPG is a strong negative allosteric effector on the oxygen-binding properties of Hb. The synthesis of BPG within the erythrocyte is a major reaction pathway for glucose consumption in the red cell. Its synthesis is critical for controlling the affinity of oxygen for Hb. Also, its synthesis yields no energy, unlike glycolysis where two ATP are the net energy gain. Anyway, here is how it works. Remember that the Taut (T) state of Hb is where it is deoxygenated. This configuration creates a cavity in the Hb structure capable of
binding 2,3-BPG. Such binding stabilizes the Hb in the T state. In contrast, less availability of 2,3-BPG means that it is not bound in the central cavity, and Hb is more readily converted to oxyhemoglobin. Therefore, like increased proton concentration and the Bohr Effect, increased 2,3-BPG favors conversion of the R form of Hb to the T form., decreasing the amount of oxygen bound by Hb at any concentration of oxygen. It is known that different hemoglobin species have different amino acid compositions and thus different 2,3-BPG-binding capabilities. HbF (fetal hemoglobin) binds 2,3-BPG more poorly than does HbA (normal adult hemoglobin). The result is that HbF in the unborn child of a pregnant woman binds oxygen with greater affinity than it does the HbA of the mother. This gives the child preferential access to oxygen from the mother’s circulatory system. Molecular Abnormalities Although I suspect that this topic is covered in some detail in Clinical Biochemistry, we should at least mention that numerous, genetic-derived molecular abnormalities arise in protein structures, including hemoglobin. In fact, today over 1500 “disease genes” have been identified where a mutant gene leads to a defective protein that is responsible for a specific pathological condition. Among these are the thalassemias and the well-described sickle cell anemia, which was the first disorder to have its molecular basis understood. In fact, in 1945, Linus Pauling first proposed that sickle cell anemia was the result of a mutant-derived hemoglobin. As the years went by, this molecular basis was more fully understood. Basically, a single gene mutation exchanges the non-polar amino acid, valine, for the polar glutamic acid at position number six in the β-subunits. This results in a polymerization of Hb molecules into fibers that essentially crystallize, such that the cytoplasm of the red cell is not able to support the cell membrane. This is because the hydrophobic protrusion from the β-subunit that normally does not occur now does occur, due to the valine. That protrusion finds a hydrophobic pocket in an adjacent Hb β-subunit, causing the polymerization. This only occurs when the Hb is in the deoxy state, and it causes the RBC membrane to collapse, and the cell assumes the shape of a crescent moon or sickle when the cells traverse the narrow capillaries and are giving up their oxygen to the tissues. Thus, they can fragment, leading to clot formation. When the disorder occurs in the heterozygous form (one parent contributes a defective gene), the sickle cell trait occurs where no significant clinical sequelae occur. This individual, however, is now a carrier of the disorder. In the homozygous form (where both parents contribute the defective gene), sickle cell anemia is life-threatening and lifeshortening, and the quality of life can be quite difficult. I trust that gene therapy will eventually lead to a reversal of this terrible disorder.
Although this topic is covered in Clinical Biochemistry, I will show you an electrophoretic pattern of various hemoglobins. This technique of electrophoresis takes advantage of charge differences in proteins to separate them by an electric current passed through a gel, to which the proteins are added, all in a buffer environment. Such an electrophoretic gel (many of which can be found on Google images) is shown below:
Antibody Structure We can all appreciate the need for an antibody to be specific against a given antigen. Humans make about 1 x 108 different antibodies, with there being five major classes of these immunoglobins. The proliferation of recognized autoimmune disorders points up the importance of an antibody neutralizing the right antigen. That is why antibodies are proteins. Proteins, due to their unique, amino acid-based, three-dimensional structures provide the specificity of structure (and thus function) that other biomolecular groups can not offer. As confirmed by X-ray diffraction studies, antibodies have pretty much the same Y-shape structure shown in the first diagram below. First notice the single stem of the Y shape. This is the end (C-terminal for carboxyl) that attaches to the surface of the immune cells. The branching N-terminal ends of the Y are where attachment to the antigen occurs. You will notice that these ends are labeled with a “V.” This stands for variable region. Thus,
it would only stand to reason that the peptide sequence would be different from antibody to antibody, in order to accommodate the need for specificity in binding to a unique antigen. This specificity is conferred by chemical complementarity between the antigen and its specific binding site, in terms of its shape, location charged, non-polar, and hydrogen-binding groups. For example, a binding site with a positively charged group might bind an antigen with a negatively charged group in the complementary position. Often, complementarity is achieved by interaction as the structures of the antigen and the binding site approach one another, influencing one another. Conformational changes in the antibody and/or antigen can then occur that will allow the complementary groups to fully interact. This is an example of induced fit (as opposed to lock and key). We will see in next week’s lecture that induced fit is a hallmark of the interaction between an enzyme and its substrate. If we think about it, there is little or no need for variability from antibody to antibody in regions of the structure that are not involved in attaching to the antigen. Thus, they are labeled “C” regions, which stands for constant. There are three constant domains in each heavy chain and one constant domain in each light chain. These need not differ greatly among the various antibodies, given their structural roles, such as attachment to the immune cells (ex. plasma cells) that produce them or macrophages that process the antigens. The “H” and the “L” designations refer to the relative molecular weights of the polypeptide chains, heavy and light, respectively. Thus, two antigen-binding sites are formed by the combination of variable domains from one light (VL) chain and one heavy chain VH. Also notice the four sets of disulfide linkages (thus involving cysteine) holding the heavy and light chains together, as well as the hinge region between the branches of the stem of the Y. There is a similar second diagram below. Can you picture what a space-filled diagram of an antibody would look like?
Collagen Collagen, of which there are at least a twenty specific kinds assembled from 33 genetically distinct chains, is an important protein found in connective tissue, whether it be the skin, the frame work of bone, tendons and ligaments, the arterial wall, or almost anywhere else you could reasonably imagine. If you look back at your amino acid structures, you will be reminded that proline is the only cyclical amino acid. This means that the carbonyl oxygen and the amine nitrogen would not point at the same directional angle that they normally would in any of the other amino acids. Hence the stabilizing hydrogen bonds that form the α-helix are not possible, with proline (and the modified proline – 4-hydroxyproline) comprising up to 30% of the collagen structure. Instead, what we have is a rather unique structure for a protein – the right handed triple helix. That means there are three polypeptide strands wound around each other to form a triple helix, with every third amino acid residue passing through the center of the triple helix. This makes for such a crowded arrangement, that only a glycine (with no side chain) can fit. Thus, every third amino acid in each polypeptide chain is glycine. We might take note that proline is hydroxylated after the polypeptide chains are formed
in the rough endoplasmic reticulum of fibroblasts, chondroblasts, osteoblasts, and other connective tissue-producing cells. Lysine is also hydroxylated in the same manner, and both hydroxylations require an oxidase enzyme that itself requires the cofactor, ascorbic acid. This explains why vitamin C-deficient individuals have abnormal connective tissues, thus affecting many tissues, including bone development. Read page 244-245 in your text for more details on this aspect of collagen synthesis, and be responsible for that information for the first exam. The hydroxylated proline contributes to triple helix stability, as does the proline. However, we must realize that collagen is secreted in small triple helix procollagen segments (about 1000 amino acids for each of the three polypeptide chains). Each of these polypeptide chains of the triple helix contains small registration peptides on the Nterminal ends. These registration peptides are removed outside the cell, where triple helix polymerization of the small procollagens can take place end to end. This lengthens the collagen. However, in order for the collagen to grow into a thick fiber useful in and as connective tissue, triple helix polymerization must also occur side to side. This occurs mainly through cross-linkage of the hydroxylysine residues. As the complete connective tissue matrix is forming, the lengthened, thickened collagen fibers become embedded in and chemically cross-linked to various various, especially glucose and galactose, some sulfated and polymerized to form carbohydrates complexes, such as chondroitin sulfate etc. This cross-linkage to carbohydrates usually occurs at the hydroxylysine residues. As collagen ages, there is less hydration associated with the mature collagen, and progressively more intramolecular (within the triple helix) and intermolecular (between triple helices) cross-links occur with time. The result is more dehydrated, tougher, lesswater-soluble, less flexible connective tissue fiber. Degradation of Collagen Normally, collagen is a stable protein molecule, with half-lives as long as several months. Yet connective tissue is dynamic, with continuous remodeling occurring. Such changes are often responses to injury or growth of the tissue. In the extracellular matrix, collagen degradation occurs by a family of collagenases that break collagen fibers into smaller segments that can be phagocytized by immune cells and further degraded by lysosomal enzymes into the amino acid components of the collagen. Collagen Disorders Genetic defects can occur in any step of collagen synthesis or maturation. Thus, collagen forms improperly, and the tensile strength normally required for a given tissue is lacking. More than 1000 mutations have been identified in 22 genes coding for the various collagen types. Look up the defects in the following two conditions and familiarize yourself with them for the first exam: Ehlers-Danlos Syndrome and Osteogenesis Imperfecta.
Insulin The importance of insulin in regulating carbohydrate metabolism can not be overestimated. However, that is for a later lecture. Here I just wish to describe the structure of this peptide hormone. In doing so, however, we can see a principle of protein synthesis we have not yet discussed. That is, many proteins are synthesized in a “pro” form that is inactive, with activation taking place after certain specific amino acids are cleaved and/or removed. Although this might seem like the same phenomenon we just discussed with removing the registration peptides from procollagen, it actually is not, because in the case of insulin, transformation to its active form creates an additional functional peptide at the same time, namely peptide C. Let us look at this by starting with the diagram shown below and taken from the Devlin text. As you look at this diagram notice the primary structure containing three sets of disulfide bonds. You can see a slashed line on the left of the diagram between glutamine and arginine and on the right, between glutamic acid and arginine. You also see a solid line between arginine and glycine on the left and arginine and threonine on the right. Enzymatic cleavage occurs at both the slashed lines and the solid lines, resulting in removal of two amino acids on the left side and two on the right. Now look below of observe the now-formed structure of insulin, comprised of an α and a β chain held together by two covalent disulfide linkages. There are a total of 51 amino acids in the two chains of insulin (the diagram labels the last amino acid in the α-chain # 20, whereas it should read # 21). By looking at the very top of the diagram, you can figure out the primary amino acid sequence of peptide C, which is what we started with, minus the insulin and the four amino acids that were cleaved away. Thus, the insulin before any cleavages was just called proinsulin, having no physiological/biochemical value as such. This change over from proinsulin to active insulin takes place within the pancreatic beta of Langerhans cells, the insulin-producing cells of the body The primary sequence is important, not only for structural assignments, but also because the primary sequence of insulin from different species varies somewhat. By observing which amino acids are replaced in different species-related insulin molecules, one can ascertain that those replacements do not cause changes that would otherwise render the insulin non-functional. Residue 30 in the β-chain is such an example that varies widely from species to species without changing the functionality of the insulin. In the clinical setting, peptide C is often used as a measure of insulin production.
The Zymogen Concept Many proteins are formed in the inactive state. Insulin just served as an example of that. Now we take one step further, because we are not only talking about synthesizing a protein in an inactive form, but also activating it at some location relatively far away. Let us use the proteolytic enzyme, trypsin, as an example. This enzyme synthesized by the exocrine cells of the pancreas, and it is actually synthesized in the form of trypsinogen, an inactive precursor of the enzyme. When it reaches the duodenum, it is activated, partly by the neutral pH environment of that first segment of the small intestine. Why would this occur? Does this seem like a waste of energy? Perhaps at first glance, you might think so. However, if we look at this proteolytic enzyme, we would soon realize that it would be devastating to the living cell if active trypsin became functional in the pancreas, where it could cause a great deal of cellular and tissue damage. However, in the duodenum, proteins fragments are present that require breakdown before final digestion can occur. Thus, an active trypsin would be ideal in the duodenal area where partially dietary protein is readily found.
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