BIOCHEMISTRY DISCUSSION

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So in the last chapter about lipids we talked a lot about membranes, but we didn't really show very much about them. Now we're going to show how these lipids do get organized into membranes, which are some of the most important elements of living organisms.

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Biochemistry: A Short Course

Fourth Edition

CHAPTER 12 Membrane

Structure and Function

Tymoczko • Berg • Gatto • Stryer

Well, first we’ll be talking about how: (1) phospholipids and glycolipids form bimolecular sheets, which are called membranes, which we've talked about before. And as mentioned in the last chapter, (2) membrane fluidity is controlled by both the fatty acid composition and the cholesterol content of the membrane. (3) Proteins are very important for carrying out membrane processes, letting things get across the membrane, helping things interact with the membrane. (4) Now, it's important also to recognize that a lipid membrane is a two dimensional structure, but things can still diffuse two dimensionally in that structure. And also (5) a lot of proteins are designed to transport things across membranes.

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Chapter 12: Outline

12.1 Phospholipids and Glycolipids Form Bimolecular Sheets

12.2 Membrane Fluidity Is Controlled by Fatty Acid Composition and Cholesterol Content

12.3 Proteins Carry Out Most Membrane Processes

12.4 Lipids and Many Membrane Proteins Diffuse Laterally in the Membrane

12.5 A Major Role of Membrane Proteins Is to Function as Transporters

THIS SECTION IS NOT DISCUSSED IN DETAIL IN THE LECTURE

So here are some basic characteristics of membranes, and some interesting points. 1) Remember that membranes are sheet-like structures. They are two molecules

thick - that is, two phospholipids thick. And they always form a closed boundary with an inside and an outside. I guess there’s occasionally some transitional states that they go through, but ...

2) membranes are composed of lipids and proteins. And both of these can be decorated with carbohydrates, and ARE decorated with carbohydrates, and that is very important for their interactions with other molecules, signaling, etc.

3) Membrane lipids are small amphipathic molecules. You may recall amphipathic means that it has a hydrophilic and a hydrophobic region. And that's why they form membranes actually, but we'll get into that in a bit.

4) Proteins are important for getting things across the membranes. 5) Membranes are fluid. They are not static, they move around just like stuff in

solution would. {but only in two dimensions! – this makes them, in general, MUCH SMALLER in volume than the cytoplasm, and has other interesting effects.}

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Characteristics of Membranes 1. Membranes are sheetlike structures, two molecules thick, that form

closed boundaries. 2. Membranes are composed of lipids and proteins, either of which can

be decorated with carbohydrates. 3. Membrane lipids are small amphipathic molecules that form closed

bimolecular sheets that prevent the movement of polar or charged molecules.

4. Proteins serve to mitigate the impermeability of membranes and allow movement of molecules and information across the cell membrane.

5. Membranes are fluid, noncovalent assemblies.

So phospholipids and glycolipids form the lipid bilayer in aqueous solutions. And that is powered by the hydrophobic effect. Also by the interaction of the hydrophilic elements with the water around it.

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Section 12.1 Phospholipids and Glycolipids Form Bimolecular Sheets

Learning objective 1: Identify the energetic force that powers the formation of membranes.

• Phospholipids and glycolipids form lipid bilayers in aqueous solutions.

• The formation of membranes is powered by the hydrophobic effect.

Figure 12.1 A section of phospholipid bilayer membrane. (A) Electron micrograph of a cell. The nucleus is surrounded by a double membrane or two lipid bilayers, with occasional pores (p. 12). Only one of the lipid bilayers is expanded. (B) Space-filling model of an idealized view showing regular structures. [Don W. Fawcett/Science Source.]

Up at the top here we see an actual electron micrograph of a membrane, and as a bilayer membrane, you can see the two thick portions that represent the two layers. And those are probably the phosphates that you're seeing there because the phosphorus has a large nucleus. The little diagram there at the bottom will show you that the polar head groups are all oriented towards the water where they will interact with the water because they're hydrophilic, and the hydrophobic tails are all in the interior. You can see here the generalized diagram of one of these phospholipids is a red ball with two green strings attached. Those green strings represent the hydrophobic tails. And recall from phospholipid structure that pretty much all of these phospholipids have got two large hydrophobic tails attached to them. Depending on what book you're reading, this picture may be wrong in the book, but this is the correct picture.

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Electron Micrograph and Space-filling Model of a Phospholipid Bilayer Membrane

Figure 12.3 The preparation of glycine-containing liposomes. Liposomes containing glycine are formed by the sonication of phospholipids in the presence of glycine. Free glycine is removed by filtration.

So membranes are a natural structure. {Like bubbles!} They self-assemble! If you take a bunch of phospholipids in the bottom of a beaker and you shake them up, the phospholipids will naturally form these closed structures, which are called vesicles. And if you have some sort of molecule in the water, like glycine, some of that glycine will be trapped inside of these vesicles. And then you can filter the rest of the water out, and if you're left with the vesicles, you'll still have vesicles that contain glycine inside of them. So it does this because membranes are a very stable structure, so they’re thermodynamically and entropically favored. And that's one of the reasons that membranes are believed to be one of the first important developments in the history of life - because these would just naturally form, and create these microenvironments where things can take place sheltered from the outside.

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Diagram of the Preparation of Glycine- Containing Liposomes

So you've got a bunch of small molecules floating around in the cytoplasm, or in the extracellular matrix, and they come across a membrane. What's going to happen? Well, if it's an ion, or a polar molecule, then it will tend to interact with the hydrophilic heads of these membrane molecules and will not be able to {or it will be hard to} get deeply into the hydrophobic portion of the membrane. Hydrophobic molecules, on the other hand will generally be stuck to a membrane to begin with because they're not soluble in water. But when two membranes come together, those molecules can actually transfer from one membrane to another, and they can easily cross to the inside and the outside of the membrane. So on the left you can see a couple examples of hydrophobic molecules that would be able to pass through a membrane fairly easily. But a sodium ion encased in water has a great deal of interactions, and all of those interactions would need to be broken in order to get that sodium ion across, and it's very unlikely that that's going to happen.

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Lipid Bilayers Are Highly Impermeable to Ions and Most Polar Molecules

Learning objective 2: Explain why membranes are impermeable to most substances. • The ability of small molecules to cross a membrane is a function of

its hydrophobicity.

• Indole is more soluble than tryptophan in membranes because it is uncharged. Ions cannot cross membranes because of the energy cost of shedding their associated water molecules.

Figure 12.4 Permeability coefficients of ions and molecules in a lipid bilayer. The ability of molecules to cross a lipid bilayer spans a wide range of values. The permeability coefficient (P), expressed in cm s-1, provides a quantitative estimate of the rate of passage of a molecule across a membrane.

So there is a level of permeability for ALL molecules. And you can see on the right nitrogen, oxygen, carbon dioxide, nitric oxide {some} steroids - those are all able to pass through the membrane fairly easily. Water, actually, is able to pass through the membrane fairly easily, compared to a lot of things {primarily because it is so small}. Indole, which is a little more hydrophobic, actually passes through fairly easily. Urea and glycerol, a little more hydrophilic there, not so easy. Tryptophan, glucose – glucose, very hydrophilic, is hard to get across. But not as difficult as the chlorine, potassium, and sodium ions, which are the charged particles. And those are the hardest ones to get across the membrane.

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Permeability Coefficients of Ions and Molecules in a Lipid Bilayer

N2 O2 CO2 NO

some steroids

As we've already mentioned, many membrane processes depend on the fluidity of the membrane - and as a matter of fact, there’s these portions of the membrane sometimes that are especially dense, and they're called lipid rafts. And those play a very important role in certain cellular processes. We'd also mentioned that fluidity is dependent on the length of the fatty acids, and the degree of unsaturation, and how cholesterol works, so I won't talk too much about that now.

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Section 12.2 Membrane Fluidity Is Controlled by Fatty Acids Composition

and Cholesterol Content

• Membrane processes depend on the fluidity of the membrane.

• Fluidity is dependent on the length of the fatty acids in the membrane lipid and the degree of cis unsaturation.

• Cholesterol helps to maintain proper membrane fluidity in membranes in animals.

Figure 12.6 The packing of fatty acid chains in a membrane. The highly ordered packing of fatty acid chains is disrupted by the presence of cis double bonds. The space-filling models show the packing of (A) three molecules of stearate (C18, saturated) and (B) a molecule of oleate (C18, unsaturated) between two molecules of stearate.

So this is just an example of why it might make more fluid membranes if you have these unsaturated fatty acids. You can see how, on the left, the fatty acids pack together very tightly; but on the right, they can't, because of the bend in the central fatty acid. Now that's an exaggerated bend, but there is a grain of truth to that, which is that that bend does in fact make it more difficult for the atoms to pack close together.

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Diagram of the Packing of Saturated Fatty Acid Chains in a Membrane

THIS IS EXAGGERATED. HYDROCARBONS ARE NOT RIGID.

Figure 12.7 Cholesterol disrupts the tight packing of the fatty acid chains. [After S. L. Wolfe, Molecular and Cellular Biology (Wadsworth, 1993).]

Cholesterols integrate into the membrane with the hydroxyl group pointing toward the outside of the membrane, and they interact with the fatty acid chains disrupting their packing - but also they provide a degree of rigidity to the membrane as well {because they themselves are fairly rigid}.

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Diagram of Cholesterol Disrupting the Tight Packing of Fatty Acid Chains

So the lipids are preventing things from getting across, but things do need to get across these membranes - and the way they get across is with the help of proteins.

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Section 12.3 Proteins Carry Out Most Membrane Processes

Learning objective 3: Describe the roles of proteins in making membranes selectively permeable.

• Although membrane lipids establish a permeability barrier, membrane proteins allow transport of molecules and information across the membrane.

So there's about three ways that proteins can become associated with the membrane. They can either be embedded completely in the membrane, or they can be attached to the polar head groups of membranes, or they can be covalently attached to something hydrophobic, which then gets inserted into the membrane.

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Proteins Associate with the Lipid Bilayer in a Variety of Ways

Proteins associate with the lipid bilayer in a variety of ways. • Integral membrane proteins are embedded in the

hydrocarbon core of the membrane. • Peripheral membrane proteins are bound to the polar head

groups of membrane lipids or to the exposed surfaces of integral membrane proteins.

• Some proteins are associated with membranes by attachment to a hydrophobic moiety that is inserted into the membrane.

Figure 12.8 Integral and peripheral membrane proteins. Integral membrane proteins (a and b) interact extensively with the hydrocarbon region of the bilayer. Most known integral membrane proteins traverse the lipid bilayer. Some peripheral membrane proteins (c) interact with the polar head groups of the lipids. Other peripheral membrane proteins (d) bind to the surfaces of integral proteins. Some proteins (e) are tightly anchored to the membrane by a covalently attached lipid molecule.

Here is an example of those. Number A would be fully inserted into the membrane. And B would also be inserted into the membrane, but D would be attached to B and that's why it's membrane-associated. C would be associated with the polar head groups, and E has got a hydrophobic tail which is embedded in the membrane.

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Diagram of Integral and Peripheral Membrane Proteins

Figure 12.9 Structure of bacteriorhodopsin. Notice that bacteriorhodopsin consists largely of membrane-spanning α helices (represented by yellow cylinders). The view is through the membrane bilayer. The interior of the membrane is green, and the head groups are red. [Drawn from 1 BRX.pdb.]

Now, one way that proteins can get across the membrane is by using alpha helices - and here you see a bundle of alpha helices. There's seven of them, and this is a very common motif, called a seven-transmembrane protein. It just so happens that this is a fairly stable arrangement. And of course, the amino acids that are {interacting with the membrane} would be hydrophobic mostly. And then the outside parts {exposed to the solution} would be amino acids that were more hydrophilic. And that's how it would find itself in this configuration.

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Structure of Bacteriorhodopsin • Membrane-spanning α helices are a common structural

feature of integral membrane proteins.

hydrophobic residues in here

hydrophilic residues out here

FIGURE 12.10 The structure of bacterial porin (from Rhodopseudomonas blastica). Notice that this membrane protein is built entirely of β strands. Only one monomer of the trimeric protein is shown. [Drawn from 1PRN.pdb.]

But beta strands can also be used to get across the membrane. Once again, it would be hydrophobic residues on the outsides of these beta strands {in this case I’m talking about the outside of the “barrel”, interacting with the membrane} and possibly hydrophilic residues on the inside {of the barrel. Loops floating around outside the membrane would also be hydrophilic}. And this is a barrel shape, so if you have hydrophilic things on the inside, it would allow water molecules and other ions to pass through.

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Structure of Bacterial Porin • Other means of embedding integral membrane proteins is

by using β strands to form a pore in the membrane or by embedding part of the protein into the membrane.

Hydrophobic residues out here

SIDE VIEW TOP VIEW

Hydrophilic residues in

here

Figure 12.11 The attachment of prostaglandin H2 synthase-1 to the membrane. Notice that prostaglandin H2 synthase-1 is held in the membrane by a set of a helices (orange) coated with hydrophobic side chains. One monomer of the dimeric enzyme is shown. [Drawn from 1 PTH.pdb.]

So a protein doesn't need to go all the way through the membrane to be associated with it. An example here is prostaglandin H2 synthase-1, and it happens to have a few alpha helices that are covered with very hydrophobic amino acid side chains - and, therefore, those hydrophobic amino acid side chains will get embedded in the membrane, whereas the rest of the protein will be sitting on the top.

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Diagram of the Attachment of Prostaglandin H2 synthase-1 to the

Membrane

So there are some proteins which facilitate the flow of small molecules, ions, polar charged molecules, across the membrane, and we call those transport proteins. There are two main types - there's passive transport, which is basically just a hole, and things can diffuse through that hole; and then there's active transport, where it actually uses energy to push molecules against their concentration gradient.

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Section 12.5 A Major Role of Membrane Proteins Is to Function as Transporters

• Transport proteins function as pumps or channels to facilitate the flow of small molecules across the cell membrane.

• Passive transport or facilitated diffusion occurs when a molecule moves down its concentration gradient through a transport protein.

• Protein pumps use energy to move a molecule against its concentration gradient in the process of active transport.

One very important example of active transport is the Na+-K+ ATPase. And that is able to pump three Na+ ions out of the cell, and two K+ ions into the cell - and as you can see {on the next page}, that would result in a net increase of positive charge outside of the cell.

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The Na+–K+ ATPase Is an Important Pump in Many Cells

• The Na+–K+ ATPase or Na+–K+ pump uses the energy of ATP hydrolysis to simultaneously pump three Na+ ions out of the cell and two K+ ions into the cell against their concentration gradients.

Figure 12.16 Energy transduction by membrane proteins. The Na+–K+ ATPase converts the free energy of phosphoryl transfer into the free energy of a Na+ ion gradient.

And here's a diagram of what that might look like. We see sodium on the outside of the cell in red - and there is a lot of sodium outside the cell, whereas there is more potassium than sodium on the inside of the cell. So normally, if you let things just diffuse, they would diffuse across and you would end up having equal amounts of sodium and potassium on the inside and the outside. But this pump will break down ATP and use that energy that it gets from that to actually pump sodium and potassium against their gradients in active transport.

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Diagram of Energy Transduction by Membrane Proteins

Secondary transporters use the concentration gradient of one substance in order to power the formation of another. There's two different main types. There's the symporters, which will come together in the same direction, and antiporters in which the molecules will flow in opposite directions.

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Secondary Transporters Use One Concentration Gradient to Power the Formation of Another

• Symporters power the transport of a molecule against its concentration gradient by coupling the movement to the movement of another molecule down its concentration gradient, with both molecules moving in the same direction.

• Antiporters also use one concentration gradient to power the formation of another, but the molecules move in opposite directions.

Figure 12.18 Antiporters and symporters. Secondary transporters can transport two substrates in opposite directions (antiporters) or two substrates in the same direction (symporters).

So you can see here, examples of that, a diagram of that. An antiporter would use the gradient of A to power the pumping out of B. And in the symporter, you would use the gradient of A to power the pumping in of B.

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Diagram of Antiporters and Symporters

Figure 12.19 Secondary transport. The ion gradient set up by the Na+–K+ ATPase can be used to move materials into the cell through the action of a secondary transporter such as the Na+-glucose symporter.

So now that we've talked about each one of those types, we can have an example of how they work together. And you see on the left, the sodium-potassium pump is creating a sodium gradient outside of the cell. And then the sodium-glucose symporter, on the right, is using that sodium gradient to drive glucose against its concentration gradient into the cell.

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Diagram of Secondary Transport • Glucose is moved into some animal cells against its

concentration gradient by a symporter that is powered by Na+ ions moving down a concentration gradient.

So channels can transport ions across membranes, and these are essentially holes that allow ions to form with their concentration gradient. So they don't hydrolyze ATP and they don't move anything against their concentration gradient, but they can be very specific as to what ions they will let pass; and also their activity can be changed by changes in voltage across a membrane, and also by attachment of ligands to these channels. Voltage would be an example where you would see with the sodium-potassium pump, for example, greater positive charge on the outside than on the inside. That would create a voltage across the membrane and might end up shutting down or activating some channels. Very common in the brain actually, that's one of the main ways that action potentials are transported down axons.

And with the ligand-activated channels, this is another example of allosteric activation, where you're acting on a certain part of the channel and causing a conformational change in the rest of the channel which is allowing ions to pass through.

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Specific Channels Can Rapidly Transport Ions Across Membrane

• Ion channels are passive transport systems that allow specific and rapid transport of ions down their concentration gradients.

• Channels can be activated by changes in the voltage across a membrane (voltage-activated channels) or by binding of specific molecules to the channels (ligand- activated channels).

FYI - Tetrodotoxin, produced by the pufferfish, is a lethal inhibitor of the Na+ channel.

We're going to talk in particular about the potassium channel - and how is it that the channel allows only potassium to come through? Well, in one sense it's very easy to imagine anything that's too large will not make it through the channel. But what about things that are a little smaller than potassium, such as sodium? How is it that the channel would be able to let potassium through but not sodium?

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The Structure of the Potassium Ion Channel Reveals the Basis of Ion Specificity

• The potassium channel selectively and rapidly transports K+ across the cell membrane. Larger ions are not transported because they are too big to enter the channel.

• Smaller ions are excluded because they cannot interact with the selectivity filter. Such ions are small enough that the energy of desolvation cannot be compensated for by interactions with the selectivity filter.

Figure 12.22 A path through a channel. A potassium ion entering the K+ channel can pass a distance of 22 Å into the membrane while remaining solvated with water (blue). At this point, the pore diameter narrows to 3 Å (yellow), and potassium ions must shed their water and interact with carbonyl groups (red) of the pore amino acids.

Well, here we have a diagram of a potassium channel. And from the cell interior there is a chamber that is quite wide - it's 10 angstroms wide. And that allows sodium and potassium ions to enter with their associated water molecules. But then it narrows, up at the top, and it's only 3 angstroms wide, and that portion of the channel is covered with oxygens.

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Diagram of a Path Through a Channel

Figure 12.23 The selectivity filter of the potassium ion channel. Potassium ions interact with the carbonyl groups of the selectivity filter, located at the 3-Å-diameter pore of the K+ channel. Only two of the four channel subunits are shown.

And as we can see on the right here, as potassium ions pass through the channel, they interact with these oxygens, and the oxygens stabilize the positive charges.

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Diagram of the Selectivity Filter of the Potassium Ion Channel

FIGURE 12.24 The energetic basis of ion selectivity. The energy cost of dehydrating a potassium ion is compensated by favorable interactions with the selectivity filter.

So I mentioned that it does take energy to remove the water molecules from the potassium ion, and it takes a certain amount of energy to do that - but that energy is compensated by the interactions of potassium with oxygen atoms as you can see on the right. And as a matter of fact, the interaction of potassium with oxygen atoms on the right is stronger than the interaction of the potassium with the water molecules. So therefore, it's favored, and potassium ions are able to enter the channel.

You can see this with the red arrow, representing the energy required to remove the water molecules, and the green arrow is the energy that you gain from interacting with the oxygens in the channel. So we start at the energy of the solvated potassium ion on the left-hand side, and that's the black horizontal bar, and the red arrow is the energy that you need to put into that system to pull those water molecules off of that potassium ion. And then on the right, the {naked} potassium ion is at a high {energy} level, but then you can gain energy by interacting with the oxygens in the channel. And so the green arrow shows the energy that is released from that interaction, and it ultimately ends up at a lower energy level than it started - and so that means it's a favored reaction.

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Diagram of the Energetic Basis of Ion Selectivity (Postassium)

FIGURE 12.24 The energetic basis of ion selectivity. Because a sodium ion is too small to interact favorably with the selectivity filter, the free energy of desolvation cannot be compensated, and the sodium ion does not pass through the channel.

Sodium ions, on the other hand, are too small to interact with all of the oxygens inside the channel at once. They can only interact with four of those oxygens instead of eight of those oxygens, for example (I don't know if those numbers are actually accurate or not). But it takes a certain amount of energy to peel away the water molecules, and you can't get that energy back by interacting with the oxygen molecules because the sodium ion is too small. And therefore, it's not energetically favored to enter the channel.

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Diagram of the Energetic Basis of Ion Selectivity (Sodium)

So a quick quiz to ask yourself, what determines the direction of flow through an ion channel? I'll give you three seconds.... It's the concentration gradient. That's the only thing that matters with an ion channel. Because an ion channel is passive diffusion, and that's controlled completely by the gradient of the ions, flowing from an area of higher concentration to an area of lower concentration.

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Quick Quiz 2

QUICK QUIZ 2 What determines the direction of flow through an ion channel?