In-Text Questions and Exercises Chapter 6

6.1 Lipid Structure and Function Lipid is a catchall term for carbon-containing compounds that are found in organisms and are largely nonpolar and hydrophobic meaning that they do not dissolve readily in water. (Recall from Chapter 2 that water is a polar solvent.) Lipids do dissolve,, however,, in liquids consisting of nonpolar organic compounds. To understand why lipids are insoluble in water, examine the five-carbon compound called isoprene, illustrated in FIGURE 6.1a. Note that isoprene consists of carbon atoms bonded to hydrogen atoms. The figure also shows the structural formula of a chain of linked isoprenes, called an isoprenoid. Molecules that contain only carbon and hydrogen are known as hydrocarbons. Hydrocarbons are nonpolar because electrons are shared equally in C-H bonds-owing to the approximately equal electronegativity of carbon and hydrogen. Since these bonds form no partial charges, hydrocarbons are hydrophobic. Thus lipids do not dissolve in water, because they have a significant hydrocarbon component. Bond Saturation is an important Aspect of Hydrocarbon Structure FIGURE 6.1b gives the structural formula of a fatty acid, a simple lipid consisting of a hydrocarbon chain bonded to a carboxyl (-COOH) functional group. Fatty acids and isoprenes are key building blocks of important lipids found in organisms. Just as subtle differences in the orientation of hydroxyls in sugars can lead to dramatic effects in their structure and function, the type of C-C bond used in hydrocarbon chains is a key factor in lipid structure. When two carbon atoms form a double bond, the attached atoms are found in a plane instead of a three-dimensional tetrahedron. The carbon atoms involved are also locked into place. They cannot rotate freely, as they do in carbon-carbon single bonds. As a result, certain double bonds between carbon atoms produce a "kink" in an otherwise straight hydrocarbon chain (Figure 6.1b, left). Hydrocarbon chains that consist of only single bonds between the carbons are called saturated. If one or more double bonds exist in the hydrocarbon chains, then they are unsaturated. The choice of terms is logical. If a hydrocarbon chain does not contain a double bond, it is saturated with the maximum number of hydrogen atoms that can attach to the carbon skeleton. If it is unsaturated, then a C-H bond is removed to form a C=C double bond, resulting in fewer than the maximum number of attached hydrogen atoms. Foods that contain lipids with many double bonds are said to be polyunsaturated and are advertised as healthier than foods with saturated fats. Recent research suggests that polyunsaturated fats help protect the heart from disease. Exactly how this occurs is under investigation. Bond saturation also profoundly affects the physical state of lipids. Highly saturated fats, such as butter, are solid at room temperature (FIGURE 6.2a). Saturated lipids that have extremely long hydrocarbon tails, like waxes do, form particularly stiff sol ids at room temperature (FIGURE 6.2b). Highly unsaturated fats are liquid at room temperature (FIGURE 6.2c). A Look at Three Types of Lipids Found in Cells Unlike amino acids, nucleotides, and monosaccharides, lipids are characterized by a physical property—their insolubility in water-instead of a shared chemical structure. This insolubility is based on the high proportion of nonpolar C-C and C-H bonds relative to polar functional groups. As a result, the structure of lip ids varies widely. For example, consider the most important types of lipids found in cells: fats, steroids, and phospholipids. Fats Fats are nonpolar molecules composed of three fatty acids that are linked to a three-carbon molecule called glycerol. Be cause of this structure, fats are also called triacylglycerols or triglycerides. When the fatty acids are polyunsaturated, they form liquid triacylglycerols called oils. In organisms, the primary role of fats is energy storage. As FIGURE 6.3a shows, fats form when a dehydration reaction occurs between a hydroxyl group of glycerol and the carboxyl group of a fatty acid. The glycerol and fatty acid molecules become joined by an ester linkage. Fats are not polymers, however, and fatty acids are not monomers. As FIGURE 6.3b shows, fatty acids are not linked together to form a macromolecule in the way that amino acids, nucleotides, and monosaccharides are. Steroids Steroids are a family of lipids distinguished by the bulky, four-ring structure shown in orange in FIGURE 6.4a. The various steroids differ from one another by the functional Groups or side groups attached to different carbons in those hydrophobic rings. The steroid shown in the figure is cholesterol, which has a hydrophilic hydroxyl group attached to the top ring and an isoprenoid "tail"" attached at the bottom. Cholesterol is an important component of plasma membranes in many organisms. Phospholipids Phospholipids consist of a glycerol that is linked to a phosphate group and two hydrocarbon chains of either isoprenoids or fatty acids. The phosphate group is also bonded to a small organic molecule that is charged or polar (FIGURE 6.4b). Phospholipids composed of fatty acids are found in the domains Bacteria and Eukarya;; phospholipids with isoprenoid lains are found in the domain Archaea. (The domains of life were introduced in Chapter 1.) In all three domains, phospholipids crucial components of the plasma membrane. The lipids found in organisms have a wide array of structures and functions.. In addition to storing chemical energy, lipids act As pigments that capture or respond to sunlight, serve as signals between cells,, form waterproof coatings on leaves and skin, and act as vitamins used in many cellular processes. The most prominent function of lipids, however, is their role in cell membranes. The Structures of Membrane Lipids Not all lipids can form membranes. Membrane-forming lipids have a polar, hydrophilic region-in addition to the nonpolar, hydrophobic region found in all lipids. To better understand this structure, take another look at the phospholipid illustrated in Figure 6.4b. Notice that the molecule has a "head" region containing highly polar covalent bonds as well as a negatively charged phosphate attached to a polar or charged group. The charges and polar bonds in the head region interact with water molecules when a phospholipid is placed in solution. In contrast, the long hydrocarbon tails of a phospholipid are nonpolar and hydrophobic. Water molecules cannot form hydrogen bonds with the hydrocarbon tail, so they do not interact extensively with this part of the molecule. Compounds that contain both hydrophilic and hydrophobic elements are amphipathic (literally, dual-sympathy"). Phospholipids are amphipathic. As Figure 6.4a shows, cholesterol is also amphipathic. Because it has a hydroxyl functional group attached to its rings, it has both hydrophilic and hydrophobic regions. If you understand these concepts, you should be able to look back at Figure 6.1b and explain why fatty acids are also amphipathic. The amphipathic nature of phospholipids is far and away their most important feature biologically. It is responsible for life's defining barrier—the plasma membrane. If the membrane defines life, then amphipathic lipids must have existed when life first originated during chemical evolution. Was that possible? Were Lipids Present during Chemical Evolution? Like amino acids, nucleic acids, and carbohydrates (Chapters 3-5), there is evidence that lipids were present during chemical evolution. Laboratory experiments have shown that simple lipids, such as fatty acids, can be synthesized from H2 and CO2 via reactions with mineral catalysts under conditions thought to be present in prebiotic hydrothermal vent systems (Chapter 2). It is also possible that lipids literally fell from the sky early in Earth's history. Modern meteorites have been found to contain not only amino acids and carbohydrates but also lipids that exhibit amphipathic qualities. For example, lipids extracted from the meteorite that struck Murchison, Australia, in 1969 spontaneously formed lipid "bubbles" that resembled small cells. Why do amphipathic lipids do this? check your understanding If you understand that • Fats, steroids, and phospholipids differ in structure and function. • Fats and oils are nonpolar; fatty acids, phospholipids, and certain steroids, like cholesterol, are amphipathic because they have both polar and nonpolar regions. • Fats store chemical energy; certain steroids and phospholipids are key components of plasma membranes. You should be able to ... 1. Compare and contrast the structure of a fat, a steroid, and a phospholipid. 2. Based on their structure, explain what makes cholesterol and phospholipids amphipathic.

(1) Fats consist of three fatty acids linked to glycerol; steroids have a distinctive four-ring structure with variable side groups attached; phospholipids have a hydrophilic, phosphate-containing "head" region and a hydrocarbon tail. (2) understand in cholesterol, the hydrocarbon steroid rings and isoprenoid chain are hydrophobic; the hydroxyl group is hydrophilic. In phospholipids, the phosphate-containing head group is hydrophilic; the hydrocarbon chains are hydrophobic.

6.2 Phospholipid Bilayers Amphipathic lipids do not dissolve when they are placed in water. Their hydrophilic heads interact with water, but their hydrophobic tails do not. Instead of dissolving in water, then, amphipathic lipids assume one of two types of structures: micelles or lipid bilayers. • Micelles (FIGURE 6.5a) are tiny droplets created when the hydrophilic heads of a set of lipids face the water and form hydrogen bonds, while the hydrophobic tails interact with each other in the interior, away from the water. A lipid bilayer is created when two sheets of lipid molecules align. As FIGURE 6.5b shows, the hydrophilic heads in each layer face the surrounding solution while the hydrophobic tails face one another inside the bilayer. In this way, the hydrophilic heads interact with water while the hydrophobic tails interact with one another. Figure 6.5 Lipids Form Micelles and Bilayers in Solution in (a) micelle or (b) a lipid bilayer, the hydrophilic heads of lipids face out, toward water; the hydrophobic tails face in, away from water. Lipid bilayers are the foundation of plasma membranes (a) lipid micelles Hydrophilic heads interact with water Hydrophobic tails interact with on another (b) lipid bilayers Hydrophilic heads interact with water Hydrophobic tails interact with one another Micelles tend to form from fatty acids or other simple amphipathic hydrocarbon chains. Bilayers tend to form from phospholipids that contain two hydrocarbon tails. For this reason, bilayers are often called phospholipid bilayers. It's critical to recognize that micelles and phospholipid bilay ers form spontaneously-no input of energy is required. This concept can be difficult to grasp because entropy clearly decreases when these structures form. The key is to recognize that micelles and lipid bilayers are much more stable energetically than are independent phospholipids in solution. Independent lipids are unstable in water because their hydrophobic tails disrupt hydrogen bonds that could otherwise form between water molecules. As a result, the tails of amphipathic molecules are forced together and participate in hydrophobic interactions (introduced in Chapter 2). This point should also remind you of the aqueous behavior of hydrophobic side chains in proteins and bases in nucleic acids. Artificial Membranes as an Experimental System When phospholipids are added to an aqueous solution and agitated, lipid bilayers spontaneously form small spherical structures. The hydrophilic heads on both sides of the bilayer remain in contact with the aqueous solution-water is present both inside and outside the vesicle. Artificial membrane-bound vesicles like these are called liposomes (FIGURE 6.6). Liposomes are Artificial Membrane-Bound Vesicles Electron micrograph of liposomes in cross section and a cross sectional diagram of the lipid bilayer in a liposome. To explore how membranes work, researchers began creating and experimenting with liposomes and planar bilayers-lipid bilayers constructed across a hole in a glass or plastic wall separating two aqueous solutions (FIGURE 6.7a) Use of Planar Bilayers in Experiments. (a) The construction of a planar bilayer across a hole in a wall separating two water-filled compartments. Planar Bilayers: artificial membrane (b) A wide variety of experiments are possible with planar bilayers; a few are suggested here. Artificial-membrane experiments How rapidly can different solutes cross the membrane (if at all) when... 1. Different types of phospholipids are used to make the membrane 2. Proteins or other molecules are added to the membrane? Some of the first questions they posed concerned the permeability of lipid bilayers. The permeability of a structure is its tendency to allow a given substance to pass through it. Using liposomes and planar bilayers, researchers can study what happens when a known ion or molecule is added to one side of a lipid bilayer (FIGURE 6.7b). Does the substance cross the membrane and show up on the other side? If so, how rapidly does the movement take place? What happens when a different type of phospholipid is used to make the artificial membrane? Does the membrane's permeability change when proteins or other types of molecules become part of it? Biologists describe such an experimental system as elegant and powerful because it gives them precise control over which factor changes from one experimental treatment to the next. Control, in turn, is why experiments are such an effective way to explore scientific questions. Recall that good experimental design allows researchers to alter one factor at a time and determine what effect, if any, each has on the process being studied (Chapter 1). Selective Permeability of Lipid Bilayers When researchers put molecules or ions on one side of a liposome or planar bilayer and measure the rate at which the molecules arrive on the other side, a clear pattern emerges: Lipid bilayers are highly selective. Selective permeability means that some substances cross a membrane more easily than other substances do. Small nonpolar molecules move across bilayers quickly. In contrast, large molecules and charged substances cross the membrane slowly, if at all. This difference in membrane permeability is a critical issue because controlling what passes between the exterior and interior environments is a key characteristic of cells. According to the data in FIGURE 6.8, small nonpolar molecules such as oxygen (O2) move across selectively permeable membranes more than a billion times faster than do chloride ions (CL-). In essence, ions cannot cross membranes at all-unless they have "help" in the form of membrane proteins introduced later in the chapter. Very small and uncharged molecules such as water (H2O) can cross membranes relatively rapidly, even if they are polar. Small polar molecules such as glycerol have intermediate permeability. The leading hypothesis to explain this pattern is that charged compounds and large polar molecules are more stable dissolved in water than they are in the nonpolar interior of membranes. If you understand this hypothesis, you should be able to predict where amino acids and nucleotides would be placed in Figure 6.8 and explain your reasoning. Figure 6.8 Lipid Bilayers Show Selective Permeability. Only certain substances cross lipid bilayers readily. Size and Polarity or charge affect the rate of diffusion across a membrane. High permeability (cm/sec) small, nonpolar molecules small, uncharged polar molecules Large, uncharged polar molecules Ions Low permeability

Amino acids have amino and carboxyl groups that are ionized in water and nucleotides have negatively charged phosphates. Due to their charge and larger size, both would be placed below the small ions at the bottom of the scale (permeability<10^-12 cm/sec)

FIGURE 6.4 Some Lipids Contain Hydrophilic and Hydrophobic Regions. (a) All steroids have the distinctive four-ring structure shown in orange. Cholesterol has a polar hydroxyl group and an isoprenoid chain attached to these rings. (b) Most phospholipids consist of two fatty acid or isoprenoid chains that are linked to glycerol, which is linked to a phosphate group, which is linked to a small organic molecule that is polar or charged. QUESTION If cholesterol and phospholipids were in solution, which part of the molecules would interact with water molecules?

At the polar hydroxyl group in cholesterol and the polar head group in phospholipids.

Figure 6.22 Membrane Channels are highly selective. Outside Cell Key residues allow water to pass but block ions and larger molecules Inside cell A cutaway view looking at the inside of a membrane channel, aquaporin. The key residues identified in the space-filling model selectively filter ions and other small molecules, allowing only water (red and white structures) to pass through Figure 6.23 Some Membrane Channels Are Highly Regulated. A model of a voltage-gated K+ channel in the closed and open configurations. The channel filter displaces water molecules that normally surround the K+ ions in an aqueous solution Outside Cell Gate blocking ions from entering channel Inside Cell When the inside of the membrane is negatively charged relative to the outside, channel is closed. Outside cell Filter allows only K+ ions to pass Inside cell If membrane charge asymmetry is reversed, channel opens Figure 6.24 Carrier Proteins Undergo Structural Changes to Move Substances 1. Unbound protein: GLUT-1 is a transmembrane transport protein, shown with its binding site facing outside the cell Outside cell Glucose GLUT-1 Inside cell 2. Glucose binding: Glucose binds to GLUT-1 from outside the cell. 3. Conformational change: Glucose binding causes a conformational change, transporting glucose to the interior. 4. Release: Glucose moves inside the cell. Steps may repeat or reverse, depending on the concentration gradient. This model suggests that GLUT-1 binds a glucose molecule, undergoes a conformational change, and releases glucose on the other side of the membrane. Figure 6.25 The Sodium-potassium pump depends on an input of Chemical Energy Stored in ATP PROCESS: How the Sodium-Potassium Pump (Na+/K+ -ATPase) Works 1. Unbound Proteins: Three binding sites within the protein have a high affinity for sodium ions Outside cell K+ ions Vacant Protein site with affinity for Na+ ions Inside cell Na+ ions 2. Sodium binding: Three sodium ions from the inside of the cell bind to these sites Outside cell K+ ions Three Na+ ions bind to protein sites Inside cell ATP Molecule 3. Shape Change: A phosphate group from ATP binds to the protein. In response, the protein changes shape Outside cell K+ ions Three Na+ ions unbind from protein site, 1 phosphate group binds to protein site Inside cell ADP Molecule 4. Release: The sodium ions leave the protein and move to the exterior of the cell. Outside cell K+ ions Na+ ions Vacant Protein Site with conformational change Inside cell Phosphate group bound to protein site 5. Unbound protein: In this conformation, the protein has binding sites with a high affinity for potassium ions Outside cell K+ ions Na+ ions Vacant Protein site with affinity for K+ ions Inside cell Phosphate group bound to protein site 6. Potassium binding: Two potassium ions bind to the pump Outside cell Na+ ions Protein site binding with K+ ions Inside cell Phosphate group bound to protein site 7. Shape Change: The phosphate group is cleaved from the protein, allowing the pump to return to its original shape. Outside cell Na+ ions Original conformation Protein site with loose K+ ions Inside cell Phosphate group cleaved from protein site 8. Release: The potassium ions leave the protein and diffuse to the interior of the cell. These 8 steps repeat. Figure 6.26 Summary of the Passive and Active Mechanisms of Membrane Transport. Complete the Chart.

Diffusion: description as given; no proteins involved. Facilitated diffusion: Passive movement of ions or molecules that cannot cross a phospholipid bilayer readily along a concentration gradient; facilitated by channel or carrier proteins. Active transport: Active movement of ions or molecules that will build a gradient; facilitated by pump proteins powered by an energy source such as ATP.

The Structures of Membrane Lipids The Structures of Membrane Lipids Not all lipids can form membranes. Membrane-forming lipids have a polar, hydrophilic region-in addition to the nonpolar, hydrophobic region found in all lipids. To better understand this structure, take another look at the phospholipid illustrated in Figure 6.4b. Notice that the molecule has a "head" region containing highly polar covalent bonds as well as a negatively charged phosphate attached to a polar or charged group. The charges and polar bonds in the head region interact with water molecules when a phospholipid is placed in solution. In contrast, the long hydrocarbon tails of a phospholipid are nonpolar and hydrophobic. Water molecules cannot form hydrogen bonds with the hydrocarbon tail, so they do not interact extensively with this part of the molecule. Compounds that contain both hydrophilic and hydrophobic elements are amphipathic (literally, "dual-sympathy"). Phospholipids are amphipathic. As Figure 6.4a shows, cholesterol is also amphipathic. Because it has a hydroxyl functional group attached to its rings, it has both hydrophilic and hydrophobic regions If you understand these concepts, you should be able to look back at Figure 6.1b and explain why fatty acids are also amphipathic.. Figure 6.1 Hydrocarbon Structure. (a) Isoprene subunits, like the one shown on the left, can be linked to each other, end to end, to form long hydrocarbon chains called isoprenoids. Isoprene Isoprenoid Hydrocarbon chain (b) Fatty acids typically contain a total of 14-20 carbon atoms, most found in their long hydrocarbon "tails." Unsaturated hydrocarbons contain carbon-carbon double bonds: saturated hydrocarbons do not. Fatty acids Carboxyl group Hydrocarbon chain In unsaturated fatty acid, cis double bonds cause kinks in hydrocarbon chains.

Fatty acids are amphipathic because their hydrocarbon tails are hydrophobic but their carboxyl functional groups are hydrophilic

Process: Osmosis Figure 6.13 Osmosis Is the Diffusion of Water. 1. Unequal concentrations across membrane: Start with more solute on one side of the lipid bilayer than the other, using a solute that cannot cross the selectively permeable membrane. 2. Water movement: Water undergoes a net movement from the region of low concentration of solute to the region of high concentration of solute. Suppose you doubled the number of solute molecules on the left of the membrane (at the start). At equilibrium, would the water level on the left side be higher or lower than what is the water level on Shown in the second drawing?

Higher, because less water would have to move to the right side to achieve equilibrium.

6.3 How Molecules Move across Lipid Bilayers: Diffusion and Osmosis Small uncharged molecules and hydrophobic compounds can cross membranes readily and spontaneously-without an input of energy. The question now is: How is this possible? What process is responsible for movement of molecules across lipid bilayers? Diffusion A thought experiment can help explain how substances can cross membranes spontaneously. Suppose you rack up a set of billiard balls in the middle of a pool table and then begin to vibrate the table. 1. Because of the vibration, the billiard balls will move about randomly. They will also bump into one another. 2. After these collisions, some balls will move outward-away from their original position. 3. As movement and collisions continue, the overall or net movement of balls will be outward. This occurs because the random motion of the balls disrupts their original, nonrandom position. As the balls move at random, they are more likely to move away from one another than to stay together. 4. Eventually, the balls will be distributed randomly across the table. The entropy of the billiard balls has increased. Recall that entropy is a measure of the randomness or disorder in a system (Chapter 2). The second law of thermodynamics states that in an isolated system, entropy always increases. This hypothetical example illustrates how vibrating billiard balls move at random. More to the point, it also explains how substances located on one side of a lipid bilayer can move to the other side spontaneously. All dissolved molecules and ions, or solutes, have thermal energy and are in constant, random motion. Movement of molecules and ions that results from their kinetic energy is known as diffusion. A difference in solute concentrations creates what is called a concentration gradient. Solutes move randomly in all directions, but when a concentration gradient exists, there is a net movement from regions of high concentration to regions of low concentration. Diffusion down a concentration gradient, or away from the higher concentration, is a spontaneous process because it results in an increase in entropy. Once the molecules or ions are randomly distributed throughout a solution, a chemical equilibrium is established. For example, consider two aqueous solutions separated by a lipid bilayer. FIGURE 6.12 shows how molecules that can pass through the bilayer diffuse to the other side. At equilibrium, these molecules continue to move back and forth across the membrane, but at equal rates-simply because they are equally likely to move in any direction. This means that there is no longer a net movement of molecules across the membrane. If you understand diffusion, you should be able to predict how a difference in temperature across a membrane would affect the concentration of a solute at equilibrium.

If there is a difference in temperature, then there would be a difference in thermal motion. The solute concentration on the side with a higher temperature would decrease because the solute particles would be moving faster and hence be more likely to move to the cooler side of the membrane, where they would slow down.

Cholesterol Reduces Membrane Permeability Cholesterol molecules are present, to varying extents, in the membranes of every cell in your body. What effect does adding cholesterol have on a membrane? Researchers have found that adding cholesterol molecules to liposomes dramatically reduces the permeability of lipid bilayers. The data behind this conclusion are presented in FIGURE 6.10. To read the graph in the "Results" section of Figure 6.10., your finger on the x-axis at the point marked 20°C, and note that permeability to glycerol is much higher at this temperature in membranes that contain no cholesterol versus 20 percent or 50 percent cholesterol. Using this procedure at other temperature points should convince you that membranes lacking cholesterol are more permeable than the other two membranes at every temperature tested in the experiment. What explains this result? Because the steroid rings in cholesterol are bulky, adding cholesterol fills gaps that would otherwise be present in the hydrophobic section of the membrane. How Does Temperature Affect the Fluidity and Permeability of Membranes? At about 25°C-or "room temperature"—the phospholipids in a plasma membrane have a consistency resembling olive oil. This fluid physical state allows individual lipid molecules to move laterally within each layer (FIGURE 6.11), a little like a person moving about in a dense crowd. By tagging individual phospholipids and following their movement, researchers have clocked average speeds of 2 micrometers (um)/second at room temperature. At these speeds, a phospholipid could travel the length of a small bacterial cell in a second. Recall that permeability is closely related to fluidity. As temperature drops, molecules in a bilayer move more slowly. As a result, the hydrophobic tails in the interior of membranes pack together more tightly. At very low temperatures. lipid bilayers even begin to solidify. As the graph in Figure 6.10 indicates, low temperatures can make membranes impervious to molecules that would normally cross them readily. Put your finger on the x-axis of that graph, just about the freezing point of water (0*C), and note that even membranes that lack cholesterol are almost completely impermeable to glycerol. Indeed, trace any of the three lines in Figure 6.10, and as you move to the right (increasing temperature), you also move up (increasing permeability). These observations on glycerol and lipid movement demonstrate that membranes are dynamic. Phospholipid molecules whiz around each layer, while water and small nonpolar molecules shoot in and out of the membrane. How quickly molecules move within and across membranes is a function of temperature, the structure of hydrocarbon tails, and the number of cholesterol molecules in the bilayer. RESEARCH QUESTION: Does adding cholesterol to a membrane affect its permeability? HYPOTHESIS: Cholesterol reduces permeability because it fills spaces in phospholipid bilayers. NULL HYPOTHESIS: Cholesterol has no effect on permeability. EXPERIMENTAL SETUP: 1. Construct liposomes: Create with no cholesterol, 20% cholesterol, and 50% cholesterol. Phospholipids Cholesterol 2. Measure Glycerol movement: Record how quickly glycerol moves across each type of membrane at different temperatures. Liposome Glycerol PREDICTION: Liposomes with higher cholesterol levels will have reduced permeability. PREDICTION OF NULL HYPOTHESIS: All liposomes will have the same permeability. RESULTS: Permeability of membrane to glycerol No cholesterol 20% of lipids = cholesterol 50% of lipids = cholesterol Temperature(*C) CONCLUSION: Adding cholesterol to membranes decreases their permeability to glycerol. The permeability of all membranes analyzed in this experiment increases with increasing temperature FIGURE 6.10 The Permeability of a Membrane Depends on Its Composition QUANTITATIVE Suppose the investigators had instead created liposomes using phospholipids with fully saturated tails and compared them to two other sets of liposomes where either 20 percent or 50 percent of the phospholipids contained polyunsaturated tails. Label the three lines on the graph above with your prediction for the three different liposomes in this new experiment.

Increasing the number of phospholipids with polyunsaturated tails would increase permeability of the liposomes. Starting from the left, the first line (no cholesterol) would represent liposomes with 50% polyunsaturated phospholipids, the second line would be 20% polyunsaturated phospholipids, and the third line would contain only saturated phospholipids

Is an ion Channel Involved in Cystic Fibrosis? To understand the types of experiments that biologists do to confirm that a membrane protein is an ion channel, consider work on the cause of cystic fibrosis. cystic fibrosis (CF) is the most common genetic disease in humans of Northern European descent. It affects cells that produce mucus, sweat, and digestive juices. Normally these secretions are thin and slippery and act as lubricants. In individuals with CF, however, the secretions become abnormally concentrated and sticky and clog passageways in organs like the lungs. Experiments published in 1983 suggested that cystic fibrosis is caused by defects in a membrane protein that allow chloride ions (Cl-) to move across plasma membranes. It was proposed that reduced chloride ion transport would account for the thick mucus. How is the transport of chloride ions involved in mucus consistency? Water movement across cell membranes is largely determined by the presence of extracellular ions like chloride. If a defective channel prevents chloride ions from leaving cells, water isn't pulled from cells by osmosis to maintain the proper mucus consistency. In effect, the disease results from the mismanagement of osmosis. Using molecular techniques introduced in Unit 3 (see Chapter 20), biologists were able to (1) find the gene that is defective in people suffering from CF and (2) use the gene to produce copies of the normal protein, which was called CFTR (short for cystic fibrosis transmembrane conductance regulator). Is CFTR a chloride channel? To answer this question, researchers inserted purified CFTR into planar bilayers and measured the flow of electric current across the membrane. Because ions carry a charge, ion movement across a membrane produces an electric current. The graphs in FIGURE 6.21, which plot the amount of current flowing across this membrane over time, show the results from this experiment. Notice that when CFTR was absent, no electric current passed through the membrane. But When CFTR was inserted into the membrane, current began to flow. This was strong evidence that CFTR was indeed a chloride ion channel. QUESTION: Is CFTR a chloride channel? Hypothesis: CFTR increases the flow of chloride ions across a membrane. Null hypothesis: CFTR has no effect on membrane permeability Experimental Setup: Membrane without CFTR Ion flow? Membrane with CFTR Ion Flow? 1. Create planar bilayers with and without CFTR. 2. Add chloride ions to one side of the planar bilayer to create an electrochemical gradient. 3. Record electrical currents to measure ion flow across the planar bilayers. PREDICTION: Ion flow will be higher in membrane with CFTR PREDICTION OF NULL HYPOTHESIS: Ion flow will be the same in both membranes. RESULTS: Current (picoamperes) with CFTR without CFTR (shortly with CFTR added) Time CONCLUSION: CFTR facilitates diffusion of chloride ions along an electrochemical gradient. CFTR is a chloride channel. FIGURE 6.21 Electric Current Measurements Indicate that Chloride Flows through CFTR. QUESTION: The researchers repeated the "with CFTR" treatment 45 times, but recorded a current in only 35 of the replicates. Does this observation negate the conclusion? Explain why or why not.

No—the 10 replicates where no current was recorded probably represent instances where the CFTR protein was damaged and not functioning properly. (In general, no experimental method works "perfectly.")

Protein Structure Determines Channel Selectivity Subsequent research has shown that cells have many different types of pore like channel proteins in their membranes, including ion channels like CFTR.. Channel proteins are selective. Each channel protein has a structure that permits only a particular type of ion or small molecule to pass through it. For example, Peter Agre and co-workers discovered channels called aquaporins ("water-pores") that allow water to cross the plasma membrane over 10 times faster than it does in the absence of aquaporins. Aquaporins admit water but not other small molecules or ions. FIGURE 6.22 shows a cutaway view from the side of an aquaporin, indicating how it fits in a plasma membrane. Like other channels that have been studied in detail, aquaporins have a pore that is lined with polar functional groups in this case, carbonyl groups that interact with water. A channels pore is hydrophilic relative to the hydrophobic residues facing the phospholipid tails of the membrane. But how can aquaporin be selective for water and not other polar molecules? The answer was found when researchers examined its structure. Key side chains in the interior of the pore function as a molecular filter. The distance between these groups across the channel allows only those substances capable of interacting with all of them to pass through to the other side. Movement Through Many Membrane Channels Is Regulated Recent research has shown that many aquaporins and ion channels are gated channels-meaning that they open or close in response to a signal, such as the binding of a particular molecule or a change in the electrical voltage across the membrane. As an example of how voltage-gated channels work, FIGURE 6.23 shows a potassium channel in closed and open configurations. The electrical charge on the membrane is normally negative on the inside relative to the outside, which causes the channel to adopt a closed shape that prevents potassium ions from passing through. When this charge asymmetry is reversed, the shape changes in a way that opens the channel and allows potassium ions to cross. The key point here is that in almost all cases, the flow of ions and small molecules through membrane channels is carefully controlled. In all cases, however, the movement of substances through channels is passive-meaning it does not require an input of energy. Passive transport is powered by diffusion along an electrochemical gradient. Channel proteins simply enable ions or polar molecules to move across lipid bilayers efficiently, in response to An existing gradient. They are responsible for facilitated diffusion: transport of substances that otherwise would not cross a membrane readily. Facilitated Diffusion via Carrier Proteins Facilitated diffusion can also occur through carrier proteins specialized membrane proteins that change shape during the transport process. Perhaps the best-studied carrier protein is one that is involved in transporting glucose into cells. The Search for a Glucose Carrier Next to ribose, the six-carbon sugar glucose is the most prevalent sugar found in organisms. Virtually all cells alive today use glucose as a building block for important macromolecules and as a source of stored chemical energy (Chapter 5). But as Figure 6.8 shows, lipid bilayers are only moderately permeable to glucose. It is reasonable to expect, then, that plasma membranes have some mechanism for increasing their permeability to this sugar. This prediction was supported in experiments on pure preparations of plasma membranes from human red blood cells. These plasma membranes turned out to be much more permeable to glucose than are pure lipid bilayers. Why? After isolating and analyzing many proteins from red blood cell membranes, researchers found one protein that specifically increases membrane permeability to glucose. When they added this purified protein to liposomes, the artificial membrane transported glucose at the same rate as a membrane from a living cell. This experiment convinced biologists that the membrane protein-now called GLUT-1 (short for glucose transporter 1)—was indeed responsible for transporting glucose across plasma membranes. How Does GLUT-1 Work? How Does GLUT-1 Work? Recall that proteins frequently change shape when they bind to other molecules and that such conformational changes are often a critical step in their function (Chapter 3). FIGURE 6.24 illustrates the current hypothesis for how GLUT-1 works to facilitate the movement of glucose. The idea is that when glucose binds to GLUT-1, it changes the shape of the protein in a way that moves the sugar through the hydrophobic region of the membrane and releases it on the other side. What powers the movement of molecules through carriers? The answer is diffusion.. GLUT-1 facilitates diffusion by allowing glucose to enter the carrier from either side of the membrane. Glucose will pass through the carrier in the direction dictated by its concentration gradient. A large variety of molecules move across plasma membranes via specific carrier proteins. Pumps Perform Active Transport Diffusion-whether it is facilitated by proteins or not-is a passive process that will move substances in either direction across a membrane to make the cell interior and exterior more similar. But it is also possible for cells to move molecules or ions in a directed manner, often against their electrochemical gradient. Accomplishing this task requires an input of energy, because the cell must counteract the decrease in entropy that occurs when molecules or ions are concentrated. It makes sense, then, that transport against an electrochemical gradient is called active transport. In cells, ATP (adenosine triphosphate) often provides the energy for active transport by transferring a phosphate group (HPO42) to an active transport protein called a pump. Recall that ATP contains three phosphate groups (Chapter 4), and that phosphate groups carry two negative charges (Chapter 2). When a phosphate group leaves ATP and binds to a pump, its negative charges interact with charged amino acid residues in the protein. As a result, the protein's potential energy increases and its shape changes. The Sodium-Potassium Pump A classic example of how structural change leads to active transport is provided in the sodium potassium pump, or more formally, Na+/K+-ATPase. The Na K* part of the name refers to the ions that are transported, ATP indicates that adenosine triphosphate is used, and -ase identifies the molecule as an enzyme. As shown in FIGURE 6.25, sodium and potassium ions move in a multistep process: Step 1 When Na+/K+-ATPase is in the conformation shown here, binding sites with a high affinity for sodium ions are available. Step 2 Three sodium ions from the inside of the cell bind to these sites and activate the ATPase activity in the pump. Step 3 A phosphate group from ATP is transferred to the pump. When the phosphate group attaches, the pump changes its shape in a way that opens the ion-binding pocket to the external environment and reduces its affinity for sodium ions. Step 4 The sodium ions leave the protein and move to the exterior of the cell. Step 5 In this conformation, the pump has binding sites with a high affinity for potassium ions facing the external environment. Step 6 Two potassium ions from outside the cell bind to the pump. Step 7 When the potassium is bound, the phosphate group is cleaved from the protein and its structure changes in response back to the original shape with the ion-binding pocket facing the interior of the cell. Step 8 In this conformation, the pump has low affinity for potassium ions. The potassium ions leave the protein and move to the interior of the cell. The cycle then repeats. Other types of pumps move protons (H+), calcium ions (Ca2+), or other ions or molecules across membranes in a directed manner, regardless of the gradients. As a result, cells can import and concentrate valuable nutrients and ions inside the cell despite their relatively low external concentration. They can also expel molecules or ions, even when a concentration gradient favors diffusion of these substances into the cell. Secondary Active Transport Approximately 30 percent of all the ATP generated in your body is used to drive the Na+/K+-ATPase cycle. Each cycle exports three Na+ ions for every two K+ ions it imports. In this way, the sodium-potassium pump converts energy from ATP to an electrochemical gradient across the mem. brane. The outside of the membrane becomes positively charged relative to the inside. This gradient favors a flow of anions out of the cell and a flow of cations into the cell. The electrochemical gradients established by the Nat/K+. ATPase represent a form of stored energy, much like the electrical energy stored in a battery. How do cells use this energy? Gradients are crucial to the function of the cell, in part because they make it possible for cells to engage in secondary active transport-also known as cotransport. When cotransport occurs, a gradient set up by a pump provides the energy required to power the movement of a different molecule against its particular gradient. Recall that GLUT-1 facilitates the movement of glucose into or out of cells in the direction of its gradient. Can glucose be moved against its gradient? The answer is yes—a cotransport protein in your gut cells uses the Na* gradient created by Na+/K+-ATPases to import glucose against its chemical gradient. When Nations bind to this cotransporter, its shape changes in a way that allows glucose to bind. Once glucose binds, another shape transports both the sodium and glucose to the inside of the cell. After dropping off sodium and glucose, the protein's original shape returns to repeat the cycle. In this way, glucose present in the food you are digesting is actively transported into your body. The glucose molecules eventually diffuse into your bloodstream and are transported to your brain, where they provide the chemical energy you need to stay awake and learn some biology. (You will learn more about secondary active transport in Units 7 and 8.) Plasma Membranes and the Intracellular Environment Taken together, the selective permeability of the lipid bilayer and the specificity of the proteins involved in passive transport and active transport enable cells to create an internal environment that is much different from the external one (FIGURE 6.26). With the evolution of membrane proteins, the early cells acquired the ability to create an internal environment that was conducive to life-one that contained the substances required for manufacturing ATP and copying ribozymes. Cells with particularly efficient and selective membrane proteins would be favored by natural selection and would come to dominate the population. Cellular life had begun. Some 3.5 billion years later, cells continue to evolve. What do today's cells look like, and how do they produce and store the chemical energy that makes life possible? Answering these and related questions is the focus of the following unit. check your understanding If you understand that ... • Membrane proteins allow substances that ordinarily do not readily cross lipid bilayers to enter or exit cells. • Substances may move across a membrane along an electrochemical gradient, via facilitated diffusion through channel or carrier proteins. Or, they may move against a gradient in response to work done by pumps. You should be able to ... Explain what is passive about passive transport, active about active transport, and "co" about cotransport

Passive transport does not require an input of energy-it happens as a result of energy already present in existing concentration or electrical gradients. Active transport is active in the sense of requiring an input of energy from, for example, ATP. In cotransport, a second ion or molecule is transported against its concentration gradient along with (i.e., "co") an ion that is transported along its concentration gradient.

PROCESS: VISUALIZING MEMBRANE PROTEINS Figure 6.18 Freeze Fracture Preparations Allow Biologists to View Membrane Proteins. 1. Fracture cell: Strike frozen cell with a knife. Fracture splits the lipid bilayer. Membrane exterior Lipid bilayer Cell Membrane interior 2. Separate the parts and prepare for scanning electron microscopy 3. Microscopy: Observe pits and mounds in the membrane interior. Mounds and pits in the middle of lipid bilayer Membrane Exterior 4. Interpretation: Image supports fluid-mosaic model of membrane structure. Question: What would be an appropriate control to show that the pits and mounds were not simply irregularities in the lipid bilayer caused by the freeze-fracture process? Process: Isolating Membrane Proteins FIGURE 6.19 Detergents Can Be Used To Isolate Proteins from Membranes 1. Addition of detergents: Detergents are small, amphipathic molecules that tend to form micelles in water 2. Binding by detergents: Detergents break up plasma membranes; they coat hydrophobic portions of membrane proteins and phospholipids. 3. Isolation of proteins Treating a plasma membrane with a detergent is an effective way to isolate membrane proteins so they can be purified and studied in detail.

Repeat the procedure using a lipid bilayer that is free of membrane proteins, such as synthetic liposomes constructed from only phospholipids. If proteins were responsible for the pits and mounds, then this control would not show these structures.

Osmosis What about water? As the data in Figure 6.8 show, water moves across lipid bilayers relatively quickly. The movement of water is a special case of diffusion that is given its own name: osmosis. Osmosis occurs only when solutions are separated by a membrane that permits water to cross, but holds back some or all of the solutes—that is, a selectively permeable membrane. It's important to note that some of the water molecules in a solution are unavailable to diffuse across the membrane. Recall that solutes form ionic or hydrogen bonds with water molecules (Chapter 2). Water molecules that are bound to a solute that can't cross the membrane are themselves prevented from crossing. Only unbound water molecules are able to diffuse across the membrane during osmosis. When these unbound water molecules move across a membrane, they flow from the solution with the lower solute concentration into the solution with the higher solute concentration. To drive this point home, let's suppose the concentration of a particular solute is higher on one side of a selectively permeable membrane than it is on the other side (FIGURE 6.13, step 1). Further, suppose that this solute cannot diffuse through the membrane to establish equilibrium. What happens? Water will move from the side with a lower concentration of solute to the side with a higher concentration of solute (Figure 6.13, step 2). Osmosis dilutes the higher concentration and equalizes the concentrations on both sides. The movement of water is spontaneous. It is driven by the increase in entropy achieved when solute concentrations are equal on both sides of the membrane. Movement of water by osmosis is important because it can swell or shrink a membrane-bound vesicle. Consider the liposomes illustrated in FIGURE 6.14. (Remember that osmosis occurs only when a solute cannot pass through a separating membrane.) Figure 6.14 Osmosis Can Shrink or Burst Membrane-Bound Vesicles Start with: Inside solution hypotonic to outside Lipid bilayer inside solution hypertonic to outside inside and outside isotonic Arrows represent the direction of net water movement via osmosis. Result: Net flow of water out of vesicle; vesicle shrinks Net flow of water into vesicle; vesicle swells of even bursts No change Left If the solution inside the membrane has a lower concentration of solutes than the exterior has, water moves out of the vesicle into the solution outside. The solution inside is said to be hypotonic ("lower-tone") relative to the outside of the vesicle. As water leaves, the vesicle shrinks and the membrane shrivels, resulting in lower vesicle firmness. Middle If the solution inside the membrane has a higher concentration of solutes than the exterior has, water moves into the vesicle via osmosis. The inside solution is said to be hypertonic ("excess-tone") relative to the outside of the vesicle. The incoming water causes the vesicle to swell and increase in firmness, or even burst. Right If solute concentrations are equal on both sides of the membrane, the liposome maintains its size. When the inside solution does not affect the membrane's shape, that solution is called isotonic ("equal-tone"). Note that the terms hypertonic, hypotonic, and isotonic are relative—they can be used only to express the relationship between a given solution and another solution separated by a membrane. Biologists also commonly use these terms to describe the solution that is exterior to the cells or vesicles. Membranes and Chemical Evolution What do diffusion and osmosis have to do with the first membranes floating in the prebiotic soup? Both processes tend to reduce differences in chemical composition between the inside and outside of membrane-bound compartments. If liposome-like structures first arose in the oceans of early Earth, their interiors probably didn't offer a radically different environment from the surrounding solution. In all likelihood, the primary importance of the first lipid bilayers was simply to provide a container for replicating RNA, the macromolecule most likely to have been the first "living" molecule (see Chapter 4). But ribonucleotide monomers would need to be available for these RNAs to replicate. Can negatively charged ribonucleotides get across lipid bilayers and inside lipid-bounded vesicles? The answer is yes. Jack Szostak and colleagues first set out to study the permeability of membranes consisting of fatty acids and other simple amphipathic lipids thought to be present in the early oceans. Like phospholipids, fatty acids will spontaneously assemble into lipid bilayers and water-filled vesicles. Their experiments showed that ions, and even ribonucleotides, can diffuse across the fatty acid vesicle membranes—meaning that monomers could have been available for RNA synthesis. Lending support to this hypothesis, the same minerals found to catalyze the polymerization of RNA from activated nucleotides (see Chapter 4) will also promote the formation of fatty acid vesicles-and in the process, often incorporate themselves and RNA inside. Simple vesicle-like structures that harbor nucleic acids are referred to as protocells (FIGURE 6.15). Most origin-of-life researchers view protocells as possible intermediates in the evolution of the cell. Laboratory simulations also showed that free lipids and micelles can become incorporated into fatty acid bilayers, causing protocells to grow. Shearing forces, as from bubbling, shaking, or wave action, cause protocells to divide. Based on these observations, it is reasonable to hypothesize that once replicating RNAs became surrounded by a lipid bilayer, this simple life form and its descendants would occupy cell-like structures that grew and divided Now let's investigate the next great innovation in the evolution of the cell: the ability to create and maintain a specialized internal environment that is conducive to life. What is necessary to construct an effective plasma membrane-one that imports ions and molecules needed for life while excluding ions and molecules that might damage it? check your understanding If you understand that ..., • Diffusion is the net movement of ions or molecules in solution from regions of high concentration to regions of low concentration. • Osmosis is the movement of water across a selectively permeable membrane, from a region of low solute concentration to a region of high solute concentration. You should be able to ... Make a concept map (see BioSkills 15 in Appendix B) that includes the boxed terms water molecules, solute molecules, osmosis, diffusion, areas of high concentration, selectively permeable membranes. concentration gradients, hypertonic solutions, hypotonic solutions, and isotonic solutions

See Figure A6.1

How Does Lipid Structure Affect Membrane Permeability? The amphipathic nature of phospholipids allows them to spontaneously form membranes. But not all phospholipid bilayers are the same. The nature of the hydrocarbon tails, in addition to the presence of cholesterol molecules, profoundly influences how a membrane behaves. Bond Saturation and Hydrocarbon Chain Length Affect Membrane Fluidity and Permeability The degree of saturation in a phospholipid-along with the length of its hydrocarbon tails affects key aspects of a lipid's behavior in a membrane. • When unsaturated hydrocarbon tails are packed into a lipid bilayer, kinks created by double bonds produce spaces among the tails. These spaces reduce the strength of the van der Waals interactions (see Chapter 3) that hold the hydrophobic tails together, weakening the barrier to solutes. Packed saturated hydrocarbon tails have fewer spaces and stronger van der Waals interactions. As the length of saturated hydrocarbon tails increases, the forces that hold them together also grow stronger, making the membrane even denser. These observations have profound impacts on membrane fluidity and permeability-two closely related properties. Figure 6.9 Fatty Acid Structure Changes the Permeability of Membranes. Lipid bilayers consisting of phospholipids containing unsaturated fatty acids should have more gaps and be more permeable than those with saturated fatty acids (1) Lipid bilayer with short and unsaturated hydrocarbon tails. Higher permeability and fluidity (2) Lipid bilayer with long and saturated hydrocarbon tails Lower Permeability and fluidity As FIGURE 6.9 shows, lipid bilayers are more permeable as well as more fluid when they contain short, kinked, unsaturated hydrocarbon tails. An unsaturated membrane allows more materials to pass because its interior is held together less tightly. Bilayers containing long, straight, saturated hydrocarbon tails are much less permeable and fluid. Experiments on liposomes have shown exactly these patterns. check your understanding If you understand that ... • In water, phospholipids form bilayers that are selectively permeable-meaning that some substances cross them much more readily than others do. Permeability is a function of the degree of saturation and the length of the hydrocarbon tails in membrane phospholipids, the amount of cholesterol in the membrane, and the temperature. You should be able to ... Fill in a chart with columns labeled Factor, Effect on permeability, and Reason and rows under the Factor column labeled Temperature, Cholesterol, Length of hydrocarbon tails, and Saturation of hydrocarbon tails.

See Table A6.1

Systems for Studying Membrane Proteins The discovery of transmembrane proteins was consistent with the hypothesis that proteins affect membrane permeability. To test this hypothesis, researchers needed some way to isolate and purify membrane proteins. FIGURE 6.19 outlines one method that researchers developed to separate proteins from membranes. The key to the technique is the use of detergents. A detergent is a small amphipathic molecule. When detergents are added to the solution surrounding a lipid bilayer, the hydrophobic tails of the detergent molecule interact with the hydrophobic tails of the lipids and with the hydrophobic portions of transmembrane proteins. These interactions displace the membrane phospholipids and end up forming water-soluble detergent-protein complexes that can be isolated Since intensive experimentation on membrane proteins began, researchers have identified three broad classes of proteins that affect membrane permeability: channels, carriers, and pumps. Let's consider each class in turn. Facilitated Diffusion via Channel Proteins As the data in Figure 6.8 show, ions almost never cross pure phospholipid bilayers on their own. But in cells, ions routinely cross membranes through specialized membrane proteins called ion channels. Ion channels form pores, or openings, in a membrane. Ions move through these pores in a predictable direction from regions of high concentration to regions of low concentration and from areas of like charge to areas of unlike charge. In FIGURE 6.20, for example, a large concentration gradient favors the movement of sodium ions from the outside of a membrane to the inside. But in addition, the inside of this cell has a net negative charge while the outside has a net positive charge. As a result, the combination of these two factors influences the final concentration of sodium ions inside cell once equilibrium has been established. Figure 6.20 An Electrochemical Gradient is a Combined Concentration and Electrical Gradient Electrochemical gradients are established when ions build up on one side of a membrane Outside cell High concentration of NA+ Net (+) Charge Electrochemical gradient for sodium ions (Na+) Inside Cell Net (-) Charge Low concentration of Na+ Ions move in response to a combined concentration and electrical gradient, or what biologists call an electrochemical gradient If you understand this concept, you should be able to add an arrow to Figure 6.20 indicating the electrochemical gradient for chloride ions

Your arrow should point out of the cell. There is no concentration gradient for chloride, but the outside has a net positive charge, which favors outward movement of negative ions