第四章 蛋白质的生物学功能

The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances.

A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands.

Proteins are flexible. Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the protein. Changes in conformation may also be more dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a protein’s function.

The binding of a protein and a ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit.

Myoglobin and hemoglobin may be the most-studied and bestunderstood proteins. They were the first proteins for which threedimensional structures were determined, and these two molecules illustrate almost every aspect of that critical biochemical process: the reversible binding of a ligand to a protein. This classic model of protein function tells us a great deal about how proteins work.

Oxygen is poorly soluble in aqueous solutions (see Table 2-2) and cannot be carried to tissues in sufficient quantity if it is simply dissolved in blood serum. Also, diffusion of oxygen through tissues is ineffective over distances greater than a few millimeters. The evolution of larger, multicellular animals depended on the evolution of proteins that could transport and store oxygen. However, none of the amino acid side chains in proteins are suited for the reversible binding of oxygen molecules. This role is filled by certain transition metals, among them iron and copper, that have a strong tendency to bind oxygen. Multicellular organisms exploit the properties of metals, most commonly iron, for oxygen transport.

Iron used in cells is therefore bound in forms that sequester it and/or make it less reactive. In multicellular organisms, iron is often incorporated into a protein-bound prosthetic group called heme (or haem). (Recall from Chapter 3 that a prosthetic group is a compound permanently associated with a protein that contributes to the protein’s function.) Heme is found in many oxygen-transporting proteins, as well as in some proteins, such as the cytochromes, that participate in oxidationreduction (electron-transfer) reactions (Chapter 19).

Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2+) state (Fig. 5-1). 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.

Iron in the Fe2+ state binds oxygen reversibly; in the Fe3+ state it does not bind oxygen. The structure of heme and the globins to which it is bound represents an evolutionary adaptation to prevent Fe2+ oxidation. Free heme molecules (heme not bound to protein) leave Fe2+ with two “open” coordination bonds. Simultaneous reaction of one O2 molecule with two free heme molecules (or two free Fe2+) can result in irreversible conversion of Fe2+ to Fe3+. The coordinated nitrogen atoms (which have an electron-donating character) help prevent conversion of the heme iron to the ferric (Fe3+) state. In hemecontaining proteins, this reaction is further prevented by sequestering each heme deep within the protein structure. Thus, access to the two open coordination bonds is restricted. In the globins that will shortly become the focus of our narrative, one of these two coordination bonds is occupied by a side-chain nitrogen of a highly conserved His residue referred to as the proximal His.

The other is the binding site for molecular oxygen (O2) (Fig. 5-2). When oxygen binds, the electronic properties of heme iron change; this accounts for the change in color from the dark purple of oxygen-depleted venous blood to the bright red of oxygen-rich arterial blood. Some small molecules, such as carbon monoxide (CO) and nitric oxide (NO), coordinate to heme iron with greater affinity than does O2.

The globins are a widespread family of proteins. Globins are commonly found in eukaryotes of all classes, as well as in archaea and bacteria. All evolved from a common ancestral protein. Their tertiary structure is highly conserved, made up of eight α-helical segments connected by bends. This folding pattern, illustrated by myoglobin (Fig. 5-3), constitutes a structural motif known as the globin fold. The primary amino acid sequence of globins is less conserved, well reflecting both the ancient origins of this protein family and the evolutionary relatedness of species from which globin comparisons might be made.

Most globins function in oxygen transport or storage, although some play a role in the sensing of oxygen, nitric oxide, or carbon monoxide. The simple nematode worm Caenorhabditis elegans has genes encoding 33 different globins. In humans and other mammals, there are at least four kinds of globins. The monomeric myoglobin facilitates oxygen diffusion in muscle tissue.

The tetrameric hemoglobin is responsible for oxygen transport in the bloodstream. The monomeric neuroglobin is expressed largely in neurons and helps to protect the brain from hypoxia (low oxygen) or ischemia (restricted blood supply). Cytoglobin, another monomeric globin, is found at high concentrations in the walls of blood vessels, where it functions to regulate levels of nitric oxide, a localized signal for muscle relaxation (see Box 12-2).

Any detailed discussion of protein function inevitably involves protein structure. Myoglobin (Mr 16,700; abbreviated Mb) is a single polypeptide of 153 amino acid residues with one molecule of heme (Fig. 5-3). About 78% of the amino acid residues in the protein are found in the eight α helices typical of the globin fold, named A through H.

An individual amino acid residue is designated either by its position in the amino acid sequence or by its location in the sequence of a particular α-helical segment. For example, the His residue coordinated to the heme in myoglobin — the proximal His — is His93 (the ninety-third residue from the amino-terminal end of the myoglobin polypeptide sequence) and is also called His F8 (the eighth residue in α helix F).

The function of myoglobin depends on the protein’s ability not only to bind oxygen but also to release it when and where it is needed. Function in biochemistry often revolves around a reversible protein-ligand interaction of this type. A quantitative description of this interaction is a central part of many biochemical investigations.

where ka and kd are rate constants (more on these below). The term Ka is an association constant (not to be confused with the Ka that denotes an acid dissociation constant; p. 57) that describes the equilibrium between the complex and the unbound components of the complex. The association constant provides a measure of the affinity of the ligand L for the protein. Ka has units of M−1; a higher value of Ka corresponds to a higher affinity of the ligand for the protein.

The equilibrium term Ka is also equivalent to the ratio of the rates of the forward (association) and reverse (dissociation) reactions that form the PL complex. The association rate is described by the rate constant ka, and dissociation is described by the rate constant kd. As discussed further in the next chapter, rate constants are proportionality constants, describing the fraction of a pool of reactant that reacts in a given amount of time. When the reaction involves one molecule, such as the dissociation reaction PL → P + L, the reaction is first order and the rate constant (kd) has units of reciprocal time (s−1). When the reaction involves two molecules, such as the association reaction P + L → PL, it is called second order, and its rate constant (ka) has units of M−1 s−1.

When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not appreciably change the concentration of free (unbound) ligand — that is, [L] remains constant. This condition is broadly applicable to most ligands that bind to proteins in cells, and it simplifies our description of the binding equilibrium.

The value of Ka can be determined from a plot of Y versus the concentration of free ligand, [L] (Fig. 5-4a). Any equation of the form x = y/(y + z) describes a hyperbola, and Y is thus found to be a hyperbolic function of [L]. The fraction of ligand-binding sites occupied approaches saturation asymptotically as [L] increases. The [L] at which half of the available ligand-binding sites are occupied (that is, Y = 0.5) corresponds to 1/Ka.

It is more common (and intuitively simpler), however, to consider the dissociation constant, Kd, which is the reciprocal of Ka (Kd = 1/Ka) and has units of molar concentration (M). Kd is the equilibrium constant for the release of ligand. The relevant expressions change to

The mathematics can be reduced to simple statements: Kd is equivalent to the molar concentration of ligand at which half of the available ligand-binding sites are occupied. At this point, the protein is said to have reached half-saturation with respect to ligand binding. The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus the lower the value of Kd. Some representative dissociation constants are given in Table 5-1; the scale shows typical ranges for dissociation constants found in biological systems.

A reported dissociation constant is valid only for the particular solution conditions under which it was measured. Kd values for a protein-ligand interaction can be altered, sometimes by several orders of magnitude, by changes in the solution’s salt concentration, pH, or other variables.

We can determine the dissociation constants by inspecting the graph. Because Y represents the fraction of binding sites occupied by ligand, the concentration of ligand at which half the binding sites are occupied — that is, the point where the binding curve crosses the line where Y = 0.5 — is the dissociation constant. For A, Kd = 2μM; for B, Kd = 6μM. Because A is half-saturated at a lower [L], it has a higher affinity for the ligand.

The binding of oxygen to myoglobin follows the patterns discussed above. However, because oxygen is a gas, we must make some minor adjustments to the equations so that laboratory experiments can be carried out more conveniently. We first substitute the concentration of dissolved oxygen for [L] in Equation 5-8 to give

In experiments using oxygen as a ligand, it is the partial pressure of oxygen (pO2) in the gas phase above the solution that is varied, because this is easier to measure than the concentration of oxygen dissolved in the solution. The concentration of a volatile substance in solution is always proportional to the local partial pressure of the gas. So, if we define the partial pressure of oxygen at [O2]0.5 as P50, substitution in Equation 5-10 gives

The binding of a ligand to a protein is rarely as simple as the above equations would suggest. The interaction is greatly affected by protein structure and is often accompanied by conformational changes. For example, the specificity with which heme binds its various ligands is altered when the heme is a component of myoglobin.

For free heme, the tighter binding by CO reflects differences in the way the orbital structures of CO and O2 interact with Fe2+. Those same orbital structures lead to different binding geometries for CO and O2 when they are bound to heme (Fig. 5-5a, b). The change in relative affinity of CO and O2 for heme when the heme is bound to a globin is mediated by the globin structure.

When heme is bound to myoglobin, its affinity for O2 is selectively increased by the presence of the distal His (His64, or His E7 in myoglobin). The Fe-O2 complex is much more polar than the Fe-CO complex. A partial negative charge is distributed across the oxygen atoms in the bound O2 due to partial oxidation of the interacting iron atom. A hydrogen bond between the imidazole side chain of His E7 and the bound O2 stabilizes this polar complex electrostatically (Fig. 5-5c). The affinity of myoglobin for O2 is thus selectively increased by a factor of about 500; there is no such effect for Fe-CO binding in myoglobin. Consequently, the 20,000-fold stronger binding affinity of free heme for CO compared with O2 declines to approximately 40-fold for heme embedded in myoglobin.

This favorable electrostatic effect on O2 binding is even more dramatic in some invertebrate hemoglobins, where two groups in the binding pocket can form strong hydrogen bonds with O2, causing the heme group to bind O2 with greater affinity than CO. This selective enhancement of O2 affinity in globins is physiologically important: it helps prevent poisoning by the CO that is generated in small amounts from metabolism and that is sometimes generated in larger amounts by our industrial-age environment.

The binding of O2 to the heme in myoglobin also depends on molecular motions, or “breathing,” in the protein structure. The heme molecule is deeply buried in the folded polypeptide, with limited direct paths for oxygen to move from the surrounding solution to the ligand-binding site. If the protein were rigid, O2 could not readily enter or leave the heme pocket.

However, rapid molecular flexing of the amino acid side chains produces transient cavities in the protein structure, and O2 makes its way in and out by moving through these cavities. Computer simulations of rapid structural fluctuations in myoglobin suggest that there are many such pathways. The distal His acts as a gate to control access to one major pocket near the heme iron. Rotation of that His residue to open and close the pocket occurs on a nanosecond (10−9 s) time scale.

Nearly all the oxygen carried by whole blood in animals is bound and transported by hemoglobin in erythrocytes (red blood cells). Normal human erythrocytes are small (6 to 9 μm in diameter), biconcave disks. They are formed from precursor stem cells called hemocytoblasts. In the maturation process, the stem cell produces daughter cells that form large amounts of hemoglobin and then lose their organelles — nucleus, mitochondria, and endoplasmic reticulum. Erythrocytes are thus incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days.

Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 54b), is relatively insensitive to small changes in the concentration of dissolved oxygen, so it functions well as an oxygen-storage protein. Hemoglobin, with its multiple subunits and O2-binding sites, is better suited to oxygen transport. As we shall see, interactions between the subunits of a multimeric protein can permit a highly sensitive response to small changes in ligand concentration.

Hemoglobin (Mr 64,500; abbreviated Hb) is roughly spherical, with a diameter of nearly 5.5 nm. It is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. Adult hemoglobin contains two types of globin: two α chains (141 residues each) and two β chains (146 residues each). The three-dimensional structures of the two types of subunits are very similar to each other and to myoglobin (Fig. 5-6), reflecting their evolution within the larger globin superfamily. However, fewer than half of the amino acid residues are identical in the polypeptide sequences of the α and β subunits, and only 27 are identical in the three polypeptides (Fig. 5-7). The helix-naming convention described for myoglobin is also applied to the hemoglobin polypeptides, except that the α subunit lacks the short D helix. The heme-binding pocket is made up largely of the E and F helices in each of the subunits.

The quaternary structure of hemoglobin features strong interactions between unlike subunits. The α1β1 interface (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 disassemble the tetramer into αβ dimers, these dimers remain intact. The α1β2 (and α2β1) interface involves 19 residues (Fig. 5-8). The hydrophobic effect plays the major role in stabilizing these interfaces, but there are also many hydrogen bonds and a few ion pairs (or salt bridges), whose importance is discussed below.

X-ray analysis has revealed two major conformations of hemoglobin: the R state and the T state. Although oxygen binds to hemoglobin in either state, it has a significantly higher affinity for hemoglobin in the R state. Oxygen binding stabilizes the R state.

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.

Max Perutz proposed that the T → R transition is triggered by changes in the positions of key amino acid side chains surrounding the heme. 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 interface.

Hemoglobin must bind oxygen efficiently in the lungs, where the pO2 is about 13.3 kPa, and it must release oxygen in the tissues, where the pO2 is about 4 kPa. Myoglobin, or any protein that binds oxygen with a hyperbolic binding curve, would be ill-suited to this function, for the reason illustrated in Figure 5-12.

Hemoglobin solves the problem by undergoing a transition from a low-affinity state (the T state) to a high-affinity state (the R state) as more O2 molecules are bound. As a result, hemoglobin has a hybrid S-shaped, or sigmoid, binding curve for oxygen (Fig. 5-12). A single-subunit protein with a single ligand-binding site cannot produce a sigmoid binding curve — even if binding elicits a conformational change — because each molecule of ligand binds independently and cannot affect ligand binding to another molecule.

In contrast, O2 binding to individual subunits of hemoglobin can alter the affinity for O2 in adjacent subunits. The first molecule of O2 that interacts with deoxyhemoglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to conformational changes that are communicated to adjacent subunits, making it easier for additional molecules of O2 to bind. In effect, the T → R transition occurs more readily in the second subunit once O2 is bound to the first subunit. The last (fourth) O2 molecule binds to a heme in a subunit that is already in the R state, and hence it binds with much higher affinity than the first molecule.

An allosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein. The term “allosteric” derives from the Greek allos, “other,” and stereos, “solid” or “shape.”

modulators for allosteric proteins may be either inhibitors or activators. When the normal ligand and modulator are identical, the interaction is termed homotropic. When the modulator is a molecule other than the normal ligand, the interaction is heterotropic. Some proteins have two or more modulators and therefore can have both homotropic and heterotropic interactions.

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. 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 multisubunit proteins. The principle of allostery extends readily to regulatory enzymes, as we shall see in Chapter 6.

Equation 5-16 is the Hill equation, and a plot of log [Y/(1 − Y)] versus log [L] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n. However, the experimentally determined slope actually reflects not the number of binding sites but the degree of interaction between them. The slope of a Hill plot is therefore denoted by nH, the Hill coefficient, which is a measure of the degree of cooperativity.

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.

The first model was proposed by Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux in 1965, and is called the MWC model or the concerted model (Fig. 5-14a). 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 it binds much more tightly to the R state. Successive binding of ligand molecules to the low-affinity T state (which is more stable in the absence of ligand) gradually shifts the equilibrium to favor the R state.

In the second model, the sequential model (Fig. 5-14b), 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 more likely, and makes the binding of a second ligand molecule more likely as well. 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. Unfortunately, the two models have proven very difficult to distinguish experimentally. In Chapter 6 we use these models to investigate allosteric enzymes.

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. As you can see from the reaction catalyzed by carbonic anhydrase, 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.

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 and the blood pH consequently rises, the affinity of hemoglobin for oxygen increases and the protein binds more O2 for transport to the peripheral tissues. This effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect, after Christian Bohr, the Danish physiologist (and father of physicist Niels Bohr) who discovered it in 1904.

where HHb+ denotes a protonated form of hemoglobin. This equation tells us that the O2-saturation curve of hemoglobin is influenced by the H+ concentration (Fig. 5-15). Both O2 and H+ are bound by hemoglobin, but with inverse affinity. When the oxygen concentration is high, as in the lungs, hemoglobin binds O2 and releases protons. When the oxygen concentration is low, as in the peripheral tissues, H+ is bound and O2 is released.

Oxygen and H+ are not bound at the same sites in hemoglobin. Oxygen binds to the iron atoms of the hemes, whereas H+ binds to any of several amino acid residues in the protein. A major contribution to the Bohr effect is made by His146 (His HC3) of the β subunits. When protonated, this residue forms one of the ion pairs — to Asp94 (Asp FG1) — that helps stabilize deoxyhemoglobin in the T state (Fig. 5-9). Protonation of the amino-terminal residues of the α subunits, certain other His residues, and perhaps other groups has a similar effect.

When the concentration of carbon dioxide is high, as in peripheral tissues, some CO2 binds to hemoglobin and the affinity for O2 decreases, causing its release. Conversely, when hemoglobin reaches the lungs, the high oxygen concentration promotes binding of O2 and release of CO2. It is the capacity to communicate ligand-binding information from one polypeptide subunit to the others that makes the hemoglobin molecule so beautifully adapted to integrating the transport of O2, CO2, and H+ by erythrocytes.

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 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.

BPG is important in the physiological adaptation to the lower pO2 at high altitudes. For a healthy human at sea level, the binding of O2 to hemoglobin is regulated such that the amount of O2 delivered to the tissues is nearly 40% of the maximum that could be carried by the blood (Fig. 5-16). Imagine that this person is suddenly transported from sea level to 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 it has a considerable effect on the release of O2 in the tissues.

As a result, the delivery of oxygen to the tissues is restored to nearly 40% of the O2 that 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 inadequate functioning of the lungs or circulatory system.

The site of BPG binding to hemoglobin is the cavity between the β subunits in the T state (Fig. 5-17). This cavity is lined with positively charged amino acid residues that interact with the negatively 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. In the absence of BPG, hemoglobin is primarily present in the R state, where it binds O2 efficiently in the lungs but fails to release it in the tissues.

Regulation of oxygen binding to hemoglobin by BPG has an important role in fetal development. Because a fetus must extract oxygen from its mother’s blood, fetal hemoglobin must have greater affinity than the maternal hemoglobin for O2. The fetus synthesizes γ subunits rather than β subunits, forming α2γ2 hemoglobin.

Carbon monoxide (CO), a colorless, odorless gas, is one of the most common known causes of death due to poisoning (in countries where such records are kept). CO has an approximately 250-fold greater affinity for hemoglobin than does oxygen. Consequently, relatively low levels of CO can have substantial and tragic effects. When CO combines with hemoglobin, the complex is referred to as carboxyhemoglobin, or COHb.

In an average, healthy individual, 1% or less of the total hemoglobin is complexed as COHb. Since CO is a product of tobacco smoke, many smokers have COHb levels in the range of 3% to 8% of total hemoglobin, and the levels can rise to 15% for chain-smokers. COHb levels equilibrate at 50% in people who breathe air containing 570 ppm of CO for several hours. Reliable methods have been developed that relate CO content in the atmosphere to COHb levels in the blood (Fig. 1). CO levels in the home of the Florida victim exceeded OSHA-mandated limits by more than 30-fold.

How is a human affected by COHb? At levels of less than 10% of total hemoglobin, symptoms are rarely observed. At 15%, the individual experiences mild headaches. At 20% to 30%, the headache is severe and is generally accompanied by nausea, dizziness, confusion, disorientation, and some visual disturbances; these symptoms are generally reversed if the individual is treated with oxygen. At COHb levels of 30% to 50%, the neurological symptoms become more severe, and at levels near 50%, the individual loses consciousness and can sink into coma. Respiratory failure may follow. With prolonged exposure, some damage becomes permanent. Death normally occurs when COHb levels rise above 60%.

It may seem surprising that the loss of half of one’s hemoglobin to COHb can prove fatal — we know that people with any of several anemic conditions manage to function reasonably well with half the usual complement of active hemoglobin. However, the binding of CO to hemoglobin does more than remove protein from the pool available to bind oxygen. It also affects the affinity of the remaining hemoglobin subunits for oxygen. As CO binds to one or two subunits of a hemoglobin tetramer, the affinity for O2 is increased substantially in the remaining subunits (Fig. 2). Thus, a hemoglobin tetramer with two bound CO molecules can efficiently bind O2 in the lungs — but it releases very little of it in the tissues. Oxygen deprivation in the tissues rapidly becomes severe.

When CO poisoning is suspected, rapid removal of the person from the CO source is essential, but this does not always result in rapid recovery. When an individual is moved from the CO-polluted site to a normal, outdoor atmosphere, O2 begins to replace the CO in hemoglobin — but the COHb level drops only slowly. The half-time is 2 to 6.5 hours, depending on individual and environmental factors. If 100% oxygen is administered with a mask, the rate of exchange can be increased about fourfold

The hereditary human disease sickle cell anemia demonstrates strikingly the importance of amino acid sequence in determining the secondary, tertiary, and quaternary structures of globular proteins, and thus their biological functions. Almost 500 genetic variants of hemoglobin are known to occur in the human population; all but a few are quite rare. Most variations consist of differences in a single amino acid residue. The effects on hemoglobin structure and function are often minor but can sometimes be extraordinary. Each hemoglobin variation is the product of an altered gene. Variant genes are called alleles. Because humans generally have two copies of each gene, an individual may have two copies of one allele (thus being homozygous for that gene) or one copy of each of two different alleles (thus heterozygous).

Sickle cell anemia occurs in individuals who inherit the allele for sickle cell hemoglobin from both parents. The erythrocytes of these individuals are fewer and also abnormal. In addition to an unusually large number of immature cells, the blood contains many long, thin, sickle-shaped erythrocytes (Fig. 5-18).

deoxygenated, it becomes insoluble and forms polymers that aggregate into tubular fibers (Fig. 5-19). Normal hemoglobin (hemoglobin A, or HbA) remains soluble upon deoxygenation. The insoluble fibers of deoxygenated HbS cause the deformed, sickle shape of the erythrocytes, and the proportion of sickled cells increases greatly as blood is deoxygenated.

The altered properties of HbS result from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two β chains. Replacement of the Glu residue by Val creates a “sticky” hydrophobic contact point at position 6 of the β chain, which is on the outer surface of the molecule. These sticky spots cause deoxyHbS molecules to associate abnormally with each other, forming the long, fibrous aggregates characteristic of this disorder.

Sickle cell anemia is life-threatening and painful. People with this disease suffer repeated crises brought on by physical exertion. They become weak, dizzy, and short of breath, and they also experience heart murmurs and an increased pulse rate. The hemoglobin content of their blood is only about half the normal value of 15 to 16 g/100 mL, because sickled cells are very fragile and rupture easily; this results in anemia (“lack of blood”). An even more serious consequence is that capillaries become blocked by the long, abnormally shaped cells, causing severe pain and interfering with normal organ function — a major factor in the early death of many people with the disease.

Investigation into this matter led to the finding that when heterozygous, the allele confers a small but significant resistance to lethal forms of malaria. The heterozygous individuals experience a milder condition called sickle cell trait; only about 1% of their erythrocytes become sickled on deoxygenation. These individuals may live completely normal lives if they avoid vigorous exercise and other stresses on the circulatory system. Natural selection has resulted in an allele population that balances the deleterious effects of the homozygous condition against the resistance to malaria afforded by the heterozygous condition.

Protein function often entails interactions with other molecules. A protein binds a molecule, known as a ligand, at its binding site. Myoglobin contains a heme prosthetic group, which binds oxygen. Heme consists of a single atom of Fe2+ coordinated within a porphyrin. Globins are a specialized family of transport proteins containing heme; most globins store oxygen. Oxygen binds to myoglobin reversibly. Reversible binding of a ligand to a protein can be described quantitatively by a dissociation constant Kd.

protein such as myoglobin, the fraction of binding sites occupied by a ligand is a hyperbolic function of ligand concentration. Proteins may undergo conformational changes when a ligand binds, a process called induced fit. In a multisubunit protein, the binding of a ligand to one subunit may affect ligand binding to other subunits. Ligand binding can be regulated. Hemoglobin transports oxygen in blood. Normal adult hemoglobin has four heme-containing subunits, two α and two β, similar in structure to each other and to myoglobin. Hemoglobin exists in two interchangeable structural states, T and R. The T state is most stable when oxygen is not bound. Oxygen binding promotes transition to the R state.

Oxygen binding to hemoglobin is both allosteric and cooperative. As O2 binds to one binding site, the hemoglobin undergoes conformational changes that affect the other binding sites — an example of allosteric behavior. Conformational changes between the T and R states, mediated by subunit-subunit interactions, result in cooperative binding; this is described by a sigmoid binding curve and can be analyzed by a Hill plot. Two major models have been proposed to explain the cooperative binding of ligands to multisubunit proteins: the concerted model and the sequential model. Hemoglobin also binds H+ and CO2, resulting in the formation of ion pairs that stabilize the T state and lessen the protein’s affinity for O2 (the Bohr effect). Oxygen binding to hemoglobin is also modulated by 2,3bisphosphoglycerate, which binds to and stabilizes the T state.

Sickle cell anemia is a genetic disease caused by a single amino acid substitution (Glu6 to Val6) in each β chain of hemoglobin. The change produces a hydrophobic patch on the surface of the hemoglobin that causes the molecules to aggregate into bundles of fibers. This homozygous condition results in serious medical complications.

a ligand is more often like the hemoglobin binding site for BPG —a cleft in the protein lined with amino acid residues, arranged to make the binding interaction highly specific. Effective discrimination between ligands is the norm at binding sites, even when the ligands have only minor structural differences.

Almost all organisms have an immune system of some type that allows them to respond to challenges presented by environmental pathogens. The emergence of vertebrates about 500 million years ago was accompanied by the evolution of an adaptive immune system based on the generation of large numbers of distinct cell clones, each expressing a protein variant that could recognize and bind to a particular type of chemical structure. The system distinguishes molecular “self” from “nonself” and then destroys what is identified as nonself. In this way, the immune system eliminates viruses, bacteria, and other pathogens and molecules that may pose a threat to the organism.

Immunity is brought about by a variety of leukocytes (white blood cells), including macrophages and lymphocytes, all of which develop from undifferentiated stem cells in the bone marrow. Leukocytes can leave the bloodstream and patrol the tissues, each cell producing one or more proteins capable of recognizing and binding to molecules that might signal an infection.

The immune response consists of two complementary systems, the humoral and cellular immune systems. The humoral immune system (Latin humor, “fluid”) is directed at bacterial infections and extracellular viruses (those found in the body fluids), but it can also respond to individual foreign proteins. The cellular immune system destroys host cells infected by viruses and also destroys some parasites and foreign tissues.

Immunoglobulins bind bacteria, viruses, or large molecules identified as foreign and target them for destruction. Making up 20% of blood protein, the immunoglobulins are produced by B lymphocytes, or B cells, so named because they complete their development in the bone marrow.

The agents at the heart of the cellular immune response are a class of T lymphocytes, or T cells (so called because the latter stages of their development occur in the thymus), known as cytotoxic T cells (TC cells, also called killer T cells). Recognition of infected cells or parasites involves proteins called T-cell receptors on the surface of TC cells. Receptors are proteins, usually found on the outer surface of cells and extending through the plasma membrane, that recognize and bind extracellular ligands, thus triggering changes inside the cell.

In addition to cytotoxic T cells, there are helper T cells (TH cells), whose function it is to produce soluble signaling proteins called cytokines, which include the interleukins. TH cells interact with macrophages. The TH cells participate only indirectly in the destruction of infected cells and pathogens, stimulating the selective proliferation of those TC and B cells that can bind to a particular antigen. This process, called clonal selection, increases the number of immune system cells that can respond to a particular pathogen. A host organism needs time, often days, to mount an immune response against a new antigen, but memory cells permit a rapid response to pathogens previously encountered.

When injected into a person, the vaccine generally does not cause an infection and illness, but it effectively “teaches” the immune system what the viral particles look like, stimulating the production of memory cells. On subsequent infection, these cells can bind to the virus and trigger a rapid immune response.

The importance of TH cells is dramatically illustrated by the epidemic produced by HIV (human immunodeficiency virus), the virus that causes AIDS (acquired immune deficiency syndrome). TH cells are the primary targets of HIV infection; elimination of these cells progressively incapacitates the entire immune system.

Each recognition protein of the immune system, either a T-cell receptor or an antibody produced by a B cell, specifically binds some particular chemical structure, distinguishing it from virtually all others. Humans are capable of producing more than 108 different antibodies with distinct binding specificities. Given this extraordinary diversity, any chemical structure on the surface of a virus or an invading cell will most likely be recognized and bound by one or more antibodies. Antibody diversity is derived from random reassembly of a set of immunoglobulin gene segments through genetic recombination mechanisms that are discussed in Chapter 25 (see Fig. 25-42).

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 several different antibodies. An individual antibody or T-cell receptor binds only a particular molecular structure within the antigen, called its antigenic determinant or epitope.

It would be unproductive for the immune system to respond to small molecules that are common intermediates and products of cellular metabolism. Molecules of Mr < 5,000 are generally not antigenic. However, when small molecules are covalently attached to large proteins in the laboratory, they can be used to elicit an immune response. These small molecules are called haptens. The antibodies produced in response to protein-linked haptens will then bind to the same small molecules in their free form. Such antibodies are sometimes used in the development of analytical tests described later in this chapter or as a specific ligand in affinity chromatography (see Fig. 3-17c).

has four polypeptide chains: two large ones, called heavy chains, and two smaller ones, called light chains, linked by noncovalent and disulfide bonds into a complex of Mr 150,000. 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. 5-20). 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 in the 1960s. 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 sequence is found. The variable domains associate to create the antigenbinding site (Fig. 5-20), allowing formation of an antigen-antibody complex (Fig. 5-21).

In many vertebrates, IgG is but one of five classes of immunoglobulins. Each class has a characteristic type of heavy chain, denoted α, δ, ε, γ, and μ for IgA, IgD, IgE, IgG, and IgM, respectively. Two types of light chain, κ and λ, occur in all classes of immunoglobulins. The overall structures of IgD and IgE are similar to that of IgG. IgM occurs either in a monomeric, membrane-bound form or in a secreted form that is a crosslinked pentamer of this basic structure (Fig. 5-22). IgA, found principally in secretions such as saliva, tears, and milk, can be a monomer, a dimer, or a trimer. IgM is the first antibody to be made by B lymphocytes and the major antibody in the early stages of a primary immune response. Some B cells soon begin to produce IgD (with the same antigen-binding site as the IgM produced by the same cell), but the particular function of IgD is less clear.

The IgG described above is the major antibody in secondary immune responses, which are initiated by a class of B cells called memory B cells. As part of the organism’s ongoing immunity to antigens already encountered and dealt with, IgG is the most abundant immunoglobulin in the blood. When IgG binds to an invading bacterium or virus, it activates certain leukocytes such as macrophages to engulf and destroy the invader, and it also activates some other parts of the immune response.

IgE plays an important role in the allergic response, interacting with basophils (phagocytic leukocytes) in the blood and with histamine-secreting cells called mast cells, which are widely distributed in tissues. This immunoglobulin binds, through its Fc region, to special Fc receptors on the basophils or mast cells. In this form, IgE serves as a receptor for antigen. If antigen is bound, the cells are induced to secrete histamine and other biologically active amines that cause the dilation and increased permeability of blood vessels. These effects on the blood vessels are thought to facilitate the movement of immune system cells and proteins to sites of inflammation.

The binding specificity of an antibody is determined by the amino acid residues in the variable domains of its heavy and light chains. Some of those residues, 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. 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 influence each other as they come closer together. Conformational changes in the antibody and/or the antigen then allow the complementary groups to interact fully. This is an example of induced fit. The complex of a peptide derived from HIV (a model antigen) and an Fab molecule, shown in Figure 5-24, illustrates some of these properties. The changes in structure observed on antigen binding are particularly striking in this example.

A typical antibody-antigen interaction is quite strong, characterized by Kd values as low as 10−10 M (recall that a lower Kd corresponds to a stronger binding interaction; see Table 5-1). The Kd reflects the energy derived from the hydrophobic effect and the various ionic, hydrogen-bonding, and van der Waals interactions that stabilize the binding. The binding energy required to produce a Kd of 10−10 M is about 65 kJ/mol.

The extraordinary binding affinity and specificity of antibodies make them valuable analytical reagents. Two types of antibody preparations are in use: polyclonal and monoclonal. Polyclonal antibodies are those produced by many different B lymphocytes responding to one antigen, such as a protein injected into an animal. Cells in the population of B lymphocytes produce antibodies that bind specific, different epitopes within the antigen.

Thus, polyclonal preparations contain a mixture of antibodies that recognize different parts of the protein. Monoclonal antibodies, in contrast, are synthesized by a population of identical B cells (a clone) grown in cell culture. These antibodies are homogeneous, all recognizing the same epitope.

The specificity of antibodies has practical uses. In a versatile analytical technique, an antibody is attached to a reagent that makes it easy to detect (Fig. 5-25). When the antibody binds the target protein, the label reveals the presence of the protein in a solution or its location in a gel, or even in a living cell (Fig. 5-25a).

In one application of this technique, an immunoblot or Western blot assay (Fig. 5-26b), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. After washing, the membrane is treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms only along the band containing the protein of interest. Immunoblotting allows the detection of a minor component in a sample and provides an approximation of its molecular weight.

The immune response is mediated by interactions among an array of specialized leukocytes and their associated proteins. T lymphocytes produce T-cell receptors. B lymphocytes produce immunoglobulins. Humans have five classes of immunoglobulins, each with different biological functions. The most abundant class is IgG, a Y-shaped protein with two heavy chains and two light chains.

The domains near the upper ends of the Y are hypervariable within the broad population of IgGs and form two antigen-binding sites. A given immunoglobulin generally binds to only a part, called the epitope, of a large antigen. Binding often involves a conformational change in the IgG, an induced fit to the antigen. The exquisite binding specificity of immunoglobulins is exploited in analytical techniques such as immunoblotting.

Organisms move. Cells move. Organelles and macromolecules within cells move. Most of these movements arise from the activity of a fascinating class of protein-based molecular motors. Fueled by chemical energy, usually derived from ATP, large aggregates of motor proteins undergo cyclic conformational changes that accumulate into a unified, directional force — the tiny force that pulls apart chromosomes in a dividing cell, and the immense force that levers a pouncing, quarter-ton jungle cat into the air.

Motor proteins underlie the migration of organelles along microtubules, the motion of eukaryotic and bacterial flagella, and the movement of some proteins along DNA. Here, we focus on the well-studied example of the contractile proteins of vertebrate skeletal muscle as a paradigm for how proteins translate chemical energy into motion.

The contractile force of muscle is generated by the interaction of two proteins, myosin and actin. These proteins are arranged in filaments that undergo transient interactions and slide past each other to bring about contraction. Together, actin and myosin make up more than 80% of the protein mass of muscle.

Myosin (Mr 520,000) has six subunits: two heavy chains (each of Mr 220,000) and four light chains (each of Mr 20,000). The heavy chains account for much of the overall structure. At their carboxyl termini, they are arranged as extended α helices, wrapped around each other in a fibrous, left-handed coiled coil similar to that of α-keratin (Fig. 5-26). At its amino terminus, each heavy chain has a large globular domain containing a site where ATP is hydrolyzed. The light chains are associated with the globular domains.

In muscle cells, molecules of myosin aggregate to form structures called thick filaments (Fig. 5-27a). These rodlike structures are the core of the contractile unit. Within a thick filament, several hundred myosin molecules are arranged with their fibrous “tails” associated to form a long, bipolar structure. The globular domains project from either end of this structure, in regular stacked arrays.

The second major muscle protein, actin, is abundant in almost all eukaryotic cells. In muscle, molecules of monomeric actin, called G-actin (globular actin; Mr 42,000), associate to form a long polymer called F-actin (filamentous actin). The thin filament consists of F-actin (Fig. 5-27b), along with the proteins troponin and tropomyosin (discussed below).

The filamentous parts of thin filaments assemble as successive monomeric actin molecules add to one end. Upon addition, each monomer binds ATP, then hydrolyzes it to ADP, so every actin molecule in the filament is complexed to ADP. This ATP hydrolysis by actin functions only in the assembly of the filaments; it does not contribute directly to the energy expended in muscle contraction. Each actin monomer in the thin filament can bind tightly and specifically to one myosin head group (Fig. 5-27c).

Skeletal muscle consists of parallel bundles of muscle fibers, each fiber a single, very large, multinucleated cell, 20 to 100 μm in diameter, formed from many cells fused together; a single fiber often spans the length of the muscle. Each fiber contains about 1,000 myofibrils, 2 μm in diameter, each consisting of a vast number of regularly arrayed thick and thin filaments complexed to other proteins (Fig. 5-28).

Examined under the electron microscope, muscle fibers reveal alternating regions of high electron density and low electron density, called the A bands and I bands (Fig. 5-28b, c). The A and I bands arise from the arrangement of thick and thin filaments, which are aligned and partially overlapping. The I band is the region of the bundle that in cross section would contain only thin filaments. The darker A band stretches the length of the thick filament and includes the region where parallel thick and thin filaments overlap.

Bisecting the I band is a thin structure called the Z disk, perpendicular to the thin filaments and serving as an anchor to which the thin filaments are attached. The A band, too, is bisected by a thin line, the M line or M disk, a region of high electron density in the middle of the thick filaments. The entire contractile unit, consisting of bundles of thick filaments interleaved at either end with bundles of thin filaments, is called the sarcomere. The arrangement of interleaved bundles allows the thick and thin filaments to slide past each other (by a mechanism discussed below), causing a progressive shortening of each sarcomere (Fig. 5-28d).

The interaction between actin and myosin, like that between all proteins and ligands, involves weak bonds. When ATP is not bound to myosin, a face on the myosin head group binds tightly to actin (Fig. 5-29). When ATP binds to myosin and is hydrolyzed to ADP and phosphate, a coordinated and cyclic series of conformational changes occurs in which myosin releases the F- actin subunit and binds another subunit farther along the thin filament.

a “power stroke” during which the conformation of the myosin head returns to the original resting state, its orientation relative to the bound actin changing so as to pull the tail of the myosin toward the Z disk. ADP is then released to complete the cycle. Each cycle generates about 3 to 4 pN (piconewtons) of force and moves the thick filament 5 to 10 nm relative to the thin filament.

Because there are many myosin heads in a thick filament, at any given moment some (probably 1% to 3%) are bound to thin filaments. This prevents thick filaments from slipping backward when an individual myosin head releases the actin subunit to which it was bound. The thick filament thus actively slides forward past the adjacent thin filaments. This process, coordinated among the many sarcomeres in a muscle fiber, brings about muscle contraction.

The interaction between actin and myosin must be regulated so that contraction occurs only in response to appropriate signals from the nervous system. The regulation is mediated by a complex of two proteins, tropomyosin and troponin (Fig. 5-30). Tropomyosin binds to the thin filament, blocking the attachment sites for the myosin head groups.

protein. A nerve impulse causes release of Ca2+ ions from the sarcoplasmic reticulum. The released Ca2+ binds to troponin (another protein-ligand interaction) and causes a conformational change in the tropomyosin-troponin complexes, exposing the myosin-binding sites on the thin filaments. Contraction follows.

Troponin is attached to the actin-tropomyosin complex at regular intervals of 38.5 nm. Troponin consists of three different subunits: I, C, and T. Troponin I prevents binding of the myosin head to actin; troponin C has a binding site for Ca2+; and troponin T links the entire troponin complex to tropomyosin.

Working skeletal muscle requires two types of molecular functions that are common in proteins: binding and catalysis. The actin-myosin interaction, a protein-ligand interaction like that of immunoglobulins with antigens, is reversible and leaves the participants unchanged. This interaction illustrates why reversibility is important; a permanent actin-myosin interaction

defines the state of rigor mortis, a state we all want to avoid as long as possible. When ATP binds myosin, however, it is hydrolyzed to ADP and Pi (inorganic phosphate). Myosin is not only an actin-binding protein, but also an ATPase — an enzyme. The function of enzymes in catalyzing chemical transformations is the topic of the next chapter.

Protein-ligand interactions achieve a special degree of spatial and temporal organization in motor proteins. Muscle contraction results from choreographed interactions between myosin and actin, coupled to the hydrolysis of ATP by myosin. Myosin consists of two heavy chains and four light chains, forming a fibrous coiled coil (tail) domain and a globular (head) domain. Myosin molecules are organized into thick filaments, which slide past thin filaments composed largely of actin.

ATP hydrolysis in myosin is coupled to a series of conformational changes in the myosin head, leading to dissociation of myosin from one F-actin subunit and its eventual reassociation with another, farther along the thin filament. The myosin thus slides along the actin filaments. Muscle contraction is stimulated by the release of Ca2+ from the sarcoplasmic reticulum. The Ca2+ binds to the protein troponin, leading to a conformational change in a troponintropomyosin complex that triggers the cycle of actin-myosin interactions.