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

Measures of Membrane Fluidity: Melting Temperature

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

As the temperature is raised, a synthetic bilayer made of one type of phospholipid undergoes aphase transition from a solid-like state to a more fluid-like state at a characteristic melting pointtemperature (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 andthere are many stabilizing interactions. As the temperature is raised, these interactions areweakened, and the lipids are in a less ordered, liquid-like state.

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

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unsaturated fatty acid chains

Membrane Composition Influences MembraneFluidity: Fatty Acid Structure

saturated fatty acid chains

lower Tm higher Tm

cis double

bond

oleic acid

OH

O O

OH

stearic acid

17 carbons

olive oil candle wax

The degree of saturation of the fatty acid chains also affects the melting temperature andmembrane fluidity. Fatty acids that are saturated, containing no double bonds, are straightand can pack more tightly than those that have double bonds. The kinks in the unsaturatedchains simply make it more difficult to pack them in an orderly manner. As a consequence, themelting temperature of bilayers containing lipids with saturated fatty acids is higher than themelting temperature of bilayers containing lipids with unsaturated fatty acids. Additionally, thefluidity of bilayers containing lipids rich in unsaturated fatty acids is greater than the fluidity ofbilayers 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 intheir 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 thanthat of stearic acid because oleic acid is unsaturated and cannot pack in as orderly a manner assaturated stearic acid.

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Membrane Composition Influences MembraneFluidity: Cholesterol Content

HO

H

H

H

H

H

Cholesterol is a major component of eukaryotic cells. The ratio of cholesterol to lipid moleculesin 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 ringstructure. Although all steroids are based on the same scaffold, they can affect differentbiological processes, ranging from the development of secondary sex characteristics(testosterone and estrogen) to inflammation (corticosteroids). Like phospholipids, cholesterolis an amphipathic molecule; it has a polar head that contains a hydroxyl group. The rest ofcholesterol 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 betweenatoms in a ring are not free to rotate as a result of the geometric constraints of being in a ring.

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Cholesterol Influences Membrane Fluidity

Cholesterol interacts with phospholipids by orienting its polar hydroxyl head group close to thepolar lipid head group. The rigid rings of cholesterol interact with and partly immobilize thefatty acid chains closest to the polar phospholipid head group. As a consequence, lipidmolecules adjacent to cholesterol are less free to adopt different conformations than those in acholesterol-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 theirpermeability to small water-soluble molecules. Therefore, cholesterol makes membranes lessfluid. Although cholesterol makes bilayers less fluid, at the high concentrations of cholesterolfound in eukaryotic cells, it also prevents fatty acid hydrocarbon chains from coming togetherand 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 membranefluidity by preventing fatty acid hydrocarbon chains from coming together and crystallizing.Under these conditions cholesterol inhibits the transition from liquid to solid (decreases themembrane freezing point). At high temperatures cholesterol decreases membrane fluidity byimmobilizing a few methylene groups in the fatty acid tails of the lipids. Therefore, under theseconditions cholesterol increases the melting point. Therefore, cholesterol acts like antifreeze -the temperature of your car engine is modulated by water circulating with antifreeze/coolantwhich 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 notoverheat.

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

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Summary of Main Points

• HIV needs host cells to replicate because its genome does not encodeall of the proteins required for living systems

• HIV recognizes host cells by interacting with specific protein receptorsfound on the surface of those cell types

• Cell membranes are bilayers composed of amphipathic phospholipidscontaining charged head groups and hydrophobic tails

• The hydrophobic effect drives the packing of lipids into structures whichminimize exposed hydrophobic groups

• Membranes are fluid because phospholipids and proteins can move inthe plane of the bilayer; fatty acid structure and cholesterol contentinfluence fluidity

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October 19, 2006

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Membrane Proteins and Membrane Transport

Alberts 389-410Lecture Readings

1. Membrane proteinsa. Association of proteins with membranesb. Transmembrane helices

2. Lipid Rafts3. Membrane transport

a. Membrane permeabilityb. Transport proteinsc. Ion distribution inside and outside cellsd. Electrochemical gradiente. Active transportf. Ion channels

4. Membrane potential

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Membrane Proteins

Although the lipid bilayer serves as a permeability barrier and provides the basic structure of allcell membranes, most membrane functions are carried out by membrane proteins, whichconstitute 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 relaythem to the inside of the cell

(4) or they can act as enzymes to catalyze reactions.

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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 masson both sides. They have hydrophobic sequences in the bilayer, interacting with thehydrophobic tails of lipids, and hydrophilic regions exposed to the aqueous environment oneither side of the membrane.

(b) Membrane associated - These are proteins located in the cytosol associated with the innerleaflet by an amphipathic alpha-helix (one side of the helix projects hydrophobic residuesand the other side projects hydrophilic ones).

(c) Lipid-linked - Some proteins lie entirely outside the bilayer, interacting with it using only acovalently attached lipid group.

(d) Protein attached - Other proteins are associated only indirectly with the membrane, heldthere by interactions with one or more membrane proteins.

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Many Membrane Proteins Cross the BilayerUsing One or More α-Helices

Many proteins that have mass on either side of bilayer cross it using alpha-helices. Helicalstructures are are well-suited for folding in the bilayer. Those that are transmembrane havehydrophobic side chains which can interact with lipid tails. Within membranes, hydrogenbonding 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 acidsare required to traverse the bilayer in an alpha-helical structure. Scientists can take advantageof knowledge of helical propensity of certain amino acids (the preference they have for oragainst being in an alpha-helical structure) and hydrophobicity to predict transmembranedomains in proteins.

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Lipid Rafts in the Plasma Membrane

protein with longertransmembrane region

enriched in cholesterol and phospholipids with long fatty acid tails

lipidbilayer

protein with fattyacid modification

As we saw in the animation, membranes are dynamic and are not uniform. Lipid rafts are amajor source of inhomogeneity in the membranes of eukaryotic cells. Lipid rafts aremicrodomains in the lipid bilayer that are established by attractive forces between hydrocarbonchains of fatty acid tails. Lipid rafts are enriched in particular lipids (with long fatty acid tailslike 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 importantbecause membrane proteins with long transmembrane segments and also containing certainfatty acid modifications concentrate in lipid rafts. The selective concentration of proteins withparticular properties is a way proteins can be segregated within the two-dimensional membranematrix. Such segregation is very important for trafficking - sorting proteins to their correctdestination in cell - and also for the functioning of certain signal transduction pathways thatcommunicate extracellular signals to the inside of the cell.

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Animation: Lipid Rafts

Animation of dynamics of lipid and protein movement in cell membranes

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MOVIE

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At time t = 50, two fluorescentlylabeled lipid membranes are photobleached at low temperature. Both membranes are identical, exceptone contains cholesterol, the otherdoes not. Which membrane containscholesterol?

A) BlueB) Red

If this experiment were conductedat high temperature, wouldyour answer change?

A) YesB) No

Breakout: Fun With FRAP

BREAKOUT!

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Breakout Answer: Fun With FRAP

At low temperatures, cholesterol increases membrane fluidity by preventing membrane lipids from packing close together.At high temperatures, cholesterol decreases membrane fluidity.

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

A) BlueB) Red

If this experiment were conductedat high temperature, wouldyour answer change?

A) YesB) No

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

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Diffusion of Molecules Across the Lipid Bilayer

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

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Ions Cross Cell Membranes Through Membrane Transport Proteins

There are many different types of membrane transport proteins, each selective for a certainclass of molecule - ions, amino acids, or sugars, for example. Many transport proteins arehighly 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 movingdown its concentration gradient (e.g. from higher concentration outside to lowerconcentration inside). Passive transport can be mediated by channel proteins, whichcreate hydrophilic pores in the membrane to allow movement of ions, or by carrierproteins, which have a binding site(s )for the molecule to be transported and undergo achange 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 concentrationgradient and movement therefore has to be coupled to a process that provides energy.Only carrier proteins can mediate active transport.

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Ion Concentrations Inside and Outside of a Cell

Na+

Na+

K+

Cl-K+

Cl-

(5-15 mM)

(140 mM)

(5-15 mM)

(145 mM)

(110 mM)

(5 mM)

inside cell

organicanions

Because of transport processes and the barrier imposed by the cell membrane, living cellsmaintain an internal ion composition that is very different from that of the externalenvironment. These differences are crucial for the cell’s function, including the activity of nervecells. The things that are important for you to remember are that sodium is the most plentifuloutside the cell and potassium is the most plentiful inside. In order to avoid the buildup of toomuch electrical charge, the amount of positive charge inside the cell must be balanced with analmost 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 highconcentration of potassium is balanced by a variety of negatively charged intracellular organicions (anions). However, tiny excesses of negative or positive charge do build up near theplasma membrane and, as we will see, they have important consequences.

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Net Driving Force for Transport is Determined by the Electrochemical Gradient

For uncharged molecules, the concentration gradient drives passive transport. If the moleculeis charged, both the concentration gradient and the charge difference across the membrane(called the membrane potential) influence transport. Together, these two forces are referredto as the electrochemical gradient. In a few slides we will discuss where the charge differenceacross the membrane comes from.

In this slide, the width of the green arrow represents the magnitude of the electrochemicalgradient for the same positively charged ion in three different situations. In (A) there is only aconcentration gradient. The cation (positively charged ion) moves down its concentrationgradient, from the outside of the cell to the inside. In (B) the concentration gradient issupplemented by a membrane potential that increases the driving force. The sameconcentration gradient exists as in (A), but now there is an additional driving force from themembrane potential, which is negative inside and therefore favors the movement of positivelycharged cations to the inside of the cell (movement of cations is favored in this case becausethe inside of the cell has a slight excess of negative charge). In (C) the membrane potentialdecreases the driving force caused by the concentration gradient. In this case, the membranepotential is opposite to (B), with a slight excess of positive charge inside the cell that willdisfavor movement of cations to the inside.

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Active Transport Can Move Molecules Againstthe Electrochemical Gradient

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

Cells carry out active transport in 3 ways:

(1) Coupled transporters couple the movement of ions against the electrochemical gradient tothe favorable movement of another molecule. These transporters use the free energyrelease during the movement of one ion down its electrochemical gradient to pumpanother ion against its gradient. In the case shown, the favorable movement of the redmolecule down its electrochemical gradient is being used to drive the movement of theyellow molecule against its electrochemical gradient.

(2) ATP-driven pumps couple movement against the electrochemical gradient to ATPhydrolysis, which is energetically favorable. In the example shown, this pump uses theenergy derived from ATP hydrolysis to move the yellow molecule into the cell, against itselectrochemical gradient.

(3) Light driven pumps, which are found mainly in bacteria and couple transport to an input ofenergy from light. In the example shown, this pump uses the energy derived from light tomove 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 unfavorablehappen by coupling it to one that is favorable, or to the expenditure of energy.

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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 throughwhich the molecule can pass. Ion channels perform this function by forming transmembrane pores that allow thepassive movement of ions across the membrane. Ion channels can move ions across the membrane very efficientlycompared to carrier proteins - more than a million ions can pass through each channel each second, a rate 1000times faster than any carrier protein. Potassium ions move through the channel down their electrochemical gradientfrom 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, eachcontaining several alpha helices that span the lipid bilayer. Two subunits are shown on this slide. The potassiumchannel 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 transportby 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 repulsionbetween these single file ions is thought to help move the ions through the pore to the outside of the cell. The endsof the four pore helices point towards the center of the vestibule, guiding potassium ions into the selectivity filterthrough interactions between the positively charged potassium ions and the negatively charged dipole at thecarboxy-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.

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K+ Specificity

The potassium channel has remarkable selectivity - it conducts potassium 10,000-fold betterthan sodium, yet these ions are spheres with similar diameters (0.133 nm vs. 0.095 nm). Asingle 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 thevestibule 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 dehydratedpotassium ion (a potassium ion that has lost its water molecules). The dehydration of thepotassium ion requires energy, which is precisely balanced by the energy regained by theinteraction of that ion with the carbonyl oxygens. The sodium ion is too small to interact withthe oxygens; therefore, it does not enter the selectivity filter because the energetic expense oflosing its interactions with water molecules is too large because these interactions are notreplaced by interactions with carbonyl oxygens.

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Ion Channels Fluctuate Between Closed and Open States

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

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Ion Channels Can Be Gated by Different Stimuli

Most ion channels are gated - that is, they are regulated so that a specific stimulus causesthem 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 theprobability that the channel will be in one state or the other.