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Cell_Biology_Alberts. Tightly Bound Small Molecules Add Extra Functions to Proteins Although we have emphasized the versatility of enzymes—and proteins in general—as chains of amino acids that perform remarkable functions, there are many instances in which the amino acids by themselves are not enough. Just as humans The reaction of the water molecule (red) completes the hydrolysis and returns the enzyme to its initial state, forming the fnal enzyme– product complex (EP).


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Cell_Biology_Alberts. The reaction of the water molecule (red) completes the hydrolysis and returns the enzyme to its initial state, forming the fnal enzyme– product complex (EP). Figure 3–51 events at the active site of lysozyme. The top left and top right drawings show the free substrate and the free products, respectively, whereas the other three drawings show the sequential events at the enzyme active site. Note the change in the conformation of sugar D in the enzyme–substrate complex; this shape change stabilizes the oxocarbenium ion-like transition states required for formation and hydrolysis of the covalent intermediate shown in the middle panel. It is also possible that a carbonium ion intermediate forms in step 2, but the covalent intermediate shown in the middle panel has been detected with a synthetic substrate (Movie 3.9). (See D.J. Vocadlo et al., Nature 412:835–838, 2001.)


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Cell_Biology_Alberts. Figure 3–52 Some general strategies of enzyme catalysis. (A) Holding substrates (A) enzyme binds to two (B) binding of substrate (C) enzyme strains the together in a precise alignment. (B) Charge substrate molecules and to enzyme rearranges bound substrate stabilization of reaction intermediates. orients them precisely to electrons in the substrate, molecule, forcing it encourage a reaction to creating partial negative toward a transition (C) Applying forces that distort bonds in the occur between them and positive charges state to favor a reaction substrate to increase the rate of a particular that favor a reaction reaction.


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Cell_Biology_Alberts. employ tools to enhance and extend the capabilities of their hands, enzymes and other proteins often use small nonprotein molecules to perform functions that would be difficult or impossible to do with amino acids alone. Thus, enzymes frequently have a small molecule or metal atom tightly associated with their active site that assists with their catalytic function. Carboxypeptidase, for example, an enzyme that cuts polypeptide chains, carries a tightly bound zinc ion in its active site. During the cleavage of a peptide bond by carboxypeptidase, the zinc ion forms a transient bond with one of the substrate atoms, thereby assisting the hydrolysis reaction. In other enzymes, a small organic molecule serves a similar purpose. Such organic molecules are often referred to as coenzymes. An example is biotin, which is found in enzymes that transfer a carboxylate group (–COO–) from one molecule to another (see Figure 2–40). Biotin participates in these reactions by forming a transient covalent


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Cell_Biology_Alberts. is biotin, which is found in enzymes that transfer a carboxylate group (–COO–) from one molecule to another (see Figure 2–40). Biotin participates in these reactions by forming a transient covalent bond to the –COO– group to be transferred, being better suited to this function than any of the amino acids used to make proteins. Because it cannot be synthesized by humans, and must therefore be supplied in small quantities in our diet, biotin is a vitamin. Many other coenzymes are either vitamins or derivatives of vitamins (Table 3–2).


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Cell_Biology_Alberts. Other proteins also frequently require specific small-molecule adjuncts to function properly. Thus, the signal receptor protein rhodopsin, which is made by the photoreceptor cells in the retina, detects light by means of a small molecule, retinal, embedded in the protein (Figure 3–53A). Retinal, which is derived from vitamin A, changes its shape when it absorbs a photon of light, and this change causes the protein to trigger a cascade of enzymatic reactions that eventually lead to an electrical signal being carried to the brain. Figure 3–53 Retinal and heme. (A) The structure of retinal, the light-sensitive molecule attached to rhodopsin in the eye. The structure shown isomerizes when it absorbs light. (B) The structure of a heme group. The carbon-containing heme ring is red and the iron atom at its center is orange. A heme group is tightly bound to each of the four polypeptide chains in hemoglobin, the oxygen-carrying protein whose structure is shown in Figure 3–19.


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Cell_Biology_Alberts. Another example of a protein with a nonprotein portion is hemoglobin (see Figure 3–19). Each molecule of hemoglobin carries four heme groups, ring-shaped molecules each with a single central iron atom (Figure 3–53B). Heme gives hemoglobin (and blood) its red color. By binding reversibly to oxygen gas through its iron atom, heme enables hemoglobin to pick up oxygen in the lungs and release it in the tissues. Sometimes these small molecules are attached covalently and permanently to their protein, thereby becoming an integral part of the protein molecule itself. We shall see in Chapter 10 that proteins are often anchored to cell membranes through covalently attached lipid molecules. And membrane proteins exposed on the surface of the cell, as well as proteins secreted outside the cell, are often modified by the covalent addition of sugars and oligosaccharides. Multienzyme Complexes Help to Increase the Rate of Cell Metabolism


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Cell_Biology_Alberts. Multienzyme Complexes Help to Increase the Rate of Cell Metabolism The efficiency of enzymes in accelerating chemical reactions is crucial to the maintenance of life. Cells, in effect, must race against the unavoidable processes of decay, which—if left unattended—cause macromolecules to run downhill toward greater and greater disorder. If the rates of desirable reactions were not greater than the rates of competing side reactions, a cell would soon die. We can get some idea of the rate at which cell metabolism proceeds by measuring the rate of ATP utilization. A typical mammalian cell “turns over” (i.e., hydrolyzes and restores by phosphorylation) its entire ATP pool once every 1 or 2 minutes. For each cell, this turnover represents the utilization of roughly 107 molecules of ATP per second (or, for the human body, about 1 gram of ATP every minute).


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Cell_Biology_Alberts. The rates of reactions in cells are rapid because enzyme catalysis is so effective. Some enzymes have become so efficient that there is no possibility of further useful improvement. The factor that limits the reaction rate is no longer the enzyme’s intrinsic speed of action; rather, it is the frequency with which the enzyme collides with its substrate. Such a reaction is said to be diffusion-limited (see Panel 3–2, pp. 142–143).


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Cell_Biology_Alberts. The amount of product produced by an enzyme will depend on the concentration of both the enzyme and its substrate. If a sequence of reactions is to occur extremely rapidly, each metabolic intermediate and enzyme involved must be present in high concentration. However, given the enormous number of different reactions performed by a cell, there are limits to the concentrations that can be achieved. In fact, most metabolites are present in micromolar (10–6 M) concentrations, and most enzyme concentrations are much lower. How is it possible, therefore, to maintain very fast metabolic rates?


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Cell_Biology_Alberts. The answer lies in the spatial organization of cell components. The cell can increase reaction rates without raising substrate concentrations by bringing the various enzymes involved in a reaction sequence together to form a large protein assembly known as a multienzyme complex (Figure 3–54). Because this assembly is organized in a way that allows the product of enzyme A to be passed directly to enzyme B, and so on, diffusion rates need not be limiting, even when the concentrations of the substrates in the cell as a whole are very low. It is perhaps not surprising, therefore, that such enzyme complexes are very common, and they are involved in nearly all aspects of metabolism—including the central genetic processes of DNA, RNA, and protein synthesis. In fact, few enzymes in eukaryotic cells diffuse freely in solution; instead, most seem to have evolved binding sites that concentrate them with other proteins of related function in particular regions of the cell, thereby increasing the


Cell_Biology_Alberts

Cell_Biology_Alberts. diffuse freely in solution; instead, most seem to have evolved binding sites that concentrate them with other proteins of related function in particular regions of the cell, thereby increasing the rate and efficiency of the reactions that they catalyze (see p. 331).


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Cell_Biology_Alberts. Eukaryotic cells have yet another way of increasing the rate of metabolic reactions: using their intracellular membrane systems. These membranes can segregate particular substrates and the enzymes that act on them into the same membrane-enclosed compartment, such as the endoplasmic reticulum or the cell nucleus. If, for example, a compartment occupies a total of 10% of the volume of TE2 2 2 1 1 4 4 5 21 4 5 3 3 3 enzyme domains (C) (E) (D) etc.5 nm C acyl carrier domain termination domain (TE) PYRUVATE DEHYDROGENASE COMPLEX FATTY ACID SYNTHASE 3 1 20 nm


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Cell_Biology_Alberts. Figure 3–54 How unstructured regions of polypeptide chain serving as tethers allow reaction intermediates to be passed from one active site to another in large multienzyme complexes. (A–C) The fatty acid synthase in mammals. (A) The location of seven protein domains with different activities in this 270 kilodalton protein. The numbers refer to the order in which each enzyme domain must function to complete each two-carbon addition step. After multiple cycles of two-carbon addition, the termination domain releases the final product once the desired length of fatty acid has been synthesized. (B) The structure of the dimeric enzyme, with the location of the five active sites in one monomer indicated. (C) How a flexible tether allows the substrate that remains linked to the acyl carrier domain (red) to be passed from one active site to another in each monomer, sequentially elongating and modifying the bound fatty acid intermediate (yellow). The five steps are repeated until the final


Cell_Biology_Alberts

Cell_Biology_Alberts. (red) to be passed from one active site to another in each monomer, sequentially elongating and modifying the bound fatty acid intermediate (yellow). The five steps are repeated until the final length of fatty acid chain has been synthesized. (Only steps 1 through 4 are illustrated here.) (D) Multiple tethered subunits in the giant pyruvate dehydrogenase complex (9500 kilodaltons, larger than a ribosome) that catalyzes the conversion of pyruvate to acetyl CoA. As in (C), a covalently bound substrate held on a flexible tether (red balls with yellow substrate) is serially passed through active sites on subunits (here labeled 1 through 3) to produce the final products. Here, subunit 1 catalyzes the decarboxylation of pyruvate accompanied by the reductive acetylation of a lipoyl group linked to one of the red balls. Subunit 2 transfers this acetyl group to CoA, forming acetyl CoA, and subunit 3 reoxidizes the lipoyl group to prepare it for the next cycle. Only one-tenth of the subunits


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Cell_Biology_Alberts. to one of the red balls. Subunit 2 transfers this acetyl group to CoA, forming acetyl CoA, and subunit 3 reoxidizes the lipoyl group to prepare it for the next cycle. Only one-tenth of the subunits labeled 1 and 3, attached to the core formed by subunit 2, are illustrated here. This important reaction takes place in the mammalian mitochondrion, as part of the pathway that oxidizes sugars to CO2 and H2O (see page 82). (A–C, adapted from T. Maier et al., Quart. Rev. Biophys. 43:373–422, 2010; D, from J.L.S. Milne et al., J. Biol. Chem. 281:4364–4370, 2006.) the cell, the concentration of reactants in that compartment may be increased by 10 times compared with a cell with the same number of enzyme and substrate molecules, but no compartmentalization. Reactions limited by the speed of diffusion can thereby be speeded up by a factor of 10.


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Cell_Biology_Alberts. The Cell Regulates the Catalytic Activities of Its Enzymes A living cell contains thousands of enzymes, many of which operate at the same time and in the same small volume of the cytosol. By their catalytic action, these enzymes generate a complex web of metabolic pathways, each composed of chains of chemical reactions in which the product of one enzyme becomes the substrate of the next. In this maze of pathways, there are many branch points (nodes) where different enzymes compete for the same substrate. The system is complex (see Figure 2–63), and elaborate controls are required to regulate when and how rapidly each reaction occurs.


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Cell_Biology_Alberts. Regulation occurs at many levels. At one level, the cell controls how many molecules of each enzyme it makes by regulating the expression of the gene that encodes that enzyme (discussed in Chapter 7). The cell also controls enzymatic activities by confining sets of enzymes to particular subcellular compartments, whether by enclosing them in a distinct membrane-bounded compartment (discussed in Chapters 12 and 14) or by concentrating them on a protein scaffold (see Figure 3–77). As will be explained later in this chapter, enzymes are also covalently modified to control their activity. The rate of protein destruction by targeted proteolysis represents yet another important regulatory mechanism (see Figure 6–86). But the most general process that adjusts reaction rates operates through a direct, reversible change in the activity of an enzyme in response to the specific small molecules that it binds.


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Cell_Biology_Alberts. The most common type of control occurs when an enzyme binds a molecule that is not a substrate to a special regulatory site outside the active site, thereby altering the rate at which the enzyme converts its substrates to products. For example, in feedback inhibition, a product produced late in a reaction pathway inhibits an enzyme that acts earlier in the pathway. Thus, whenever large quantities of the final product begin to accumulate, this product binds to the enzyme and slows down its catalytic action, thereby limiting the further entry of substrates into that reaction pathway (Figure 3–55). Where pathways branch or intersect, there are usually multiple points of control by different final products, each of which works to regulate its own synthesis (Figure 3–56). Feedback inhibition can work almost instantaneously, and it is rapidly reversed when the level of the product falls.


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Cell_Biology_Alberts. Figure 3–55 Feedback inhibition of a single biosynthetic pathway. The end product Z inhibits the first enzyme that is unique to its synthesis and thereby controls its own level in the cell. This is an example of negative regulation. Figure 3–56 Multiple feedback inhibition. In this example, which shows the biosynthetic pathways for four different amino acids in bacteria, the red lines indicate positions at which products feed back to inhibit enzymes. Each amino acid controls the first enzyme specific to its own synthesis, thereby controlling its own levels and avoiding a wasteful, or even dangerous, buildup of intermediates. The products can also separately inhibit the initial set of reactions common to all the syntheses; in this case, three different enzymes catalyze the initial reaction, each inhibited by a different product.


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Cell_Biology_Alberts. Feedback inhibition is negative regulation: it prevents an enzyme from acting. Enzymes can also be subject to positive regulation, in which a regulatory molecule stimulates the enzyme’s activity rather than shutting the enzyme down. Positive regulation occurs when a product in one branch of the metabolic network stimulates the activity of an enzyme in another pathway. As one example, the accumulation of ADP activates several enzymes involved in the oxidation of sugar molecules, thereby stimulating the cell to convert more ADP to ATP.


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Cell_Biology_Alberts. A striking feature of both positive and negative feedback regulation is that the regulatory molecule often has a shape totally different from the shape of the substrate of the enzyme. This is why the effect on a protein is termed allostery (from the Greek words allos, meaning “other,” and stereos, meaning “solid” or “three-dimensional”). As biologists learned more about feedback regulation, they recognized that the enzymes involved must have at least two different binding sites on their surface—an active site that recognizes the substrates, and a regulatory site that recognizes a regulatory molecule. These two sites must somehow communicate so that the catalytic events at the active site can be influenced by the binding of the regulatory molecule at its separate site on the protein’s surface.


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Cell_Biology_Alberts. The interaction between separated sites on a protein molecule is now known to depend on a conformational change in the protein: binding at one of the sites causes a shift from one folded shape to a slightly different folded shape. During feedback inhibition, for example, the binding of an inhibitor at one site on the protein causes the protein to shift to a conformation that incapacitates its active site located elsewhere in the protein.


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Cell_Biology_Alberts. It is thought that most protein molecules are allosteric. They can adopt two or more slightly different conformations, and a shift from one to another caused by the binding of a ligand can alter their activity. This is true not only for enzymes but also for many other proteins, including receptors, structural proteins, and motor proteins. In all instances of allosteric regulation, each conformation of the protein has somewhat different surface contours, and the protein’s binding sites for ligands are altered when the protein changes shape. Moreover, as we discuss next, each ligand will stabilize the conformation that it binds to most strongly, and thus—at high enough concentrations—will tend to “switch” the protein toward the conformation that the ligand prefers.


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Cell_Biology_Alberts. The effects of ligand binding on a protein follow from a fundamental chemical principle known as linkage. Suppose, for example, that a protein that binds glucose also binds another molecule, X, at a distant site on the protein’s surface. If the binding site for X changes shape as part of the conformational change in the protein induced by glucose binding, the binding sites for X and for glucose are said to be coupled. Whenever two ligands prefer to bind to the same conformation of an allosteric protein, it follows from basic thermodynamic principles that each ligand must increase the affinity of the protein for the other. For example, if the shift of a protein to a conformation that binds glucose best also causes the binding site for X to fit X better, then the protein will bind glucose more tightly when X is present than when X is absent. In other words, X will positively regulate the protein’s binding of glucose (Figure 3–57).


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Cell_Biology_Alberts. Conversely, linkage operates in a negative way if two ligands prefer to bind to different conformations of the same protein. In this case, the binding of the first ligand discourages the binding of the second ligand. Thus, if a shape change caused by glucose binding decreases the affinity of a protein for molecule X, the binding of X must also decrease the protein’s affinity for glucose (Figure 3–58). The linkage relationship is quantitatively reciprocal, so that, for example, if glucose has a very large effect on the binding of X, X has a very large effect on the binding of glucose.


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Cell_Biology_Alberts. The relationships shown in Figures 3–57 and 3–58 apply to all proteins, and they underlie all of cell biology. The principle seems so obvious in retrospect that we now take it for granted. But the discovery of linkage in studies of a few enzymes in the 1950s, followed by an extensive analysis of allosteric mechanisms in proteins in the early 1960s, had a revolutionary effect on our understanding of biology. Since molecule X in these examples binds at a site on the enzyme that is distinct from the site where catalysis occurs, it need not have any chemical relationship to the substrate that binds at the active site. Moreover, as we have just seen, for enzymes that are regulated in this way, molecule X can either turn the enzyme on (positive regulation) or turn it off (negative regulation). By such a mechanism, allosteric proteins serve as general switches that, in principle, can allow one molecule in a cell to affect the fate of any other.


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Cell_Biology_Alberts. A single-subunit enzyme that is regulated by negative feedback can at most decrease from 90% to about 10% activity in response to a 100-fold increase in the concentration of an inhibitory ligand that it binds (Figure 3–59, red line). Responses of this type are apparently not sharp enough for optimal cell regulation, and most enzymes that are turned on or off by ligand binding consist of symmetric assemblies of identical subunits. With this arrangement, the binding of a molecule of ligand to a single site on one subunit can promote an allosteric change in the entire assembly that helps the neighboring subunits bind the same ligand. As a result, a cooperative allosteric transition occurs (Figure 3–59, blue line), allowing


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Cell_Biology_Alberts. Figure 3–57 Positive regulation caused by conformational coupling between two separate binding sites. In this example, both glucose and molecule X bind best to the closed conformation of a protein with two domains. Because both glucose and molecule X drive the protein toward its closed conformation, each ligand helps the other to bind. Glucose and molecule X are therefore said to bind cooperatively to the protein. Figure 3–58 negative regulation caused by conformational coupling between two separate binding sites. The scheme here resembles that in the previous figure, but here molecule X prefers the open conformation, while glucose prefers the closed conformation. Because glucose and molecule X drive the protein toward opposite conformations (closed and open, respectively), the presence of either ligand interferes with the binding of the other.


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Cell_Biology_Alberts. a relatively small change in ligand concentration in the cell to switch the whole assembly from an almost fully active to an almost fully inactive conformation (or vice versa). The principles involved in a cooperative “all-or-none” transition are the same for all proteins, whether or not they are enzymes. Thus, for example, they are critical for the efficient uptake and release of O2 by hemoglobin in our blood. But they are perhaps easiest to visualize for an enzyme that forms a symmetric dimer. In the example shown in Figure 3–60, the first molecule of an inhibitory ligand binds with great difficulty since its binding disrupts an energetically favorable interaction between the two identical monomers in the dimer. A second molecule of inhibitory ligand now binds more easily, however, because its binding restores the energetically favorable monomer–monomer contacts of a symmetric dimer (this also completely inactivates the enzyme).


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Cell_Biology_Alberts. As an alternative to this induced fit model for a cooperative allosteric transition, we can view such a symmetric enzyme as having only two possible conformations, corresponding to the “enzyme on” and “enzyme off” structures in Figure 3–60. In this view, ligand binding perturbs an all-or-none equilibrium between these two states, thereby changing the proportion of active molecules. Both models represent true and useful concepts. Proteins are regulated by more than the reversible binding of other molecules. A second method that eukaryotic cells use extensively to regulate a protein’s function is the covalent addition of a smaller molecule to one or more of its amino acid side chains. The most common such regulatory modification in higher eukaryotes is the addition of a phosphate group. We shall therefore use protein phosphorylation to illustrate some of the general principles involved in the control of protein function through the modification of amino acid side chains.


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Cell_Biology_Alberts. A phosphorylation event can affect the protein that is modified in three important ways. First, because each phosphate group carries two negative charges, the enzyme-catalyzed addition of a phosphate group to a protein can cause a major conformational change in the protein by, for example, attracting a cluster of positively charged amino acid side chains. This can, in turn, affect the binding of ligands elsewhere on the protein surface, dramatically changing the


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Cell_Biology_Alberts. Figure 3–60 a cooperative allosteric transition in an enzyme composed of two identical subunits. This diagram illustrates how the conformation of one subunit can influence that of its neighbor. The binding of a single molecule of an inhibitory ligand (yellow) to one subunit of the enzyme occurs with difficulty because it changes the conformation of this subunit and thereby disrupts the symmetry of the enzyme. Once this conformational change has occurred, however, the energy gained by restoring the symmetric pairing interaction between the two subunits makes it especially easy for the second subunit to bind the inhibitory ligand and undergo the same conformational change. Because the binding of the first molecule of ligand increases the affinity with which the other subunit binds the same ligand, the response of the enzyme to changes in the concentration of the ligand is much steeper than the response of an enzyme with only one subunit (see Figure 3–59 and Movie 3.10).


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Cell_Biology_Alberts. Figure 3–59 enzyme activity versus the concentration of inhibitory ligand for single-subunit and multisubunit allosteric enzymes. For an enzyme with a single subunit (red line), a drop from 90% enzyme activity to 10% activity (indicated by the two dots on the curve) requires a 100-fold increase in the concentration of inhibitor. The enzyme activity is calculated from the simple equilibrium relationship K = [IP]/[I][P], where P is active protein, I is inhibitor, and IP is the inactive protein bound to inhibitor. An identical curve applies to any simple binding interaction between two molecules, A and B. In contrast, a multisubunit allosteric enzyme can respond in a switchlike manner to a change in ligand concentration: the steep response is caused by a cooperative binding of the ligand molecules, as explained in Figure 3–60. Here, the green line represents the idealized result expected for the cooperative binding of two inhibitory ligand molecules to an allosteric enzyme with two


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Cell_Biology_Alberts. molecules, as explained in Figure 3–60. Here, the green line represents the idealized result expected for the cooperative binding of two inhibitory ligand molecules to an allosteric enzyme with two subunits, and the blue line shows the idealized response of an enzyme with four subunits. As indicated by the two dots on each of these curves, the more complex enzymes drop from 90% to 10% activity over a much narrower range of inhibitor concentration than does the enzyme composed of a single subunit.


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Cell_Biology_Alberts. protein’s activity. When a second enzyme removes the phosphate group, the protein returns to its original conformation and restores its initial activity. Second, an attached phosphate group can form part of a structure that the binding sites of other proteins recognize. As previously discussed, the SH2 domain binds to a short peptide sequence containing a phosphorylated tyrosine side chain (see Figure 3–40B). More than ten other common domains provide binding sites for attaching their protein to phosphorylated peptides in other protein molecules, each recognizing a phosphorylated amino acid side chain in a different protein context. Third, the addition of a phosphate group can mask a binding site that otherwise holds two proteins together, and thereby disrupt protein–protein interactions. As a result, protein phosphorylation and dephosphorylation very often drive the regulated assembly and disassembly of protein complexes (see, for example, Figure 15–11).


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Cell_Biology_Alberts. Reversible protein phosphorylation controls the activity, structure, and cellular localization of both enzymes and many other types of proteins in eukaryotic cells. In fact, this regulation is so extensive that more than one-third of the 10,000 or so proteins in a typical mammalian cell are thought to be phosphorylated at any given time—many with more than one phosphate. As might be expected, the addition and removal of phosphate groups from specific proteins often occur in response to signals that specify some change in a cell’s state. For example, the complicated series of events that takes place as a eukaryotic cell divides is largely timed in this way (discussed in Chapter 17), and many of the signals mediating cell–cell interactions are relayed from the plasma membrane to the nucleus by a cascade of protein phosphorylation events (discussed in Chapter 15). A Eukaryotic Cell Contains a Large Collection of Protein Kinases and Protein Phosphatases


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Cell_Biology_Alberts. Protein phosphorylation involves the enzyme-catalyzed transfer of the terminal phosphate group of an ATP molecule to the hydroxyl group on a serine, threonine, or tyrosine side chain of the protein (Figure 3–61). A protein kinase catalyzes this reaction, and the reaction is essentially unidirectional because of the large amount of free energy released when the phosphate–phosphate bond in ATP is broken to produce ADP (discussed in Chapter 2). A protein phosphatase catalyzes the reverse reaction of phosphate removal, or dephosphorylation. Cells contain hundreds of different protein kinases, each responsible for phosphorylating a different protein or set of proteins. There are also many different protein phosphatases; some are highly specific and remove phosphate groups from only one or a few proteins, whereas others act on a broad range of proteins and are targeted to specific substrates by regulatory subunits. The state of phosphorylation of a protein at any moment, and thus its


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Cell_Biology_Alberts. a few proteins, whereas others act on a broad range of proteins and are targeted to specific substrates by regulatory subunits. The state of phosphorylation of a protein at any moment, and thus its activity, depends on the relative activities of the protein kinases and phosphatases that modify it.


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Cell_Biology_Alberts. The protein kinases that phosphorylate proteins in eukaryotic cells belong to a very large family of enzymes that share a catalytic (kinase) sequence of about 290 amino acids. The various family members contain different amino acid sequences on either end of the kinase sequence (for example, see Figure 3–10), and often have short amino acid sequences inserted into loops within it. Some of these additional amino acid sequences enable each kinase to recognize the specific set of proteins it phosphorylates, or to bind to structures that localize it in specific regions of the cell. Other parts of the protein regulate the activity of each kinase, so it can be turned on and off in response to different specific signals, as described below.


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Cell_Biology_Alberts. By comparing the number of amino acid sequence differences between the various members of a protein family, we can construct an “evolutionary tree” that is thought to reflect the pattern of gene duplication and divergence that gave rise to the family. Figure 3–62 shows an evolutionary tree of protein kinases. Kinases with related functions are often located on nearby branches of the tree: the protein kinases involved in cell signaling that phosphorylate tyrosine side chains, for example, are all clustered in the top left corner of the tree. The other kinases shown


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Cell_Biology_Alberts. Figure 3–61 Protein phosphorylation. Many thousands of proteins in a typical eukaryotic cell are modified by the covalent addition of a phosphate group. (A) The general reaction transfers a phosphate group from ATP to an amino acid side chain of the target protein by a protein kinase. Removal of the phosphate group is catalyzed by a second enzyme, a protein phosphatase. In this example, the phosphate is added to a serine side chain; in other cases, the phosphate is instead linked to the –OH group of a threonine or a tyrosine in the protein. (B) The phosphorylation of a protein by a protein kinase can either increase or decrease the protein’s activity, depending on the site of phosphorylation and the structure of the protein. phosphorylate either a serine or a threonine side chain, and many are organized into clusters that seem to reflect their function—in transmembrane signal transduction, intracellular signal amplification, cell-cycle control, and so on.


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Cell_Biology_Alberts. As a result of the combined activities of protein kinases and protein phosphatases, the phosphate groups on proteins are continually turning over—being added and then rapidly removed. Such phosphorylation cycles may seem wasteful, but they are important in allowing the phosphorylated proteins to switch rapidly from one state to another: the more rapid the cycle, the faster a population of protein molecules can change its state of phosphorylation in response to a sudden change in its phosphorylation rate (see Figure 15–14). The energy required to drive this phosphorylation cycle is derived from the free energy of ATP hydrolysis, one molecule of which is consumed for each phosphorylation event. The Regulation of the Src Protein Kinase Reveals How a Protein Can Function as a Microprocessor


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Cell_Biology_Alberts. The Regulation of the Src Protein Kinase Reveals How a Protein Can Function as a Microprocessor The hundreds of different protein kinases in a eukaryotic cell are organized into complex networks of signaling pathways that help to coordinate the cell’s activities, drive the cell cycle, and relay signals into the cell from the cell’s environment. Many of the extracellular signals involved need to be both integrated and amplified by the cell. Individual protein kinases (and other signaling proteins) serve as input–output devices, or “microprocessors,” in the integration process. An important part of the input to these signal-processing proteins comes from the control that is exerted by phosphates added and removed from them by protein kinases and protein phosphatases, respectively.


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Cell_Biology_Alberts. The Src family of protein kinases (see Figure 3–10) exhibits such behavior. The Src protein (pronounced “sarc” and named for the type of tumor, a sarcoma, that its deregulation can cause) was the first tyrosine kinase to be discovered. It is now known to be part of a subfamily of nine very similar protein kinases, which are found only in multicellular animals. As indicated by the evolutionary tree in Figure 3–62, sequence comparisons suggest that tyrosine kinases as a group were a relatively late innovation that branched off from the serine/threonine kinases, with the Src subfamily being only one subgroup of the tyrosine kinases created in this way. The Src protein and its relatives contain a short N-terminal region that becomes covalently linked to a strongly hydrophobic fatty acid, which anchors the kinase at the cytoplasmic face of the plasma membrane. Next along the linear sequence of


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Cell_Biology_Alberts. Figure 3–62 an evolutionary tree of selected protein kinases. A higher eukaryotic cell contains hundreds of such enzymes, and the human genome codes for more than 500. Note that only some of these, those discussed in this book, are shown. Figure 3–64 The activation of a Src-type protein kinase by two sequential events. As described in the text, the requirement for multiple upstream events to trigger these processes allows the kinase to serve as a signal integrator (Movie 3.11). (Adapted from S.C. Harrison et al., Cell 112:737–740, 2003. With permission from Elsevier.) Figure 3–65 How a Src-type protein kinase acts as a signal-integrating device. A disruption of the inhibitory interaction illustrated for the SH3 domain (green) occurs when its binding to the indicated orange linker region is replaced with its higher-affinity binding to an activating ligand.


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Cell_Biology_Alberts. change that inactivates the protein. The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shown in Figure 3–67. The Ras protein has an important role in cell signaling (discussed in Chapter 15). In its GTP-bound form, it is active and stimulates a cascade of protein phosphorylations in the cell. Most of the time, however, the protein is in its inactive, GDP-bound form. It becomes active when it exchanges its GDP for a GTP molecule in response to extracellular signals, such as growth factors, that bind to receptors in the plasma membrane (see Figure 15–47). Regulatory Proteins GAP and GEF Control the Activity of GTP-Binding Proteins by Determining Whether GTP or GDP Is Bound


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Cell_Biology_Alberts. GTP-binding proteins are controlled by regulatory proteins that determine whether GTP or GDP is bound, just as phosphorylated proteins are turned on and off by protein kinases and protein phosphatases. Thus, Ras is inactivated by a GTPase-activating protein (GAP), which binds to the Ras protein and induces Ras to hydrolyze its bound GTP molecule to GDP—which remains tightly bound— and inorganic phosphate (Pi), which is rapidly released. The Ras protein stays in its inactive, GDP-bound conformation until it encounters a guanine nucleotide exchange factor (GEF), which binds to GDP-Ras and causes Ras to release its GDP. Because the empty nucleotide-binding site is immediately filled by a GTP molecule (GTP is present in large excess over GDP in cells), the GEF activates Ras by indirectly adding back the phosphate removed by GTP hydrolysis. Thus, in a sense, the roles of GAP and GEF are analogous to those of a protein phosphatase and a protein kinase, respectively (Figure 3–68).


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Cell_Biology_Alberts. Proteins Can Be Regulated by the Covalent Addition of Other Proteins


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Cell_Biology_Alberts. Cells contain a special family of small proteins whose members are covalently attached to many other proteins to determine the activity or fate of the second protein. In each case, the carboxyl end of the small protein becomes linked to the amino group of a lysine side chain of a “target” protein through an isopeptide bond. The first such protein discovered, and the most abundantly used, is ubiquitin (Figure 3–69A). Ubiquitin can be covalently attached to target proteins in a variety of ways, each of which has a different meaning for cells. The major form of ubiquitin addition produces polyubiquitin chains in which—once the first ubiquitin molecule is attached to the target—each subsequent ubiquitin molecule links to Lys48 of the previous ubiquitin, creating a chain of Lys48-linked ubiquitins that are attached to a single lysine side chain of the target protein. This form of polyubiquitin directs the target protein to the interior of a proteasome, where it is digested to small


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Cell_Biology_Alberts. ubiquitins that are attached to a single lysine side chain of the target protein. This form of polyubiquitin directs the target protein to the interior of a proteasome, where it is digested to small peptides (see Figure 6–84). In other circumstances, only single molecules of ubiquitin are added to proteins. In addition, some target proteins are has this binding been disrupted? has this phosphate been added? has this phosphate been removed? P P


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Cell_Biology_Alberts. Src-type protein kinase activity turns on fully only if the answers to all of the above questions are yes Figure 3–66 GTP-binding proteins as molecular switches. The activity of a GTP-binding protein (also called a GTPase) generally requires the presence of a tightly bound GTP molecule (switch “on”). Hydrolysis of this GTP molecule by the GTP-binding protein produces GDP and inorganic phosphate (Pi), and it causes the protein to convert to a different, usually inactive, conformation (switch “off”). Resetting the switch requires that the tightly bound GDP dissociates. This is a slow step that is greatly accelerated by specific signals; once the GDP has dissociated, a molecule of GTP is quickly rebound. modified with a different type of polyubiquitin chain. These modifications have different functional consequences for the protein that is targeted (Figure 3–69B).


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Cell_Biology_Alberts. modified with a different type of polyubiquitin chain. These modifications have different functional consequences for the protein that is targeted (Figure 3–69B). Related structures are created when a different member of the ubiquitin family, such as SUMO (small ubiquitin-related modifier), is covalently attached to a lysine side chain of target proteins. Not surprisingly, all such modifications are reversible. Cells contain sets of ubiquitylating and deubiquitylating (and sumoylating and desumoylating) enzymes that manipulate these covalent adducts, thereby playing roles analogous to the protein kinases and phosphatases that add and remove phosphates from protein side chains. An Elaborate Ubiquitin-Conjugating System Is Used to Mark Proteins


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Cell_Biology_Alberts. An Elaborate Ubiquitin-Conjugating System Is Used to Mark Proteins How do cells select target proteins for ubiquitin addition? As an initial step, the carboxyl end of ubiquitin needs to be activated. This activation is accomplished when a protein called a ubiquitin-activating enzyme (E1) uses ATP hydrolysis energy to attach ubiquitin to itself through a high-energy covalent bond (a thioester). E1 then passes this activated ubiquitin to one of a set of ubiquitin-conjugating (E2) enzymes, each of which acts in conjunction with a set of accessory (E3) proteins called ubiquitin ligases. There are roughly 30 structurally similar but distinct E2 enzymes in mammals, and hundreds of different E3 proteins that form complexes with specific E2 enzymes.


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Cell_Biology_Alberts. Figure 3–70 illustrates the process used to mark proteins for proteasomal degradation. [Similar mechanisms are used to attach ubiquitin (and SUMO) to other types of target proteins.] Here, the ubiquitin ligase binds to specific degradation signals, called degrons, in protein substrates, thereby helping E2 to form a polyubiquitin chain linked to a lysine of the substrate protein. This polyubiquitin chain on a target protein will then be recognized by a specific receptor in the proteasome, causing the target protein to be destroyed. Distinct ubiquitin ligases recognize different degradation signals, thereby targeting distinct subsets of intracellular proteins for destruction, often in response to specific signals (see Figure 6–86).


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Cell_Biology_Alberts. Figure 3–67 The structure of the Ras protein in its GTP-bound form. This monomeric GTPase illustrates the structure of a GTP-binding domain, which is present in a large family of GTP-binding proteins. The red regions change their conformation when the GTP molecule is hydrolyzed to GDP and inorganic phosphate by the protein; the GDP remains bound to the protein, while the inorganic phosphate is released. The special role of the “switch helix” in proteins related to Ras is explained in the text (see Figure 3–72 and Movie 15.7). Figure 3–68 a comparison of two major intracellular signaling mechanisms in eukaryotic cells. In both cases, a signaling protein is activated by the addition of a phosphate group and inactivated by the removal of this phosphate. Note that the addition of a phosphate to a protein can also be inhibitory. (Adapted from E.R. Kantrowitz and W.N. Lipscomb, Trends Biochem. Sci. 15:53–59, 1990.)


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Cell_Biology_Alberts. Protein Complexes with Interchangeable Parts Make Efficient Use of Genetic Information


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Cell_Biology_Alberts. The SCF ubiquitin ligase is a protein complex that binds different “target proteins” at different times in the cell cycle, covalently adding polyubiquitin polypeptide chains to these targets. Its C-shaped structure is formed from five protein subunits, the largest of which serves as a scaffold on which the rest of the complex is built. The structure underlies a remarkable mechanism (Figure 3–71). At one end of the C is an E2 ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, a subunit known as an F-box protein. These two subunits are separated by a gap of about 5 nm. When this protein complex is activated, the F-box protein binds to a specific site on a target protein, positioning the protein in the gap so that some of its lysine side chains contact the ubiquitin-conjugating enzyme. The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3–71C), producing polyubiquitin chains that mark the target proteins for rapid


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Cell_Biology_Alberts. enzyme. The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3–71C), producing polyubiquitin chains that mark the target proteins for rapid destruction in a proteasome.


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Cell_Biology_Alberts. Figure 3–69 The marking of proteins by ubiquitin. (A) The three-dimensional structure of ubiquitin, a small protein of 76 amino acids. A family of special enzymes couples its carboxyl end to the amino group of a lysine side chain in a target protein molecule, forming an isopeptide bond. (B) Some modification patterns that have specific meanings to the cell. Note that the two types of polyubiquitylation differ in the way the ubiquitin molecules are linked together. Linkage through Lys48 signifies degradation by the proteasome (see Figure 6–84), whereas that through Lys63 has other meanings. Ubiquitin markings are “read” by proteins that specifically recognize each type of modification.


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Cell_Biology_Alberts. Figure 3–70 The marking of proteins with ubiquitin. (A) The C-terminus of ubiquitin is initially activated by being linked via a high-energy thioester bond to a cysteine side chain on the E1 protein. This reaction requires ATP, and it proceeds via a covalent AMP-ubiquitin intermediate. The activated ubiquitin on E1, also known as the ubiquitin-activating enzyme, is then transferred to the cysteine on an E2 molecule. (B) The addition of a polyubiquitin chain to a target protein. In a mammalian cell, there are several hundred distinct E2–E3 complexes. The E2s are called ubiquitinconjugating enzymes. The E3s are referred to as ubiquitin ligases. (Adapted from D.R. Knighton et al., Science 253:407–414, 1991.) two of many polyubiquitylated possible protein targeted substrate-binding for destruction arms


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Cell_Biology_Alberts. In this manner, specific proteins are targeted for rapid destruction in response to specific signals, thereby helping to drive the cell cycle (discussed in Chapter 17). The timing of the destruction often involves creating a specific pattern of phosphorylation on the target protein that is required for its recognition by the F-box subunit. It also requires the activation of an SCF ubiquitin ligase that carries the appropriate substrate-binding arm. Many of these arms (the F-box subunits) are interchangeable in the protein complex (see Figure 3–71B), and there are more than 70 human genes that encode them.


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Cell_Biology_Alberts. As emphasized previously, once a successful protein has evolved, its genetic information tends to be duplicated to produce a family of related proteins. Thus, for example, not only are there many F-box proteins—making possible the recognition of different sets of target proteins—but there is also a family of scaffolds (known as cullins) that give rise to a family of SCF-like ubiquitin ligases. A protein machine like the SCF ubiquitin ligase, with its interchangeable parts, makes economical use of the genetic information in cells. It also creates opportunities for “rapid” evolution, inasmuch as new functions can evolve for the entire complex simply by producing an alternative version of one of its subunits. Ubiquitin ligases form a diverse family of protein complexes. Some of these complexes are far larger and more complicated than SCF, but their underlying enzymatic function remains the same (Figure 3–71D).


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Cell_Biology_Alberts. Detailed structures obtained for one of the GTP-binding protein family members, the EF-Tu protein, provide a good example of how allosteric changes in protein conformations can produce large movements by amplifying a small, local conformational change. As will be discussed in Chapter 6, EF-Tu is an abundant molecule that serves as an elongation factor (hence the EF) in protein synthesis, loading each aminoacyl-tRNA molecule onto the ribosome. EF-Tu contains a Ras-like domain (see Figure 3–67), and the tRNA molecule forms a tight complex with its GTP-bound form. This tRNA molecule can transfer its amino acid to the growing


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Cell_Biology_Alberts. Figure 3–71 The structure and mode of action of an SCF ubiquitin ligase. (A) The structure of the five-protein ubiquitin ligase complex that includes an E2 ubiquitinconjugating enzyme. Four proteins form the E3 portion. The protein denoted here as adaptor protein 1 is the Rbx1/Hrt1 protein, adaptor protein 2 is the Skp1 protein, and the cullin is the Cul1 protein. One of the many different F-box proteins completes the complex. (B) Comparison of the same complex with two different substrate-binding arms, the F-box proteins Skp2 (top) and β-trCP1 (bottom), respectively. (C) The binding and ubiquitylation of a target protein by the SCF ubiquitin ligase. If, as indicated, a chain of ubiquitin molecules is added to the same lysine of the target protein, that protein is marked for rapid destruction by the proteasome. (D) Comparison of SCF (bottom) with a low-resolution electron microscopy structure of a ubiquitin ligase called the anaphase-promoting complex (APC/C; top) at the same scale.


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Cell_Biology_Alberts. by the proteasome. (D) Comparison of SCF (bottom) with a low-resolution electron microscopy structure of a ubiquitin ligase called the anaphase-promoting complex (APC/C; top) at the same scale. The APC/C is a large, 15-protein complex. As discussed in Chapter 17, its ubiquitylations control the late stages of mitosis. It is distantly related to SCF and contains a cullin subunit (green) that lies along the side of the complex at right, only partly visible in this view. E2 proteins are not shown here, but their binding sites are indicated in orange, along with substrate-binding sites in purple. (A and B, adapted from G. Wu et al., Mol. Cell 11:1445–1456, 2003. With permission from Elsevier; D, adapted from


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Cell_Biology_Alberts. P. da Fonseca et al., Nature 470:274–278, 2011. With permission from Macmillan Publishers Ltd.)


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Cell_Biology_Alberts. Figure 3–72 The large conformational change in eF-Tu caused by GTP hydrolysis. (A and B) The three-dimensional structure of EF-Tu with GTP bound. The domain at the top has a structure similar to the Ras protein, and its red α helix is the switch helix, which moves after GTP hydrolysis. (C) The change in the conformation of the switch helix in domain 1 allows domains 2 and 3 to rotate as a single unit by about 90 degrees toward the viewer, which releases the tRNA that was bound to this structure (see also Figure 3–73). (A, adapted from H. Berchtold et al., Nature 365:126–132, 1993. With permission from Macmillan Publishers Ltd. B, courtesy of Mathias Sprinzl and Rolf Hilgenfeld. PDB code: 1EFT.) (B) domain 1 P P P GTP-binding site switch helix domain 3 domain 2 PP bound GDP release of tRNA GTP hydrolysis site of tRNA binding G G HOOC NH2 switch helix GTP (A) polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociating the EF-Tu. Since this GTP hydrolysis is triggered


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Cell_Biology_Alberts. GTP hydrolysis site of tRNA binding G G HOOC NH2 switch helix GTP (A) polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociating the EF-Tu. Since this GTP hydrolysis is triggered by a proper fit of the tRNA to the mRNA molecule on the ribosome, the EF-Tu serves as a factor that discriminates between correct and incorrect mRNA–tRNA pairings (see Figure 6–65).


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Cell_Biology_Alberts. By comparing the three-dimensional structure of EF-Tu in its GTP-bound and GDP-bound forms, we can see how the repositioning of the tRNA occurs. The dissociation of the inorganic phosphate group (Pi), which follows the reaction GTP →GDP + Pi, causes a shift of a few tenths of a nanometer at the GTP-binding site, just as it does in the Ras protein. This tiny movement, equivalent to a few times the diameter of a hydrogen atom, causes a conformational change to propagate along a crucial piece of αhelix, called the switch helix, in the Ras-like domain of the protein. The switch helix seems to serve as a latch that adheres to a specific site in another domain of the molecule, holding the protein in a “shut” conformation. The conformational change triggered by GTP hydrolysis causes the switch helix to detach, allowing separate domains of the protein to swing apart, through a distance of about 4 nm (Figure 3–72). This releases the bound tRNA molecule, allowing its attached amino acid to be


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Cell_Biology_Alberts. helix to detach, allowing separate domains of the protein to swing apart, through a distance of about 4 nm (Figure 3–72). This releases the bound tRNA molecule, allowing its attached amino acid to be used (Figure 3–73).


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Cell_Biology_Alberts. Notice in this example how cells have exploited a simple chemical change that occurs on the surface of a small protein domain to create a movement 50 times larger. Dramatic shape changes of this type also cause the very large movements that occur in motor proteins, as we discuss next.


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Cell_Biology_Alberts. We have seen that conformational changes in proteins have a central role in enzyme regulation and cell signaling. We now discuss proteins whose major function is to move other molecules. These motor proteins generate the forces responsible for muscle contraction and the crawling and swimming of cells. Motor proteins also power smaller-scale intracellular movements: they help to move chromosomes to opposite ends of the cell during mitosis (discussed in Chapter 17), to move organelles along molecular tracks within the cell (discussed in Chapter 16), and to move enzymes along a DNA strand during the synthesis of a new DNA molecule (discussed in Chapter 5). All these fundamental processes depend on proteins with moving parts that operate as force-generating machines.


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Cell_Biology_Alberts. How do these machines work? In other words, how do cells use shape changes in proteins to generate directed movements? If, for example, a protein is required to walk along a narrow thread such as a DNA molecule, it can do this by undergoing a series of conformational changes, such as those shown in Figure 3–74. But with nothing to drive these changes in an orderly sequence, they are perfectly


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Cell_Biology_Alberts. Figure 3–73 an aminoacyl tRna molecule bound to eF-Tu. Note how the bound protein blocks the use of the tRNA-linked amino acid (green) for protein synthesis until GTP hydrolysis triggers the conformational changes shown in Figure 3–72C, dissociating the protein-tRNA complex. EF-Tu is a bacterial protein; however, a very similar protein exists in eukaryotes, where it is called EF-1 (Movie 3.12). (Coordinates determined by P. Nissen et al., Science 270:1464–1472, 1995. PDB code: 1B23.) 162 Chapter 3: Proteins reversible, and the protein can only wander randomly back and forth along the thread. We can look at this situation in another way. Since the directional movement of a protein does work, the laws of thermodynamics (discussed in Chapter 2) demand that such movement use free energy from some other source (otherwise the protein could be used to make a perpetual motion machine). Therefore, without an input of energy, the protein molecule can only wander aimlessly.


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Cell_Biology_Alberts. How can the cell make such a series of conformational changes unidirectional? To force the entire cycle to proceed in one direction, it is enough to make any one of the changes in shape irreversible. Most proteins that are able to walk in one direction for long distances achieve this motion by coupling one of the conformational changes to the hydrolysis of an ATP molecule that is tightly bound to the protein. The mechanism is similar to the one just discussed that drives allosteric protein shape changes by GTP hydrolysis. Because ATP (or GTP) hydrolysis releases a great deal of free energy, it is very unlikely that the nucleotide-binding protein will undergo the reverse shape change needed for moving backward— since this would require that it also reverse the ATP hydrolysis by adding a phosphate molecule to ADP to form ATP.


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Cell_Biology_Alberts. In the model shown in Figure 3–75A, ATP binding shifts a motor protein from conformation 1 to conformation 2. The bound ATP is then hydrolyzed to produce ADP and inorganic phosphate (Pi), causing a change from conformation 2 to conformation 3. Finally, the release of the bound ADP and Pi drives the protein back to conformation 1. Because the energy provided by ATP hydrolysis drives the transition 2 → 3, this series of conformational changes is effectively irreversible. Thus, the entire cycle goes in only one direction, causing the protein molecule to walk continuously to the right in this example. Many motor proteins generate directional movement through the use of a similar unidirectional ratchet, including the muscle motor protein myosin, direction of Figure 3–74 an allosteric “walking” protein. Although its three different conformations allow it to wander randomly back and forth while bound to a thread or a filament, the protein cannot move uniformly in a single direction.


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Cell_Biology_Alberts. Figure 3–75 How a protein can walk in one direction. (A) An allosteric motor protein driven by ATP hydrolysis. The transition between three different conformations includes a step driven by the hydrolysis of a bound ATP molecule, creating a “unidirectional ratchet” that makes the entire cycle essentially irreversible. By repeated cycles, the protein therefore moves continuously to the right along the thread. (B) Direct visualization of a walking myosin motor protein by high-speed atomic force microscopy; the elapsed time between steps was less than 0.5 sec (see Movie 16.3). (B, modified from


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Cell_Biology_Alberts. N. Kodera et al., Nature 468:72–76, 2010. With permission from Macmillan Publishers Ltd.) which walks along actin filaments (Figure 3–75B), and the kinesin proteins that walk along microtubules (both discussed in Chapter 16). These movements can be rapid: some of the motor proteins involved in DNA replication (the DNA helicases) propel themselves along a DNA strand at rates as high as 1000 nucleotides per second. Membrane-Bound Transporters Harness Energy to Pump Molecules Through Membranes We have thus far seen how proteins that undergo allosteric shape changes can act as microprocessors (Src family kinases), as assembly factors (EF-Tu), and as generators of mechanical force and motion (motor proteins). Allosteric proteins can also harness energy derived from ATP hydrolysis, ion gradients, or electron-transport processes to pump specific ions or small molecules across a membrane. We consider one example here that will be discussed in more detail in Chapter 11.


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Cell_Biology_Alberts. The ABC transporters (ATP-binding cassette transporters) constitute an important class of membrane-bound pump proteins. In humans, at least 48 different genes encode them. These transporters mostly function to export hydrophobic molecules from the cytoplasm, serving to remove toxic molecules at the mucosal surface of the intestinal tract, for example, or at the blood–brain barrier. The study of ABC transporters is of intense interest in clinical medicine, because the overproduction of proteins in this class contributes to the resistance of tumor cells to chemotherapeutic drugs. In bacteria, the same types of proteins primarily function to import essential nutrients into the cell.


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Cell_Biology_Alberts. A typical ABC transporter contains a pair of membrane-spanning subunits linked to a pair of ATP-binding subunits located just below the plasma membrane. As in other examples we have discussed, the hydrolysis of the bound ATP molecules drives conformational changes in the protein, transmitting forces that cause the membrane-spanning subunits to move their bound molecules across the lipid bilayer (Figure 3–76). Humans have invented many different types of mechanical pumps, and it should not be surprising that cells also contain membrane-bound pumps that


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Cell_Biology_Alberts. Humans have invented many different types of mechanical pumps, and it should not be surprising that cells also contain membrane-bound pumps that Figure 3–76 The abC (aTP-binding cassette) transporter, a protein machine that pumps molecules through a membrane. (A) How this large family of transporters pumps molecules into the cell in bacteria. As indicated, the binding of two molecules of ATP causes the two ATP-binding domains to clamp together tightly, opening a channel to the cell exterior. The binding of a substrate molecule to the extracellular face of the protein complex then triggers ATP hydrolysis followed by ADP release, which opens the cytoplasmic gate; the pump is then reset for another cycle.


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Cell_Biology_Alberts. (B) As discussed in Chapter 11, in eukaryotes an opposite process occurs, causing selected substrate molecules to be pumped out of the cell. (C) The structure of a bacterial ABC transporter (see Movie 11.5). (C, from R.J. Dawson and K.P. Locher, Nature 443:180–185, 2006. With permission from Macmillan Publishers Ltd; PDB code: 2HYD). function in other ways. Among the most notable are the rotary pumps that couple the hydrolysis of ATP to the transport of H+ ions (protons). These pumps resemble miniature turbines, and they are used to acidify the interior of lysosomes and other eukaryotic organelles. Like other ion pumps that create ion gradients, they can function in reverse to catalyze the reaction ADP + Pi → ATP, if the gradient across their membrane of the ion that they transport is steep enough.


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Cell_Biology_Alberts. One such pump, the ATP synthase, harnesses a gradient of proton concentration produced by electron-transport processes to produce most of the ATP used in the living world. This ubiquitous pump has a central role in energy conversion, and we shall discuss its three-dimensional structure and mechanism in Chapter 14. Proteins Often Form Large Complexes That Function as Protein Machines


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Cell_Biology_Alberts. Large proteins formed from many domains are able to perform more elaborate functions than small, single-domain proteins. But large protein assemblies formed from many protein molecules linked together by noncovalent bonds perform the most impressive tasks. Now that it is possible to reconstruct most biological processes in cell-free systems in the laboratory, it is clear that each of the central processes in a cell—such as DNA replication, protein synthesis, vesicle budding, or transmembrane signaling—is catalyzed by a highly coordinated, linked set of 10 or more proteins. In most such protein machines, an energetically favorable reaction such as the hydrolysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered series of conformational changes in one or more of the individual protein subunits, enabling the ensemble of proteins to move coordinately. In this way, each enzyme can be moved directly into position, as the machine catalyzes successive reactions in a series


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Cell_Biology_Alberts. protein subunits, enabling the ensemble of proteins to move coordinately. In this way, each enzyme can be moved directly into position, as the machine catalyzes successive reactions in a series (Figure 3–77). This is what occurs, for example, in protein synthesis on a ribosome (discussed in Chapter 6)—or in DNA replication, where a large multiprotein complex moves rapidly along the DNA (discussed in Chapter 5).


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Cell_Biology_Alberts. Cells have evolved protein machines for the same reason that humans have invented mechanical and electronic machines. For accomplishing almost any task, manipulations that are spatially and temporally coordinated through linked processes are much more efficient than the use of many separate tools. Scaffolds Concentrate Sets of Interacting Proteins


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Cell_Biology_Alberts. Scaffolds Concentrate Sets of Interacting Proteins As scientists have learned more of the details of cell biology, they have recognized an increasing degree of sophistication in cell chemistry. Thus, not only do we now know that protein machines play a predominant role, but it has also become clear that they are very often localized to specific sites in the cell, being assembled and activated only where and when they are needed. As one example, when extracellular signaling molecules bind to receptor proteins in the plasma membrane, the activated receptors often recruit a set of other proteins to the inside surface of the plasma membrane to form a large protein complex that passes the signal on (discussed in Chapter 15).


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Cell_Biology_Alberts. The mechanisms frequently involve scaffold proteins. These are proteins with binding sites for multiple other proteins, and they serve both to link together specific sets of interacting proteins and to position them at specific locations inside a cell. At one extreme are rigid scaffolds, such as the cullin in SCF ubiquitin ligase (see Figure 3–71). At the other extreme are the large, flexible scaffold proteins that often underlie regions of specialized plasma membrane. These include the Figure 3–77 How “protein machines” carry out complex functions.


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Cell_Biology_Alberts. Figure 3–77 How “protein machines” carry out complex functions. These machines are made of individual proteins that collaborate to perform a specific task (Movie 3.13). The movement of these proteins is often coordinated by the hydrolysis of a bound nucleotide such as ATP or GTP. Directional allosteric conformational changes of proteins that are driven in this way often occur in a large protein assembly in which the activities of several different protein molecules are coordinated by such movements within the complex.


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Cell_Biology_Alberts. Discs-large protein (Dlg), a protein of about 900 amino acids that is concentrated in special regions beneath the plasma membrane in epithelial cells and at synapses. Dlg contains binding sites for at least seven other proteins, interspersed with regions of more flexible polypeptide chain. An ancient protein, conserved in organisms as diverse as sponges, worms, flies, and humans, Dlg derives its name from the mutant phenotype of the organism in which it was first discovered; the cells in the imaginal discs of a Drosophila embryo with a mutation in the Dlg gene fail to stop proliferating when they should, and they produce unusually large discs whose epithelial cells can form tumors.


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Cell_Biology_Alberts. Although incompletely studied, Dlg and a large number of similar scaffold proteins are thought to function like the protein that is schematically illustrated in Figure 3–78. By binding a specific set of interacting proteins, these scaffolds can enhance the rate of critical reactions, while also confining them to the particular region of the cell that contains the scaffold. For similar reasons, cells also make extensive use of scaffold RNA molecules, as discussed in Chapter 7. Many Proteins Are Controlled by Covalent Modifications That Direct Them to Specific Sites Inside the Cell We have thus far described only a few ways in which proteins are post-translationally modified. A large number of other such modifications also occur, more than 200 distinct types being known. To give a sense of the variety, Table 3–3 presents


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Cell_Biology_Alberts. Figure 3–78 How the proximity created by scaffold proteins can greatly speed reactions in a cell. In this example, long unstructured regions of polypeptide chain in a large scaffold protein connect a series of structured domains that bind a set of reacting proteins. The unstructured regions serve as flexible “tethers” that greatly speed reaction rates by causing a rapid, random collision of all of the proteins that are bound to the scaffold. (For specific examples of protein tethering, see Figure 3–54 and Figure 16–18; for scaffold RNA molecules, see Figure 7–49B.) a few of the modifying groups with known regulatory roles. As in phosphate and ubiquitin additions described previously, these groups are added and then removed from proteins according to the needs of the cell.


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Cell_Biology_Alberts. A large number of proteins are now known to be modified on more than one amino acid side chain, with different regulatory events producing a different pattern of such modifications. A striking example is the protein p53, which plays a central part in controlling a cell’s response to adverse circumstances (see Figure 17–62). Through one of four different types of molecular additions, this protein can be modified at 20 different sites. Because an enormous number of different combinations of these 20 modifications are possible, the protein’s behavior can in principle be altered in a huge number of ways. Such modifications will often create a site on the modified protein that binds it to a scaffold protein in a specific region of the cell, thereby connecting it—via the scaffold—to the other proteins required for a reaction at that site.


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Cell_Biology_Alberts. One can view each protein’s set of covalent modifications as a combinatorial regulatory code. Specific modifying groups are added to or removed from a protein in response to signals, and the code then alters protein behavior—changing the activity or stability of the protein, its binding partners, and/or its specific location within the cell (Figure 3–79). As a result, the cell is able to respond rapidly and with great versatility to changes in its condition or environment. A Complex Network of Protein Interactions Underlies Cell Function


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Cell_Biology_Alberts. A Complex Network of Protein Interactions Underlies Cell Function There are many challenges facing cell biologists in this information-rich era when a large number of complete genome sequences are known. One is the need to dissect and reconstruct each one of the thousands of protein machines that exist in an organism such as ourselves. To understand these remarkable protein complexes, each will need to be reconstituted from its purified protein parts, so that we can study its detailed mode of operation under controlled conditions in a test tube, free from all other cell components. This alone is a massive task. But we now know that each of these subcomponents of a cell also interacts with other sets of macromolecules, creating a large network of protein–protein and protein–nucleic acid interactions throughout the cell. To understand the cell, therefore, we will need to analyze most of these other interactions as well.


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Cell_Biology_Alberts. Figure 3–79 Multisite protein modification and its effects. (A) A protein that carries a post-translational addition to more than one of its amino acid side chains can be considered to carry a combinatorial regulatory code. Multisite modifications are added to (and removed from) a protein through signaling networks, and the resulting combinatorial regulatory code on the protein is read to alter its behavior in the cell. (B) The pattern of some covalent modifications to the protein p53. We can gain some idea of the complexity of intracellular protein networks from a particularly well-studied example described in Chapter 16: the many dozens of proteins that interact with the actin cytoskeleton to control actin filament behavior (see Panel 16–3, p. 905).


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Cell_Biology_Alberts. The extent of such protein–protein interactions can also be estimated more generally. An enormous amount of valuable information is now freely available in protein databases on the Internet: tens of thousands of three-dimensional protein structures plus tens of millions of protein sequences derived from the nucleotide sequences of genes. Scientists have been developing new methods for mining this great resource to increase our understanding of cells. In particular, computer-based bioinformatics tools are being combined with robotics and other technologies to allow thousands of proteins to be investigated in a single set of experiments. Proteomics is a term that is often used to describe such research focused on the analysis of large sets of proteins, analogous to the term genomics describing the large-scale analysis of DNA sequences and genes.


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Cell_Biology_Alberts. A biochemical method based on affinity tagging and mass spectroscopy has proven especially powerful for determining the direct binding interactions between the many different proteins in a cell (discussed in Chapter 8). The results are being tabulated and organized in Internet databases. This allows a cell biologist studying a small set of proteins to readily discover which other proteins in the same cell are likely to bind to, and thus interact with, that set of proteins. When displayed graphically as a protein interaction map, each protein is represented by a box or dot in a two-dimensional network, with a straight line connecting those proteins that have been found to bind to each other.


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Cell_Biology_Alberts. When hundreds or thousands of proteins are displayed on the same map, the network diagram becomes bewilderingly complicated, serving to illustrate the enormous challenges that face scientists attempting to understand the cell (Figure 3–80). Much more useful are small subsections of these maps, centered on a few proteins of interest.


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Cell_Biology_Alberts. We have previously described the structure and mode of action of the SCF ubiquitin ligase, using it to illustrate how protein complexes are constructed from interchangeable parts (see Figure 3–71). Figure 3–81 shows a network of protein– protein interactions for the five proteins that form this protein complex in a yeast cell. Four of the subunits of this ligase are located at the bottom right of this figure. The remaining subunit, the F-box protein that serves as its substrate-binding arm, appears as a set of 15 different gene products that bind to adaptor protein 2 (the Skp1 protein). Along the top and left of the figure are sets of additional protein interactions marked with yellow and green shading: as indicated, these protein sets function at the origin of DNA replication, in cell cycle regulation, in methionine synthesis, in the kinetochore, and in vacuolar H+-ATPase assembly. We shall use this figure to explain how such protein interaction maps are used, and what they do and do


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Cell_Biology_Alberts. regulation, in methionine synthesis, in the kinetochore, and in vacuolar H+-ATPase assembly. We shall use this figure to explain how such protein interaction maps are used, and what they do and do not mean.


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Cell_Biology_Alberts. 1. Protein interaction maps are useful for identifying the likely function of previously uncharacterized proteins. Examples are the products of the genes that have thus far only been inferred to exist from the yeast genome sequence, which are the three proteins in the figure that lack a simple three-letter abbreviation (white letters beginning with Y). The three in this diagram are F-box proteins that bind to Skp1; these are therefore likely to function as part of the ubiquitin ligase, serving as substrate-binding arms that recognize different target proteins. However, as we discuss next, neither assignment can be considered certain without additional data. 2.


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Cell_Biology_Alberts. 2. Protein interaction networks need to be interpreted with caution because, as a result of evolution making efficient use of each organism’s genetic information, the same protein can be used as part of different protein complexes that have different types of functions. Thus, although protein A binds to protein B and protein B binds to protein C, proteins A and C need not function in the same process. For example, we know from detailed biochemical studies that the functions of Skp1 in the kinetochore and in Figure 3–80 a network of protein-binding interactions in a yeast cell. Each line connecting a pair of dots (proteins) indicates a protein–protein interaction. (From A. Guimerá and M. Sales–Pardo, Mol. Syst. Biol. 2:42, 2006. With permission from Macmillan Publishers Ltd.) Figure 3–81 a map of some protein–protein interactions of the SCF ubiquitin ligase and other proteins in the yeast


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Cell_Biology_Alberts. Figure 3–81 a map of some protein–protein interactions of the SCF ubiquitin ligase and other proteins in the yeast S. cerevisiae. The symbols and/or colors used for the five proteins of the ligase are those in Figure 3–71. Note that 15 different F-box proteins are shown (purple); those with white lettering (beginning with Y) are known from the genome sequence as open reading frames. For additional details, see text. (Courtesy of Peter Bowers and David Eisenberg, UCLA-DOE Institute for Genomics and Proteomics, UCLA.) vacuolar H+-ATPase assembly (yellow shading) are separate from its function in the SCF ubiquitin ligase. In fact, only the remaining three functions of Skp1 illustrated in the diagram—methionine synthesis, cell cycle regulation, and origin of replication (green shading)—involve ubiquitylation.


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Cell_Biology_Alberts. 3. In cross-species comparisons, those proteins displaying similar patterns of interactions in the two protein interaction maps are likely to have the same function in the cell. Thus, as scientists generate more and more highly detailed maps for multiple organisms, the results will become increasingly useful for inferring protein function. These map comparisons will be a particularly powerful tool for deciphering the functions of human proteins, because a vast amount of direct information about protein function can be obtained from genetic engineering, mutational, and genetic analyses in experimental organisms—such as yeast, worms, and flies—that are not feasible in humans.


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Cell_Biology_Alberts. What does the future hold? There are likely to be on the order of 10,000 different proteins in a typical human cell, each of which interacts with 5 to 10 different partners. Despite the enormous progress made in recent years, we cannot yet claim to understand even the simplest known cells, such as the small Mycoplasma bacterium formed from only about 500 gene products (see Figure 1–10). How then can we hope to understand a human? Clearly, a great deal of new biochemistry will be essential, in which each protein in a particular interacting set is purified so that its chemistry and interactions can be dissected in a test tube. But in addition, more powerful ways of analyzing networks will be needed based on mathematical and computational tools not yet invented, as we shall emphasize in Chapter 8. Clearly, there are many wonderful challenges that remain for future generations of cell biologists.


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Cell_Biology_Alberts. Proteins can form enormously sophisticated chemical devices, whose functions largely depend on the detailed chemical properties of their surfaces. Binding sites for ligands are formed as surface cavities in which precisely positioned amino acid side chains are brought together by protein folding. In this way, normally unreactive amino acid side chains can be activated to make and break covalent bonds. Enzymes are catalytic proteins that greatly speed up reaction rates by binding the high-energy transition states for a specific reaction path; they also can perform acid catalysis and base catalysis simultaneously. The rates of enzyme reactions are often so fast that they are limited only by diffusion. Rates can be further increased only if enzymes that act sequentially on a substrate are joined into a single multienzyme complex, or if the enzymes and their substrates are attached to protein scaffolds, or otherwise confined to the same part of the cell.


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Cell_Biology_Alberts. Proteins reversibly change their shape when ligands bind to their surface. The allosteric changes in protein conformation produced by one ligand affect the binding of a second ligand, and this linkage between two ligand-binding sites provides a crucial mechanism for regulating cell processes. Metabolic pathways, for example, are controlled by feedback regulation: some small molecules inhibit and other small molecules activate enzymes early in a pathway. Enzymes controlled in this way generally form symmetric assemblies, allowing cooperative conformational changes to create a steep response to changes in the concentrations of the ligands that regulate them.


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Cell_Biology_Alberts. The expenditure of chemical energy can drive unidirectional changes in protein shape. By coupling allosteric shape changes to ATP hydrolysis, for example, proteins can do useful work, such as generating a mechanical force or moving for long distances in a single direction. The three-dimensional structures of proteins have revealed how a small local change caused by nucleoside triphosphate hydrolysis is amplified to create major changes elsewhere in the protein. By such means, these proteins can serve as input–output devices that transmit information, as assembly factors, as motors, or as membrane-bound pumps. Highly efficient protein machines are formed by incorporating many different protein molecules into larger assemblies that coordinate the allosteric movements of the individual components. Such machines perform most of the important reactions in cells.


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Cell_Biology_Alberts. Proteins are subjected to many reversible, post-translational modifications, such as the covalent addition of a phosphate or an acetyl group to a specific amino acid side chain. The addition of these modifying groups is used to regulate the activity of a protein, changing its conformation, its binding to other proteins, and its location inside the cell. A typical protein in a cell will interact with more than five different partners. Through proteomics, biologists can analyze thousands of proteins in one set of experiments. One important result is the production of detailed protein interaction maps, which aim at describing all of the binding interactions between the thousands of distinct proteins in a cell. However, understanding these networks will require new biochemistry, through which small sets of interacting proteins can be purified and their chemistry dissected in detail. In addition, new computational techniques will be required to deal with the enormous complexity.


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Cell_Biology_Alberts. What are the functions of the surprisingly large amount of unfolded polypeptide chain found in proteins? How many types of protein functions remain to be discovered? What are the most promising approaches for discovering them? When will scientists be able to take any amino acid sequence and accurately predict both that protein’s three-dimensional conformations and its chemical properties? What breakthroughs will be needed to accomplish this important goal? Are there ways to reveal the detailed workings of a protein machine that do not require the purification of each of its component parts in large amounts, so that the machine’s functions can be reconstituted and dissected using chemical techniques in a test tube? What are the roles of the dozens of different types of covalent modifications of proteins that have been found in addition to those listed in Table 3–3? Which ones are critical for cell function and why?


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Cell_Biology_Alberts. Why is amyloid toxic to cells and how does it contribute to neurodegenerative diseases such as Alzheimer’s disease? Which statements are true? explain why or why not. 3–1 Each strand in a β sheet is a helix with two amino acids per turn. 3–2 Intrinsically disordered regions of proteins can be identified using bioinformatic methods to search genes for encoded amino acid sequences that possess high hydrophobicity and low net charge. 3–3 Loops of polypeptide that protrude from the surface of a protein often form the binding sites for other molecules. 3–4 An enzyme reaches a maximum rate at high substrate concentration because it has a fixed number of active sites where substrate binds. 3–5 Higher concentrations of enzyme give rise to a higher turnover number. 3–6 Enzymes that undergo cooperative allosteric transitions invariably consist of symmetric assemblies of multiple subunits.


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Cell_Biology_Alberts. 3–6 Enzymes that undergo cooperative allosteric transitions invariably consist of symmetric assemblies of multiple subunits. 3–7 Continual addition and removal of phosphates by protein kinases and protein phosphatases is wasteful of energy—since their combined action consumes ATP— but it is a necessary consequence of effective regulation by phosphorylation. Discuss the following problems.


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Cell_Biology_Alberts. Discuss the following problems. 3–8 Consider the following statement. “To produce one molecule of each possible kind of polypeptide chain, 300 amino acids in length, would require more atoms than exist in the universe.” Given the size of the universe, do you suppose this statement could possibly be correct? Since counting atoms is a tricky business, consider the problem from the standpoint of mass. The mass of the observable universe is estimated to be about 1080 grams, give or take an order of magnitude or so. Assuming that the average One such kelch repeat domain is shown in Figure Q3–1. Would you classify this domain as an “in-line” or “plug-in” type domain? ˜7 Figure Q3–1 The kelch repeat domain of D. dendroides (Problem 3–10). The seven individual β propellers are color coded and labeled. The Nand C-termini are indicated by N and C.


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Cell_Biology_Alberts. ˜7 Figure Q3–1 The kelch repeat domain of D. dendroides (Problem 3–10). The seven individual β propellers are color coded and labeled. The Nand C-termini are indicated by N and C. 3–11 Titin, which has a molecular weight of about 3 × 106, is the largest polypeptide yet described. Titin molecules extend from muscle thick filaments to the Z disc; they are thought to act as springs to keep the thick filaments centered in the sarcomere. Titin is composed of a large number of repeated immunoglobulin (Ig) sequences of 89 amino acids, each of which is folded into a domain about 4 nm in length (Figure Q3–2A).


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Cell_Biology_Alberts. You suspect that the springlike behavior of titin is caused by the sequential unfolding (and refolding) of individual Ig domains. You test this hypothesis using the atomic force microscope, which allows you to pick up one end of a protein molecule and pull with an accurately measured force. For a fragment of titin containing seven repeats of the Ig domain, this experiment gives the sawtooth force-versus-extension curve shown in Figure Q3–2B. If the experiment is repeated in a solution of 8 M urea (a protein denaturant), the peaks disappear and the measured extension becomes much longer for a given force. If the experiment is repeated after the protein has been cross-linked by treatment with glutaraldehyde, once again the peaks disappear but the extension becomes much smaller for a given force.


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Cell_Biology_Alberts. mass of an amino acid is 110 daltons, what would be the mass of one molecule of each possible kind of polypeptide chain 300 amino acids in length? Is this greater than the mass of the universe? homologous proteins is to search the database using a short signature sequence indicative of the particular protein function. Why is it better to search with a short sequence than with a long sequence? Do you not have more chances 0 for a “hit” in the database with a long sequence? 3–10 The so-called kelch motif consists of a four-stranded β sheet, which forms what is known as a β pro- Figure Q3–2 Springlike behavior of titin (Problem 3–11). (A) The peller. It is usually found to be repeated four to seven times, structure of an individual Ig domain. (B) Force in piconewtons versus forming a kelch repeat domain in a multidomain protein. extension in nanometers obtained by atomic force microscopy. a.


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Cell_Biology_Alberts. a. Are the data consistent with your hypothesis that titin’s springlike behavior is due to the sequential unfolding of individual Ig domains? Explain your reasoning. b. Is the extension for each putative domain-unfolding event the magnitude you would expect? (In an extended polypeptide chain, amino acids are spaced at intervals of 0.34 nm.) C. Why is each successive peak in Figure Q3–2B a little higher than the one before? D. Why does the force collapse so abruptly after each peak?


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Cell_Biology_Alberts. C. Why is each successive peak in Figure Q3–2B a little higher than the one before? D. Why does the force collapse so abruptly after each peak? 3–12 Rous sarcoma virus (RSV) carries an oncogene called Src, which encodes a continuously active protein tyrosine kinase that leads to unchecked cell proliferation. Normally, Src carries an attached fatty acid (myristoylate) group that allows it to bind to the cytoplasmic side of the plasma membrane. A mutant version of Src that does not allow attachment of myristoylate does not bind to the membrane. Infection of cells with RSV encoding either the normal or the mutant form of Src leads to the same high level of protein tyrosine kinase activity, but the mutant Src does not cause cell proliferation. a.


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Cell_Biology_Alberts. a. Assuming that the normal Src is all bound to the plasma membrane and that the mutant Src is distributed throughout the cytoplasm, calculate their relative concentrations in the neighborhood of the plasma membrane. For the purposes of this calculation, assume that the cell is a sphere with a radius (r) of 10 μm and that the mutant Src is distributed throughout the cell, whereas the normal Src is confined to a 4-nm-thick layer immediately beneath the membrane. [For this problem, assume that the membrane has no thickness. The volume of a sphere is (4/3)πr3.] b. The target (X) for phosphorylation by Src resides in the membrane. Explain why the mutant Src does not cause cell proliferation.


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Cell_Biology_Alberts. 3–13 An antibody binds to another protein with an equilibrium constant, K, of 5 × 109 M–1. When it binds to a second, related protein, it forms three fewer hydrogen bonds, reducing its binding affinity by 11.9 kJ/mole. What is the K for its binding to the second protein? (Free-energy change is related to the equilibrium constant by the equation ΔG° = –2.3 RT log K, where R is 8.3 × 10–3 kJ/(mole K) and T is 310 K.) 3–14 The protein SmpB binds to a special species of tRNA, tmRNA, to eliminate the incomplete proteins made from truncated mRNAs in bacteria. If the binding of SmpB to tmRNA is plotted as fraction tmRNA bound versus SmpB concentration, one obtains a symmetrical S-shaped curve as shown in Figure Q3–3. This curve is a visual display of a very useful relationship between Kd and concentration, which has broad applicability. The general expression for fraction of ligand bound is derived from the equation for Kd(Kd = [Pr][L]/[Pr–L]) by substituting ([L]TOT – [L]) for [Pr–L] and


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Cell_Biology_Alberts. which has broad applicability. The general expression for fraction of ligand bound is derived from the equation for Kd(Kd = [Pr][L]/[Pr–L]) by substituting ([L]TOT – [L]) for [Pr–L] and rearranging. Because the total concentration of ligand ([L]TOT) is equal to the free ligand ([L]) plus bound ligand ([Pr–L]), 1.0


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Cell_Biology_Alberts. Figure Q3–3 Fraction of tmRNA bound versus SmpB concentration (Problem 3–14). concentration of SmpB (M) For SmpB and tmRNA, the fraction bound = [SmpB– tmRNA]/[tmRNA]TOT = [SmpB]/([SmpB] + Kd). Using this relationship, calculate the fraction of tmRNA bound for SmpB concentrations equal to 104 Kd, 103 Kd, 102 Kd, 101 Kd, Kd, 10–1 Kd, 10–2 Kd, 10–3 Kd, and 10–4 Kd. 3–15 Many enzymes obey simple Michaelis–Menten kinetics, which are summarized by the equation where Vmax = maximum velocity, [S] = concentration of substrate, and Km = the Michaelis constant. It is instructive to plug a few values of [S] into the equation to see how rate is affected. What are the rates for [S] equal to zero, equal to Km, and equal to infinite concentration? 3–16 The enzyme hexokinase adds a phosphate to D-glucose but ignores its mirror image, L-glucose. Suppose that you were able to synthesize hexokinase entirely from D-amino acids, which are the mirror image of the normal L-amino acids. a.


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Cell_Biology_Alberts. a. Assuming that the “D” enzyme would fold to a stable conformation, what relationship would you expect it to bear to the normal “L” enzyme? b. Do you suppose the “D” enzyme would add a phosphate to L-glucose, and ignore D-glucose? 3–17 How do you suppose that a molecule of hemoglobin is able to bind oxygen efficiently in the lungs, and yet release it efficiently in the tissues? 3–18 Synthesis of the purine nucleotides AMP and GMP proceeds by a branched pathway starting with ribose 5-phosphate (R5P), as shown schematically in Figure Q3–4. Using the principles of feedback inhibition, propose a regulatory strategy for this pathway that ensures an adequate supply of both AMP and GMP and minimizes the buildup of the intermediates (A–I) when supplies of AMP and GMP are adequate. 0 0.25 0.5 0.75 Figure Q3–4 Schematic diagram of the metabolic pathway for synthesis of AMP and GMP from R5P (Problem 3–18).


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Cell_Biology_Alberts. 0 0.25 0.5 0.75 Figure Q3–4 Schematic diagram of the metabolic pathway for synthesis of AMP and GMP from R5P (Problem 3–18). Berg JM, Tymoczko JL & Stryer L (2011) Biochemistry, 7th ed. New York: WH Freeman. Branden C & Tooze J (1999) Introduction to Protein Structure, 2nd ed. New York: Garland Science. Dickerson RE (2005) Present at the Flood: How Structural Molecular Biology Came About. Sunderland, MA: Sinauer. Kuriyan J, Konforti B & Wemmer D (2013) The Molecules of Life: Physical and Chemical Principles. New York: Garland Science. Perutz M (1992) Protein Structure: New Approaches to Disease and Therapy. New York: WH Freeman. Petsko GA & Ringe D (2004) Protein Structure and Function. London: New Science Press. Williamson M (2011) How Proteins Work. New York: Garland Science. The Shape and Structure of Proteins Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181, 223–230.


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Cell_Biology_Alberts. Williamson M (2011) How Proteins Work. New York: Garland Science. The Shape and Structure of Proteins Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181, 223–230. Caspar DLD & Klug A (1962) Physical principles in the construction of regular viruses. Cold Spring Harb. Symp. Quant. Biol. 27, 1–24. Dill KA & MacCallum JL (2012) The protein-folding problem, 50 years on. Science 338, 1042–1046. Eisenberg D (2003) The discovery of the alpha-helix and beta-sheet, the principal structural features of proteins. Proc. Natl Acad. Sci. USA 100, 11207–11210. Fraenkel-Conrat H & Williams RC (1955) Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc. Natl Acad. Sci. USA 41, 690–698. Goodsell DS & Olson AJ (2000) Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 29, 105–153.


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Cell_Biology_Alberts. Goodsell DS & Olson AJ (2000) Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 29, 105–153. Greenwald J & Riek R (2010) Biology of amyloid: structure, function, and regulation. Structure 18, 1244–1260. Harrison SC (1992) Viruses. Curr. Opin. Struct. Biol. 2, 293–299. Hudder A, Nathanson L & Deutscher MP (2003) Organization of mammalian cytoplasm. Mol. Cell. Biol. 23, 9318–9326. Kato M, Han TW, Xie S et al. (2012) Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767. Koga N, Tatsumi-Koga R, Liu G et al. (2012) Principles for designing ideal protein structures. Nature 491, 222–227. Li P, Banjade S, Cheng H-C et al. (2012) Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340.