Measures of Membrane Fluidity: Melting Temperature

• Tm (melting temperature) is a phase transition, a change from a more rigid solid-like state to a fluid-like state

The fluidity - ease with which lipids move in the plane of the bilayer - of cell membranes has to be precisely regulated because many biological processes (e.g. membrane transport and some enzyme activities) cease when the bilayer fluidity is reduced too much. The fluidity of cell membranes depends on their chemical composition and also on temperature.

As the temperature is raised, a synthetic bilayer made of one type of phospholipid undergoes a phase transition from a solid-like state to a more fluid-like state at a characteristic melting point temperature (abbreviated as Tm). At low temperatures the lipids within the bilayer are well- ordered, packed into a crystal-like arrangement in which the lipids are not very mobile and there are many stabilizing interactions. As the temperature is raised, these interactions are weakened, and the lipids are in a less ordered, liquid-like state.

Lipids with longer fatty acid chains have more interactions between the hydrophobic fatty acid tails (predominantly van der Waals interactions), stabilizing the crystal-like state and therefore increasing the melting temperature and making the membrane less fluid.

1 Membrane Composition Influences Membrane Fluidity: Fatty Acid Structure unsaturated fatty acid chains saturated fatty acid chains

lower Tm higher Tm

OH OH O O 17 carbons

cis double bond

olive oil candle wax

oleic acid stearic acid

The degree of saturation of the fatty acid chains also affects the melting temperature and membrane fluidity. Fatty acids that are saturated, containing no double bonds, are straight and can pack more tightly than those that have double bonds. The kinks in the unsaturated chains simply make it more difficult to pack them in an orderly manner. As a consequence, the melting temperature of bilayers containing lipids with saturated fatty acids is higher than the melting temperature of bilayers containing lipids with unsaturated fatty acids. Additionally, the fluidity of bilayers containing lipids rich in unsaturated fatty acids is greater than the fluidity of bilayers containing lipids rich in saturated fatty acids.

An example of this difference in fluidity and melting temperature is oleic and stearic acid. These two fatty acids have the same number of carbons (17), but they differ dramatically in their fluidity and melting temperature. At room temperature oleic acid is fluid-like (olive oil) and stearic acid is solid-like (candle wax); the melting temperature of oleic acid is lower than that of stearic acid because oleic acid is unsaturated and cannot pack in as orderly a manner as saturated stearic acid.

2 Membrane Composition Influences Membrane Fluidity: Cholesterol Content

HO

H H H H H

Cholesterol is a major component of eukaryotic cells. The ratio of cholesterol to lipid molecules in a membrane can be as high as 1:1.

Cholesterol is a member of a class of natural products called steroids, characterized by a 4 ring structure. Although all steroids are based on the same scaffold, they can affect different biological processes, ranging from the development of secondary sex characteristics (testosterone and estrogen) to inflammation (corticosteroids). Like phospholipids, cholesterol is an amphipathic molecule; it has a polar head that contains a hydroxyl group. The rest of cholesterol is hydrophobic, consisting of a rigid 4 ring steroid structure and a hydrocarbon tail. The 4 ring structure makes most of the cholesterol molecule very rigid because bonds between atoms in a ring are not free to rotate as a result of the geometric constraints of being in a ring.

3 Cholesterol Influences Membrane Fluidity

Cholesterol interacts with phospholipids by orienting its polar hydroxyl head group close to the polar lipid head group. The rigid rings of cholesterol interact with and partly immobilize the fatty acid chains closest to the polar phospholipid head group. As a consequence, lipid molecules adjacent to cholesterol are less free to adopt different conformations than those in a cholesterol-free membrane region. By decreasing the mobility of a few methylene groups (CH2) in the fatty acids tails, cholesterol makes lipid bilayers less deformable and lessens their permeability to small water-soluble molecules. Therefore, cholesterol makes membranes less fluid. Although cholesterol makes bilayers less fluid, at the high concentrations of cholesterol found in eukaryotic cells, it also prevents fatty acid hydrocarbon chains from coming together and crystallizing. Therefore, cholesterol prevents fatty acid chains from ordering into a crystal- like state.

Cholesterol inhibits phase transitions in lipids. At low temperatures it increases membrane fluidity by preventing fatty acid hydrocarbon chains from coming together and crystallizing. Under these conditions cholesterol inhibits the transition from liquid to solid (decreases the membrane freezing point). At high temperatures cholesterol decreases membrane fluidity by immobilizing a few methylene groups in the fatty acid tails of the lipids. Therefore, under these conditions cholesterol increases the melting point. Therefore, cholesterol acts like antifreeze - the temperature of your car engine is modulated by water circulating with antifreeze/coolant which lowers the freezing point of the antifreeze/coolant so it does not freeze in the winter. The antifreeze/coolant also raises the boiling point in the summer so that your engine does not overheat.

The influence of cholesterol on membrane properties is critical for the normal functioning of eukaryotic cells.

4 Summary of Main Points

• HIV needs host cells to replicate because its genome does not encode all of the proteins required for living systems • HIV recognizes host cells by interacting with specific protein receptors found on the surface of those cell types • Cell membranes are bilayers composed of amphipathic phospholipids containing charged head groups and hydrophobic tails • The hydrophobic effect drives the packing of lipids into structures which minimize exposed hydrophobic groups • Membranes are fluid because phospholipids and proteins can move in the plane of the bilayer; fatty acid structure and cholesterol content influence fluidity

5 October 19, 2006

6 Membrane Proteins and Membrane Transport

1. Membrane proteins a. Association of proteins with membranes b. Transmembrane helices 2. Lipid Rafts 3. Membrane transport a. Membrane permeability b. Transport proteins c. Ion distribution inside and outside cells d. Electrochemical gradient e. Active transport f. Ion channels 4. Membrane potential

Lecture Readings Alberts 389-410

7 Membrane Proteins

Although the lipid bilayer serves as a permeability barrier and provides the basic structure of all cell membranes, most membrane functions are carried out by membrane proteins, which constitute 50% of the mass of most plasma membranes.

Membrane proteins perform many different functions: (1) they can transport ions and metabolites across the membrane (2) anchor the membrane to other proteins that play a roll in cell shape and structure (3) they can function as receptors to detect chemical signals in the environment and relay them to the inside of the cell (4) or they can act as enzymes to catalyze reactions.

8 Proteins Associate with Membranes in Different Ways

Proteins can associate with the lipid bilayer in various ways: (a) Transmembrane proteins - These proteins extend through the bilayer and have some mass on both sides. They have hydrophobic sequences in the bilayer, interacting with the hydrophobic tails of lipids, and hydrophilic regions exposed to the aqueous environment on either side of the membrane. (b) Membrane associated - These are proteins located in the cytosol associated with the inner leaflet by an amphipathic alpha-helix (one side of the helix projects hydrophobic residues and the other side projects hydrophilic ones). (c) Lipid-linked - Some proteins lie entirely outside the bilayer, interacting with it using only a covalently attached lipid group. (d) Protein attached - Other proteins are associated only indirectly with the membrane, held there by interactions with one or more membrane proteins.

9 Many Membrane Proteins Cross the Bilayer Using One or More α-Helices

Many proteins that have mass on either side of bilayer cross it using alpha-helices. Helical structures are are well-suited for folding in the bilayer. Those that are transmembrane have hydrophobic side chains which can interact with lipid tails. Within membranes, hydrogen bonding in the polypeptide backbone is maximized because water is absent from the bilayer. As a consequence, there is no competition with water for hydrogen bonding. ~20 amino acids are required to traverse the bilayer in an alpha-helical structure. Scientists can take advantage of knowledge of helical propensity of certain amino acids (the preference they have for or against being in an alpha-helical structure) and hydrophobicity to predict transmembrane domains in proteins.

10 Lipid Rafts in the Plasma Membrane

protein with fatty protein with longer acid modification transmembrane region

lipid bilayer

enriched in cholesterol and phospholipids with long fatty acid tails

As we saw in the animation, membranes are dynamic and are not uniform. Lipid rafts are a major source of inhomogeneity in the membranes of eukaryotic cells. Lipid rafts are microdomains in the lipid bilayer that are established by attractive forces between hydrocarbon chains of fatty acid tails. Lipid rafts are enriched in particular lipids (with long fatty acid tails like sphingolipids) and cholesterol. A typical lipid raft is ~70 nm in diameter.

Because the lipids concentrated in rafts are longer and straighter than most membranes lipids, rafts are thicker than the rest of bilayer. This difference in membrane thickness is important because membrane proteins with long transmembrane segments and also containing certain fatty acid modifications concentrate in lipid rafts. The selective concentration of proteins with particular properties is a way proteins can be segregated within the two-dimensional membrane matrix. Such segregation is very important for trafficking - sorting proteins to their correct destination in cell - and also for the functioning of certain signal transduction pathways that communicate extracellular signals to the inside of the cell.

11 Animation: Lipid Rafts

Animation of dynamics of lipid and protein movement in cell membranes

12 MOVIE

13 Breakout: Fun With FRAP

At time t = 50, two fluorescently labeled lipid membranes are photobleached at low temperature. Both membranes are identical, except one contains cholesterol, the other does not. Which membrane contains cholesterol?

A) Blue BREAKOUT!B) Red

If this experiment were conducted at high temperature, would your answer change?

A) Yes B) No

14 Breakout Answer: Fun With FRAP At time t = 50, two fluorescently labeled lipid membranes are photobleached at low temperature. Both lipid membranes are identical, Except one contains cholesterol, the other does not. Which membrane contains cholesterol?

A) Blue B) Red

If this experiment were conducted at high temperature, would your answer change?

A) Yes B) No At low temperatures, cholesterol increases membrane fluidity by preventing membrane lipids from packing close together. At high temperatures, cholesterol decreases membrane fluidity.

At low temperature cholesterol disrupts the orderly, crystalline packing of lipids into a solidlike state, increasing membrane fluidity. When fluidity is greater, lipids are more mobile, and fluorescence will recover faster following photobleaching. Therefore, the blue line with faster recovery corresponds to the membrane containing cholesterol. At high temperatures cholesterol has the opposite affect on membrane fluidity - it decreases fluidity by immobilizing the first few methylene groups in fatty acids tails through interactions with the rigid 4 ring structure. Therefore, the answer would change if the experiment was conducted at high temperature.

15 Diffusion of Molecules Across the Lipid Bilayer

Cells need to exchange molecules with their environment in order to function normally, but the plasma membrane provides a barrier that controls the movement of molecules into and out of the cell. Small hydrophobic and nonpolar molecules can freely diffuse through lipid bilayers, as can small uncharged polar molecules such as water. However, membranes are impermeable to larger polar molecules and all charged molecules. The charge on molecules prevents them from entering the hydrocarbon phase of the bilayer. Molecules that are charged must be moved across membranes using specialized transport proteins.

16 Ions Cross Cell Membranes Through Membrane Transport Proteins

There are many different types of membrane transport proteins, each selective for a certain class of molecule - ions, amino acids, or sugars, for example. Many transport proteins are highly selective, transporting, for example, only potassium ions, but not sodium.

Transport through membrane proteins can be: (1) passive, requiring no input of energy because the molecule being transported is moving down its concentration gradient (e.g. from higher concentration outside to lower concentration inside). Passive transport can be mediated by channel proteins, which create hydrophilic pores in the membrane to allow movement of ions, or by carrier proteins, which have a binding site(s )for the molecule to be transported and undergo a change in conformation to release the molecule on the other side of the membrane. (2) active - this means that the molecule being transported is moving against its concentration gradient and movement therefore has to be coupled to a process that provides energy. Only carrier proteins can mediate active transport.

17 Ion Concentrations Inside and Outside of a Cell Na+ (145 mM)

Na+ K+ (5-15 mM) Cl- (5 mM) (5-15 mM) K+ (140 mM) organic Cl- anions (110 mM) inside cell

Because of transport processes and the barrier imposed by the cell membrane, living cells maintain an internal ion composition that is very different from that of the external environment. These differences are crucial for the cell’s function, including the activity of nerve cells. The things that are important for you to remember are that sodium is the most plentiful outside the cell and potassium is the most plentiful inside. In order to avoid the buildup of too much electrical charge, the amount of positive charge inside the cell must be balanced with an almost exactly equal amount of negative charge; the same is true for the outside environment. Outside the cell, the high concentration of sodium is balanced by chloride. Inside, the high concentration of potassium is balanced by a variety of negatively charged intracellular organic ions (anions). However, tiny excesses of negative or positive charge do build up near the plasma membrane and, as we will see, they have important consequences.

18 Net Driving Force for Transport is Determined by the Electrochemical Gradient

For uncharged molecules, the concentration gradient drives passive transport. If the molecule is charged, both the concentration gradient and the charge difference across the membrane (called the membrane potential) influence transport. Together, these two forces are referred to as the electrochemical gradient. In a few slides we will discuss where the charge difference across the membrane comes from. In this slide, the width of the green arrow represents the magnitude of the electrochemical gradient for the same positively charged ion in three different situations. In (A) there is only a concentration gradient. The cation (positively charged ion) moves down its concentration gradient, from the outside of the cell to the inside. In (B) the concentration gradient is supplemented by a membrane potential that increases the driving force. The same concentration gradient exists as in (A), but now there is an additional driving force from the membrane potential, which is negative inside and therefore favors the movement of positively charged cations to the inside of the cell (movement of cations is favored in this case because the inside of the cell has a slight excess of negative charge). In (C) the membrane potential decreases the driving force caused by the concentration gradient. In this case, the membrane potential is opposite to (B), with a slight excess of positive charge inside the cell that will disfavor movement of cations to the inside.

19 Active Transport Can Move Molecules Against the Electrochemical Gradient

Active transport of ions against the electrochemical gradient is crucial for maintaining the internal ion composition of cells and to import ions that are at a lower concentration outside the cell than inside.

Cells carry out active transport in 3 ways: (1) Coupled transporters couple the movement of ions against the electrochemical gradient to the favorable movement of another molecule. These transporters use the free energy release during the movement of one ion down its electrochemical gradient to pump another ion against its gradient. In the case shown, the favorable movement of the red molecule down its electrochemical gradient is being used to drive the movement of the yellow molecule against its electrochemical gradient. (2) ATP-driven pumps couple movement against the electrochemical gradient to ATP hydrolysis, which is energetically favorable. In the example shown, this pump uses the energy derived from ATP hydrolysis to move the yellow molecule into the cell, against its electrochemical gradient. (3) Light driven pumps, which are found mainly in bacteria and couple transport to an input of energy from light. In the example shown, this pump uses the energy derived from light to move the yellow molecule into the cell, against its electrochemical gradient.

In this course you will see many more examples of making a process that is unfavorable happen by coupling it to one that is favorable, or to the expenditure of energy.

20 Ion Channels - K+ Channel

The simplest way to move ions from one side of the membrane to the other is to create a hydrophilic channel through which the molecule can pass. Ion channels perform this function by forming transmembrane pores that allow the passive movement of ions across the membrane. Ion channels can move ions across the membrane very efficiently compared to carrier proteins - more than a million ions can pass through each channel each second, a rate 1000 times faster than any carrier protein. Potassium ions move through the channel down their electrochemical gradient from the cytoplasm to the outside of the cell.

The structure of a potassium channel from bacteria has been well studied and consists of 4 identical subunits, each containing several alpha helices that span the lipid bilayer. Two subunits are shown on this slide. The potassium channel has the following important structural features: (1) Negatively charged amino acids are positioned at the entrance to the pore (in the cytoplasm) to attract cations (positively charged) and repel anions (negatively charged). (2) From the cytoplasmic side, the pore opens into a vestibule in the middle of the membrane which facilitates transport by allowing ions to remain hydrated (interacting with water molecules) even inside the membrane. (3) Potassium ions travel in single file through the narrow selectivity filter to the outside of the cell. Mutual repulsion between these single file ions is thought to help move the ions through the pore to the outside of the cell. The ends of the four pore helices point towards the center of the vestibule, guiding potassium ions into the selectivity filter through interactions between the positively charged potassium ions and the negatively charged dipole at the carboxy-terminus of the helix. This helix dipole arises from the polarity of hydrogen bonds, which cause the amino- terminus to be more positively charged and the carboxy-terminus to be more negatively charged.

21 K+ Specificity

The potassium channel has remarkable selectivity - it conducts potassium 10,000-fold better than sodium, yet these ions are spheres with similar diameters (0.133 nm vs. 0.095 nm). A single amino acid substitution in the selectivity filter of the channel can destroy this selectivity.

So what is the basis for the high selectivity and high conductance of this channel? In the vestibule the ions are hydrated - they interact with water molecules. In the selectivity filter, carbonyl oxygens from the protein are positioned precisely to interact with a dehydrated potassium ion (a potassium ion that has lost its water molecules). The dehydration of the potassium ion requires energy, which is precisely balanced by the energy regained by the interaction of that ion with the carbonyl oxygens. The sodium ion is too small to interact with the oxygens; therefore, it does not enter the selectivity filter because the energetic expense of losing its interactions with water molecules is too large because these interactions are not replaced by interactions with carbonyl oxygens.

22 Ion Channels Fluctuate Between Closed and Open States

Ion channels are not continuously open. This would be disastrous for the cell because the differences between the inside and the outside would quickly disappear if all channels were open all of the time. Ion channels generally fluctuate between open and closed states. Only in the open state is the channel in a conformation (structure) that allows the passage of ions.

23 Ion Channels Can Be Gated by Different Stimuli

Most ion channels are gated - that is, they are regulated so that a specific stimulus causes them to switch between closed and open states. Common mechanisms of gating include: (A) changes in voltage across the membrane (e.g. in neurons) (B) binding of ligands to the outside of the channel (e.g. neurotransmitter) (C) binding of ligands to the inside of the channel (e.g. nucleotide or ions) (D) gating by mechanical stimulation (e.g. hair cells of ear).

Ion channels fluctuate randomly between open and closed states; stimuli change the probability that the channel will be in one state or the other.

24