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19 Cards in this Set
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Describe the structure of protoporphyrin ring.
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Protoporphyrin ring is composed of four modified pyrrole subunits interconnected at their α carbon atoms via methene bridges. The iron atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring system and two perpendicular to the porphyrin. Protoporphyrin acts as a flexible cage within which iron is bound in its ferrous (+2) state. The flexibility of the cage allows for heme’s function. The coordinated nitrogen atoms (which have an electron-donating character) help prevent conversion of the heme iron to the ferric (Fe3+) state. |
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Describe the relationship of the Fe in the protoporphyrin within the myoglobin (Mb) and hemoglobin (Hb) peptides. To this answer do describe the structure of these proteins.
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In the heme molecule, iron is in the Fe2+ state and binds oxygen reversibly; when it is free, oxygen binding is irreversible and iron remains in the ferric (Fe3+ ) state. In heme-containing proteins, the reaction is prevented by sequestering of the heme deep within the protein structure where access to the two open coordination bonds is restricted. One of these two coordination bonds is occupied by a side-chain nitrogen of a His residue. One of the coordination bonds of iron is bound by the proximal F8 His, while the other coordination bond is reserved for oxygen binding. Myoglobin is a globular single subunit protein that is conjugated with a heme group in a central crevice flanked by alpha-helix F and alpha-helix E. Hemoglobin is a tetrameric globular protein consisting of 4 subunits (2 alpha and 2 beta subunits) with ion pairs at the interfaces of alpha1-beta2 and alpha2-beta1 subunits (this conformation allows for interaction and cooperative ligand binding). |
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Discuss the difference between lock and key and induced fit structure of proteins and binding ligand.
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The lock and key model of ligand binding is rooted in the idea that: 1) a protein is a fixed and non-flexible structure, and 2) a protein and its ligand are complementary to one another in size, shape, charge, and hydrophobic/hydrophilic character. Since proteins have a high specificity for particular ligands the lock and key model had been the prevailing hypothesis in protein kinetics in the time of Emil Fisher in 1894. However, we now know that proteins (and thus enzymes) are flexible and non-fixed structures. In 1958, Daniel Koshland suggested that conformational changes occur upon ligand binding bringing in a new model to enzyme kinetics, Induced Fit. In this model, both the ligand and the protein change their conformations allowing for tighter binding of the ligand and higher affinity for different ligands Also, an enzyme is thought to be complementary to the enzyme-substrate complex.
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What is the role of the two conserved histidine residues in both protein structures?
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The proximal Histidine, His93 or His F8, binds to the central iron atom through a coordinate bond stabilizing the porphyrin ring when bound by O2. The distal Histidine, His64 or His E7, is too far away to coordinate with the heme iron, but does interact with any ligand bound to heme. While the book calls this interaction a “hydrogen bond,” this interaction is more correctly defined as a molecular orbital stabilization. This MO stabilization is the reason why CO adopts a slight angle when bound myoglobin iron, thus weakening CO’s binding to it. The distal His93 (F8) is necessary for the cooperativity of subunits within hemoglobin, because it causes the conformational change in which the alpha1-beta2 (and a2-b1) subunits slide past each other - narrowing the central pore thus creating a proper microenvironment for the other heme-containing/ O2 binding crevices (active sites). |
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Describe the reversible binding of ligand to protein, base your answer on the terms Ka and Kd? Looking at Table 5-1 might be helpful.
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Reversible binding in ligand-protein interactions is necessary for when a protein must not only bind its ligand but also release it at some other point in a molecular pathway. This reversibility can be described by a simple equilibrium expression P+L ⇔ PL (with equilibrium arrows) Given that this is an equilibrium expression, it can be defined by a variable Ka, the association constant, such that Ka=[PL]/([P][L]) This constant represents the amount of ligand bound to the protein at equilibrium. The inverse of Ka, Kd ;(Ka (Kd =1/Ka)), is the dissociation constant and, more specifically, the equilibrium constant for the release of the ligand. The Kd can be used to determine the affinity of the protein to a ligand(the lower the Kd, the higher the affinity of protein to ligand). When concentration of ligand is equal to Kd, half of the ligand-binding sites are occupied. So the lower the magnitude of Kd the fewer moles of ligand are required to occupy half of the binding sites of a particular amount of protein.
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Explain how O2 is bound into the Mb cleft. Is it the same for Hb? Include the oxidation/reduction of Fe as an e- donor in your answer.
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Mb is a single-subunit protein - it does not have other subunits to interact with and therefore O2 binding follows a rectangular hyperbolic curve and is thus best suitable for ligand storing. Meanwhile, Hb is a tetrameric protein with 2 types of subunits (2 polypeptides each) - there exist interactions between each and can be found in two states (T and R). This means that O2 can bind a subunit in either of the two states and this binding is cooperative, meaning binding to one subunit leads to increased affinity of adjacent subunits to O2. This results in a sigmoidal O2 binding curve, such as that represented by the Hill plot. When O2 is bound to heme in the Mb cleft, the surrounding protein structure of Mb interacts with the binding site through sterics, orbital interactions and rotational interference from the His E7, and fluid changes in protein structure, “breathing.” Although Hb exists as a quaternary cooperatively binding protein, the way in which it binds oxygen to its cleft is very similar to that of Mb.
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Describe the structural and amino acid sequence homology of Mb and Hb (α and β peptides).
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All three molecules share the a heme group and 27 similar amino acids in their primary sequence. While the other amino acids in the primary sequence differ, the functional amino acids, for example the proximal and distal histidines, maintain the same general arrangement so that they can maintain the same functionality in binding O2. The other amino acids are mainly used in ensuring the proper arrangement of the functional amino acids and other active subunits. Myoglobin is a typical globular protein consisting of 153 amino acids; it is made up of 8 right handed alpha-helical structures that are connected by bends. Hemoglobin is a roughly spherical structure with 4 subunits, all relatively similar to myoglobin, the subunits are labeled alpha1, alpha2, beta1, beta2. Alpha1 and beta2 bond goes through the most conformational changes during O2 binding. |
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Explain the R and T states of conformation of Hb.
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Hemoglobin subunits exist in two states: R and T. It has a significantly higher affinity for hemoglobin in the R state due to increased stability. When oxygen is absent, the T state is more stable and is thus the predominant conformation of deoxyhemoglobin. T and R originally denoted “tense” and “relaxed,” respectively, because the T state is stabilized by a greater number of ion pairs, many of which lie at the α1β2 (and α2β1) interface (Fig. 5–9). The binding of O2 to a hemoglobin subunit in the T state triggers a change in conformation to the R state. When the entire protein undergoes this transition, the structures of the individual subunits change little, but the αβ subunit pairs slide past each other and rotate, narrowing the pocket between the β subunits (Fig. 5–10). In this process, some of the ion pairs that stabilize the T state are broken and some new ones are formed. In the T state, the porphyrin is slightly puckered, causing the heme iron to protrude somewhat on the proximal His (His F8) side. The binding of O2 causes the heme to assume a more planar conformation, shifting the position of the proximal His and the attached F helix (Fig. 5–11). These changes lead to adjustments in the ion pairs at the α1β2 and α2β1 interface.
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How do the 4 subunits of Hb bind together to form the quaternary of the hemoglobin molecule.
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The quaternary structure of hemoglobin features strong interactions between unlike subunits. The α1β1 (and its α2β2 counterpart) involves more than 30 residues, and its interaction is sufficiently strong that although mild treatment of hemoglobin with urea tends to cause the tetramer to disassemble into αβ dimers, these dimers remain intact. The α1β2 (and α2β1) interface involves 19 residues (Fig. 5–8). Hydrophobic interactions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs (sometimes referred to as salt bridges), whose importance is discussed below.
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Explain cooperativity in terms of the binding of O2 to the Hb molecule. In the answer you should refer to the Hill plot data in Figure 5-14. Compare the binding curve of Mb and the binding curve of Hb.
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The slope of a Hill plot is therefore denoted by nH, the Hill coefficient, which is a measure of the degree of cooperativity. If nH equals 1, ligand binding is not cooperative, a situation that can arise even in a multisubunit protein if the subunits do not communicate. An nH of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for nH is reached when nH =n. In this case the binding would be completely cooperative: all binding sites on the protein would bind ligand simultaneously, and no protein molecules partially saturated with ligand would be present under any conditions. This limit is never reached in practice, and the measured value of nH is always less than the actual number of ligand-binding sites in the protein. An nH of less than 1 indicates negative cooperativity, in which the binding of one molecule of ligand impedes the binding of others. Well-documented cases of negative cooperativity are rare. Hb does this by undergoing a transition from the low-affinity T state to the high-affinity R state as more O2 molecules are bound. The first oxygen binds weakly to the T state subunit of deoxyhemoglobin but its binding leads to conformational changes that are communicated to adjacent subunits, making it easier for additional oxygen to be bound. In effect, the T to R transition occurs more readily in the second subunit than the first. The 4th subunit is already in its R state. |
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Compare the Sequential vs. Concert models of ligand binding.
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The concerted model assumes that the subunits of a cooperatively binding protein are functionally identical, that each subunit can exist in (at least) two conformations, and that all subunits undergo the transition from one conformation to the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium. The ligand can bind to either conformation, but binds each with different affinity. Successive binding of ligand molecules to the low-affinity conformation (which is more stable in the absence of ligand) makes a transition to the high-affinity conformation more likely. In the second model, the sequential model (Fig. 5–15b), proposed in 1966 by Daniel Koshland and colleagues, 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, as well as the binding of a second ligand molecule, more likely. There are more potential intermediate states in this model than in the concerted model. The two models are not mutually exclusive; the concerted model may be viewed as the “all-or-none” limiting case of the sequential model. Actual Kd nature of hemoglobin is between concerted and sequential models of O2 binding. |
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. At the molecular level what happens to the binding of the peptides following the binding of the first O2 molecule?
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Cooperative binding of a ligand to a multimeric protein, such as we observe with the binding of O2 to hemoglobin, is a form of allosteric binding often observed in multimeric proteins. The binding of one ligand affects the affinities of any remaining unfilled binding sites, and O2 can be considered as both a ligand and an activating homotropic modulator. There is only one binding site for O2 on each subunit, so the allosteric effects giving rise to cooperativity are mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions. A sigmoid binding curve is diagnostic of cooperative binding. It permits a much more sensitive response to ligand concentration and is important to the function of many multi-subunit proteins. Cooperative conformational changes depend on variations in the structural stability of different parts of a protein. The binding sites of an allosteric protein typically consist of stable segments in proximity to relatively unstable segments, with the latter capable of frequent changes in conformation or disorganized motion (Fig. 5–13). When a ligand binds, the moving parts of the protein’s binding site may be stabilized in a particular conformation, affecting the conformation of adjacent polypeptide subunits. If the entire binding site were highly stable, then few structural changes could occur in this site or be propagated to other parts of the protein when a ligand binds. |
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How does 2,3bisphosphoglycerate stabilize the deoxy-Hb. How is it a heterotropic allosteric modulator? Us the data in Figure 5-17 in your answer.
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BPG is present in relatively high concentrations in erythrocytes. When hemoglobin is isolated, it contains substantial amounts of bound BPG, which can be difficult to remove completely. In fact, the O2-binding curves for hemoglobin that we have examined to this point were obtained in the presence of bound BPG. 2,3-Bisphospho- glycerate is known to greatly reduce the affinity of hemoglobin for oxygen—there is an inverse relationship between the binding of O2 and the binding of BPG. We can therefore describe another binding process for hemoglobin: BPG binds at a site distant from the oxygen-binding site and regulates the O2-binding affinity of hemoglobin in relation to the pO2 in the lungs. BPG plays an important role in the physiological adaptation to the lower pO2 available at high altitudes. For a healthy human strolling by the ocean, the binding of O2 to hemoglobin is regulated such that the amount of O2 delivered to the tissues is equivalent to nearly 40% of the maximum that could be carried by the blood (Fig. 5–17). Imagine that this person is quickly transported to a mountainside at an altitude of 4,500 meters, where the pO2 is considerably lower. The delivery of O2 to the tissues is now reduced. However, after just a few hours at the higher altitude, the BPG concentration in the blood has begun to rise, leading to a decrease in the affinity of hemoglobin for oxygen. This adjustment in the BPG level has only a small effect on the binding of O2 in the lungs but a considerable effect on the release of O2 in the tissues. As a result, the delivery of oxygen to the tissues is re- stored to nearly 40% of that which can be transported by the blood. The situation is reversed when the person returns to sea level. The BPG concentration in erythrocytes also increases in people suffering from hypoxia, lowered oxygenation of peripheral tissues due to in- adequate functioning of the lungs or circulatory system.The site of BPG binding to hemoglobin is the cavity between the subunits in the T state (Fig. 5–18). This cavity is lined with positively charged amino acid residues that interact with the n2egatively charged groups of BPG. Unlike O2, only one molecule of BPG is bound to each hemoglobin tetramer. BPG lowers hemoglobin’s affinity for oxygen by stabilizing the T state. The transition to the R state narrows the binding pocket for BPG, precluding BPG binding. In the absence of BPG, hemoglobin is converted to the R state more easily. A heterotropic allosteric modulator is a regulatory molecule that is not also the enzyme's substrate. It may be either an activator or an inhibitor of the enzyme. BPG affects binding affinity hemoglobin has for O. |
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How does Hb transport CO2 and H+?
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In addition to carrying nearly all the oxygen required by cells from the lungs to the tissues, hemoglobin carries two end products of cellular respiration— H+ and CO2— from the tissues to the lungs and the kidneys, where they are excreted. The CO2, produced by oxidation of organic fuels in mitochondria, is hydrated to form bicarbonate: This reaction is catalyzed by carbonic anhydrase, an enzyme particularly abundant in erythrocytes. Carbon dioxide is not very soluble in aqueous solution, and bubbles of CO2 would form in the tissues and blood if it were not converted to bicarbonate. The hydration of CO2 results in an increase in the H+ concentration (a decrease in pH) in the tissues. The binding of oxygen by hemoglobin is profoundly influenced by pH and CO2 concentration, so the interconversion of CO2 and bicarbonate is of great importance to the regulation of oxygen binding and release in the blood. Hemoglobin transports about 40% of the total H+ and 15% to 20% of the CO2 formed in the tissues to the lungs and the kidneys. (The remainder of the H+ is absorbed by the plasma’s bicarbonate buffer; the remainder of the CO2 is transported as dissolved HCO3- and CO2.) The binding of H+ and CO2 is inversely related to the binding of oxygen. At the relatively low pH and high CO2 concentration of peripheral tissues, the affinity of hemoglobin for oxygen decreases as H+ and CO2 are bound, and O2 is released to the tissues. Conversely, in the capillaries of the lung, as CO2 is excreted |
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The antibody molecule (Immunoglobulin) has several domains explain the domains and their functions.
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Immunoglobulin G (IgG) is the major class of antibody molecule and one of the most abundant proteins in the blood serum. IgG has four polypeptide chains: two large ones, called heavy chains, and two light chains, linked by noncovalent and disulfide bonds into a complex of MM 150,000 g/mol. The heavy chains of an IgG molecule interact at one end, then branch to interact separately with the light chains, forming a Y-shaped molecule (Fig. 5–23). At the “hinges” separating the base of an IgG molecule from its branches, the immunoglobulin can be cleaved with proteases. Cleavage with the protease papain liberates the basal fragment, called Fc because it usually crystallizes readily, and the two branches, called Fab, the antigen-binding fragments. Each branch has a single antigen-binding site.The fundamental structure of immunoglobulins was first established by Gerald Edelman and Rodney Porter. Each chain is made up of identifiable domains; some are constant in sequence and structure from one IgG to the next, others are variable. The constant domains have a characteristic structure known as the immunoglobulin fold, a well-conserved structural motif in the all β class of proteins (Chapter 4). There are three of these constant domains in each heavy chain and one in each light chain. The heavy and light chains also have one variable domain each, in which most of the variability in amino acid residue sequence is found. The variable domains associate to create the antigen-binding site |
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What are the CDR regions of the antibody molecule?
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CDR stands for the complementary determining region of the antibody. In the amino acid sequence of a variable domain of an antigen receptor there are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively. Since the antigen receptors are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen receptor that can collectively come into contact with the antigen. A single antibody molecule has two antigen receptors and therefore contains twelve CDRs. Sixty CDRs can be found on a pentameric IgM molecule. Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, all of diversity (D, heavy chains only) and joining (J) regions.[1] CDR3 is the most variable. They are associated with the Fab region. |
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Explain the hypervariable region.
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Since most sequence variation associated with immunoglobulins and T cell receptors is associated with CDRs, these regions are sometimes referred to as hypervariable regions.[2] Among these, CDR3 shows the greatest variability as it is encoded by a recombination of the VJ in the case of a light chain region and VDJ in the case of heavy chain regions. Since most sequence variation associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable region |
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. Explain what is an antigen determinant (epitope)?
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Some properties of the interactions between antibodies or T-cell receptors and the molecules they bind are unique to the immune system, and a specialized lexicon is used to describe them. Any molecule or pathogen capable of eliciting an immune response is called an antigen. An antigen may be a virus, a bacterial cell wall, or an individual protein or other macromolecule. A complex antigen may be bound by a number of different antibodies. An individual antibody or T-cell receptor binds only a particular molecular structure within the antigen, called its antigenic determinant or epitope. |
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Explain the antibody binding site in terms of the Induced Fit model of protein for the binding of antigen. |
The binding specificity of an antibody is determined by the amino acid residues in the variable domains of its heavy and light chains. Many residues in these domains are variable, but not equally so. Some, particularly those lining the antigen-binding site, are hypervariable— especially likely to differ. Specificity is conferred by chemical complementarity between the antigen and its specific binding site, in terms of shape and the location of charged, nonpolar, and hydrogen-bonding groups. For example, a binding site with a negatively charged group may bind an antigen with a positive charge in the complementary position. In many instances, complementarity is achieved interactively as the structures of antigen and binding site are influenced by each other during the approach of the ligand. Conformational changes in the antibody and/or the antigen then occur that allow the complementary groups to interact fully. This is an example of induced fit |
