biol 4004 exam 1

One benefit and one challenge that the cell membranes present for cells

Membranes are beneficial for cells because they allow cells to maintain their internal composition. (It allows cell to maintain solute concentrations in its cytosol which differs from extracellular fluid and organells)

One challenge they present, however, is that the lipid bilayer is impermeable to most substances and thereby presents an obstacle to the uptake of nutrients and excretion of waste products. (Cells need to move specific water-soluble molecules and ions across membranes to ingest nutrients, excrete waste products, and regulate ion concentrations). they use membrane transfer proteins to do this. the 2 main classes of MPs include: transporters (undergo conformational changes to transport specific small molecules across membranes) and channels (form narrow pores allowing passive transport mainly for water and small ions)

Protein-free Lipid Bilayers (Synthetic bilayers) are impermeable to ions (why? which mols/ions are permeable?)

Any molecule will diffuse across a protein-free bilayer down its concentration gradient. the rate depends on size and (mostly) hydrophobicity.

Small, nonpolar molecules diffuse fastest (O2 and Co2 diffuse readily)

Small, uncharged polar molecules diffuse much more slowly

Bilayers are impermeable to charged molecules of all sizes due to their charge and high degree of hydration preventing them from entering the hydrophobic region

Define Membrane Transport Protein and the 2 major classes

Membrane Transport Proteins - transfer solutes across cell membranes. they are multipass transmembrane proteins because their polypeptide chain crosss the bilayer many times which forms a protein-lined path across membrane and enables specific hydrophilic solutes to cross membrane while avoiding hydrophobic region of bilayer

* Transporters and channels are the 2 major classes of MTPs

Define Transporters

Transporters - bind specific solutes and undergo conformation changes that alternately expose solute-binding sites on 1 side of the membrane and then on the other to transfer solutes across it.

Define Channels

Channels - form continuous pores that extend across bilayer which, when open, allows specific solutes (ions and small molecules) to pass through

* much faster rate of transport than by transporters

* fig 11-3 p599

List some general features of Transporters compared to Channels

Transporters can recognize a myriad of different solutes with a high degree of specificity. There are transporters that recognize particular amino acids and others that recognize sugars. With the exception of ion channels, most channels are not very specific.

* Transporters are usually slower than channels. This is because a transporter must undergo a conformational change to allow the solute across the membrane.

* Certain types of transporters can promote the active transport of a solute across a membrane (whereas channels are always passive diffusion)

Define Passive transport/passive diffusion

transport of a solute directly through the membrane without the aid of a protein.

(passive=no protein and thus no energy input)

* all channels and many transporters allow solutes to cross the membrane passively (down its gradient without aid of a protein)

~ Define concentration gradient

Concentration gradient is the difference in concentration on the 2 sides of the membrane

* it drives passive transport of uncharged molecules and determines its direction

*

~ electrochemical gradient

for charged molecules, both the concentration gradient & the electrical potential difference across the membrane - called the membrane potential - influence its transport

* conc. gradient and membrane potential together form the electrochemical gradient - a net driving force for solutes across membranes

* most cells have a neg. charge compared to outside (-70mV resting potential)

* For charged substances, such as ions and charged molecules, the gradient of the solute is determined by two factors: the concentration of the solute across the membrane and the distribution of charge across the membrane. The two factors together define an electrochemical gradient. For example, if the concentration of Na+ ions were 100mM outside the cell and 5 mM inside the cell, this would be a chemical gradient. Sodium ions would want to flow into the cell due to the chemical gradient. If there were more positive charge outside the cell compared to inside, this would be an electrical gradient. In this case, Na+ would also want to flow into the cell because of the electrical gradient.

Facilitated diffusion

facilitated diffusion - transport of a solute across the membrane via aid of a membrane protein (MP) down its gradient. this requires no energy input

Active Transport

Active Transport - transport of a solute across the membrane via the aid of a membrane protein, against its gradient. This requires an input of energy.

* active transport is mediated by transporters coupled to an energy source

* fig 11-4

read summary

p.600

The 4 common ways (except 1) that transport is coupled to energy

Transport may be coupled to:

* ATP hydrolysis

* an ion-electrochemical gradient (uses the E stores in CGs to couple the uphill transport of 1 solute across the M to the downhill xport of another)

* electron movement ("very imp mechanism)

* light (very rare, only found in a few bacteria species)

*fig 11-7

Define Uniport

Uniport occurs when the transport of a solute is not coupled to the transport of another solute. The transport passively transfers a single solute across M at a rate determined by Vmax and Km (xporters charac. affinity for solute)

* fig 11-8

the 2 types of coupled transporters and their definitions

Symporters - involves the transport of 2 solutes in the same direction

Antiporters - couples the xport of 2 solute mols in opposite directions

~

* coupling occurs because transporter harvests the E stores in the electrochemical gradient of 1 solute - usually an ion - to xport the other

* the free E released during movement of an ion down its EC gradient is used to pump other solute uphill against its EC gradient

* Na+ is usually the cotransported ion bc its pumped out of cell by an ATP driven Na+/K+ pump in the PM

Define Primary Active Transport

Primary active transport - when energy (ATP, light, or electrical) is directly coupled to active transport

* ex: ATP-driven pumps where the free E of ATP hydrolysis drives xport of a solute against its gradient

ex: fig 11-9 Na+/glucose symporter can move glucose against its chemical gradient if a favorable Na+ gradient is present. In this case, Na+ is moving down its electrochemical gradient, enabling glucose to be transported against a gradient.

Define Secondary Active Transport

Secondary active transport - when active transport is coupled to a favorable ion-electrochemical gradient

Define Transcellular transport

Transcellular transport is the movement of a solute into a cell and out the other side. It occurs across several types of cells such as intestinal cells.

~[* occurs in epithelial cells such as those absorbing nutrients from the gut. solutes are moved across epithelial cell layer into EX fluid where they pass into blood. this is done via Na+ linked symporters located in the apical/absorptive domain of PM which actively xports nutrients into cell building up gradients in cells while uniporters at basolateral side of cell/faces blood levave cell passively down their CGs]

Ex: Figure 11-11, glucose is driven into the cell against a gradient via a Na+-coupled symporter. Due to symport, glucose can accumulate to high intracellular levels. At the other side of the cell (i.e., the side facing the blood), glucose is transported, via a uniporter, to the outside of the cell. The uniporter transports glucose in a downhill direction.

List the 4 types of ATP-driven pumps

* P-type pumps - hydrolyze ATP and establish/maintain ion (Na+, K+, Ca2+, H+) gradients across membranes. (~they phosphorylate themselves during the pumping cycle. )

* ABC Transporters (~ATP Binding Casette Transporters) couples the hydrolysis of ATP w/ the transport of small molecules across membranes.

* V-type pumps - hydrolyze ATP and pump H+ ions into organells (ex. lysosomes) to acifidy their interior/lumen of the organelle

*F-type pumps: couple the transport of H+ ions across membranes with the synthesis of ATP. F-type ATPases are found in mitochondria and bacteria. (~they work opposite as V-type pumps because they synthesize ATP from ADP+P. The H+ gradient was generated during electron transport steps during oxidation)

*Fig 11-12

P-Type Atpases pump Ca2+ into the Sarcoplasmic Reticulum (SR, out of the cell) in muscle cells

Define features of it

Ca2+-ATPases are found in the plasma membrane of most living cells where they pump Ca2+ out of the cell to maintain a low Ca2+ concentration in the cytosol.

* fig 11-13: the calcium pump contains ten transmembrane segments. Two calcium ion-binding sites are located within the transmembrane region. The ATP binding site and phosphorylation site are not in the transmembrane region. Rather, they are found in the part of the protein that projects into the cytoplasm. Somehow, phosphorylation transmits a conformational change that alters calcium ion accessibility across the membrane. In this regard, it is interesting to note that transmembrane segment 5 comes out of the membrane and comes close to the phosphorylation site. This is a critical region that transmits the conformational change from the phosphorylation site to the transmembrane region.

(* the Ca2+ pump is a P-type transport ATPasewhich have similar structures:

* 10 transmembrane alpha-helices connected to 3 cytosol domains

* aa side chains form 2 central binding sites for Ca2+

* in the pump's ATP-bound nonphosphorylated state, binding sites are only availavble in cytosolic side of the SR membrane

* Ca2+ binding triggers conformational changes which close the passageway and activate rxn where a P from ATP is transferred to an aspartate (in a cytosol domain)

*ADP dissociates and replace w/ a new ATP, causing a conf. change that opens passageway to SR lumen where the 2 Ca2+ ions exit)

*the 3 domains are: activator, phosphorylation, nucleotide-binding

How the Na+.K+ pump works to establish Na+ and K+ gradients across the membrane

*All living cells maintain an ion electrochemical gradient across their plasma membrane. In animal cells, a strong Na+ gradient is typically present. This gradient is generated by a transporter called the Na+, K+-ATPase or simply the sodium pump. For each ATP it hydrolyzes, three sodium ions are pumped out and two potassium ions are pumped into the cell

*fig 11-15

* the series of steps that occur for this transporter to work:

* 1st, three sodium ions bind to the protein from within the cell. ATP is hydrolyzed, and a phosphate group is temporarily attached to the protein covalently. The protein then undergoes a conformational change such that the sodium ions are exposed to the outside of the cell. The sodium ions are released and then two potassium ions bind. This promotes the dephosphorylation of the protein, which then causes a conformational change so that the potassium ions are exposed to the inside of the cell. The potassium ions are released and then the cycle can start over again.

(* it actively pumps Na+/K+ against their CGs so that Na+ gradient can then be used to pump nutrients into cell. the pump is electrogenic - creates a net electric current across the M, creating an electric potential with inside of cell more negative than outside)

The functional role of the sodium pump in animal cells?

One role is to drive sodium-coupled symporters as described previously in Figure 11-8. A second role is to maintain cell volume by controlling the amount of ions across the membrane. The sodium pump transports three ions out and two ions in. If the cell is swelling due to osmosis, the sodium pump will speed up to remove excess intracellular ions. If the cell is shrinking, it will slow down.

Define ABC Transporters

ABC transporters are members of a gene family that transport a diverse array of solutes including ions, sugars, and amino acids.

* ABC transporters have a modular structure. They have two transmembrane regions, each composed of six transmembrane segments, and two ATP binding domains that project into the cytosol.

* fig 11-18

Give some examples of ABC Transporters

ex: 1 eukaryotic ABC Transporter, miltidrug resistance protein (MRP) pumps hydrophobic drugs out of the cytosol. it is present at elevated levels in cancer cells and makes them resistant to a variety of cytotoxic drugs

ex: Plasmodium falciparum - a protist that causes malaria has developed resistance to the antimalarial drug chloroquine via an amplified ABC Xporter gene

ex: an ABC transporter in the ER membrane actively pumps a variety of peptides from the cytosol into the ER lumen produced by protein degradation in proteasomes and are carried from ER to cell surface for inspection by T lymphocytes which destroy the cell if the peptides are from microbes

ex: the cystic fibrosis transmembrane conductance regulator protein (CFTR) may have a mutation causing cystic fibrosis. it xports cl- across M. atp binding and hrolysis dont drive it to xport but control the opening and closing of a continuous channel. so abc transporters can function as transporters and gated channels.

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Aquaporins

Aquaporins are proteins that form very narrow channels, which allow water molecules to traverse the membrane in single file. Aquaporins, therefore, allow the passage of water molecules but completely block the passage of ions.

Define Ion Channels

Ion channels allow the movement of specific types of ions (have ion-selectivity) across the membrane. (the ion must have the right size and charge to pass through the narrow pores)

* Nearly all ion channels are gated, which means they can open and close. The stimulus that causes opening and closing differs among different ion channels

* ions shed their H2O mols and pass single file thru the narrowest part of the channel aka the selectivity filter

*fig 11-22

* they are gated, meaning they open briefly and close. with prolonged stimulation, they go into an inactivated state where they are refractory until stimulus is removed

Identify 3 gated mechanisms

* voltage gated- opening depends on the electrical gradient across the membrane

* ligand gated- opening depends on the binding of a molecule to the channel.

* mechanically gated- opening depends on physical changes such as pressure, sound waves, etc.

Define membrane potential and its role in creating electrochemical gradients (ECGs)

Another name for an electrical gradient across a membrane is the membrane potential. Differences in ion concentrations across a membrane are responsible for creating ion electrochemical gradients.

(The Membrane Potential in Animal Cells Depends Mainly on K+ Leak Channels and the K+ Gradient Across the Membrane)

how electrochemical equilibrium may occur for K+ in a cell

With regard to ion electrochemical gradients, it's interesting to examine the two components separately. As an example, let's consider K+ ions. In most animal cells, the chemical gradient is outwardly directed. There is a higher concentration of K+ ions in the cell. By comparison, the electrical gradient is inwardly directed. There tends to be more positive charge on the outside of the cell compared to the inside. It's possible for the two gradients to balance each other and create a equilibrium. The outwardly directed chemical gradient precisely counteracts the inwardly directed electrical gradient and therefore, there is no net movement of K+ ions in either direction.

define the Nernst equation

The Nernst equation is a formula that quantifies the conditions that create equilibrium. For a univalent cation such as potassium at 37oC, this equation is:

V = 61.5 mV log10 [K+out]/K+ in]

where V is the electrical gradient across the membrane (internal minus external)

As an example, let's suppose that the K+ concentration outside the cell is 10 mM and the concentration inside is 100 mM. The log10 of 10/100 equals -1. Under these conditions, V = -61.5 mV. If the membrane potential were -61.5 mV (inside negative), K+ ions would be in equilibrium.

How is a channel selective to a specific ion?

Many ion channels are highly selective for particular types of ions. For example, certain K+ channels can transport K+ ions 10,000-fold better than Na+ even though sodium ions are smaller. So how does a channel allow the faster transport of a larger ion compared to a smaller one? The answer lies in a structure of the channel called the selectivity filter

* fig 11-24

* When dissolved in water, ions interact with water molecules such that a hydration shell of water molecules surrounds the ion. This is an energetically favorable arrangement. The selectivity filter contains electronegative oxygen atoms that mimic the hydration shell that normally surrounds ions. As seen in Figure 11-25, K+ and Na+ ions are surrounded by four water molecules. To penetrate the selectivity filter, these water molecules must be stripped away, and the oxygen atoms of the selectivity filter must substitute for the oxygen atoms of water. The four oxygen atoms in the selectivity filter of a K+ channel are the correct distance apart to substitute for the hydration shell around K+ ions. Therefore, water molecules can be stripped from K+ as it moves through the filter. By comparison, the four oxygens in this selectivity filter are not the correct distance apart for all of them to interact with Na+. Therefore, the water cannot be completely stripped from Na+ and this presents an energy barrier to the movement of Na+ ions through the channel.

* fig 11-25

define action potential

A wave of excitation that is propagated from the cell body to the terminal branches of the axon

* fig 11-28

How Voltage-gated Cation Channels Generate Action Potentials

* fig 11-30

Two different voltage-gated channels, Na+ channels and K+ channels, are found

along the length of the axon. At the normal resting potential of a nerve, which is around -

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70mV, both types of channels are closed. Let's consider the sequence of events that occurs when the resting potential is changed to -50mV, as described in the graph of Figure 11-30:

1. Na+ channels open when the membrane potential changes to -50mV. According to the Nernst equation, Na+ is very far out of equilibrium. Some Na+ ions rush into the cell, and establish equilibrium at approximately +50mV. At +50mV, the Na+ channels close and exist in an inactivated conformation that cannot reopen until the resting potential is restored.

2. The K+ channels were also stimulated to open when the membrane potential reached -50 mV. However, these channels open more slowly. They don't get open until the membrane potential is at +50mV. Under these conditions, K+ ions are not in equilibrium. A few K+ ions rush out of the cell to establish equilibrium. This brings us back to the resting potential of -70mV.

Figure 11-31 describes the events as they occur along the length of the axon. You should understand part B of this figure. Na+ channels undergo a temporal change from closed to opened to inactivated to closed. This causes the action potential to be propagated from left to right. You should also be familiar with the three conformational states of cation channels as shown in Figure 11-30C.

Describe Voltage-Gated Cation Channels

They are Evolutionarily and Structurally Related.

Na+ and K+ channels are members of a gene family. In mammals, each channel has 24 transmembrane segments that are organized into modules of 6 transmembrane segments each.

How Transmitter-gated Ion Channels Convert Chemical Signals into Electrical Ones at Chemical Synapses

For the moment, let's skip ahead and take a look at Figure 11-39. When an action potential reaches the terminal tip of the axon, this opens voltage-gated calcium ion channels. This causes calcium ions to rush into the cell, which promotes the fusion of vesicles with the membrane. The vesicles shown in this figure contain acetylcholine, which is a neurotransmitter that activates a ligand-gated channel. The acetylcholine diffuses across the neuromuscular junction and binds to a channel called the acetylcholine-gated channel, or simply the acetylcholine receptor. This opens the channel and cations rush into the cell and depolarize the membrane. Via Na+ channels, the depolarization is propagated to calcium channels in the sarcoplasmic reticulum membrane causing calcium ions to be released into the cytosol. As discussed later in the course, calcium ions then promote muscle contraction.

* fig 11-39

How Chemical Synapses Can Be Excitatory or Inhibitory

* The main point here is what makes a neurotransmitter excitatory vs inhibitory.

On the whole, if a neurotransmitter causes membrane depolarization, it is excitatory. If it dampens membrane depolarization, it is inhibitory.

(* excitatory neurotransmitters open cation channels causing an influx of Na+ and Ca2+ which depolarizes the postsynaptic membrane toward the threshold potential for firing an action potential.

* inhibitory neurotransmitters open either Cl- or K+ channels which suppresses firing by making it harder for excitatory neurotransmitters to depolarize the postsynaptic membrane)

How Acetycholine Receptors work at the neuromuscular junction as transmitter-gated cation channels

* The acetylcholine receptor is found in the plasma membrane of certain muscle cells. It receives a signal from a nerve cell (i.e., via the binding of acetylcholine) and promotes a signal that ultimately causes muscle contraction.

* This channel opens in response to the binding of two ligand molecules (i.e., two acetylcholine molecules). Once open, it allows the passage of cations. This channel does not have a precise selectivity filter like the sodium and potassium channels. The acetylcholine receptors allow the passage of many cations including Na+, K+, and Ca2+. When it opens, primarily Na+ passes through the channel because the extracellular concentration of Na+ is by far the highest. When the channel opens, the Na+ ions are out of equilibrium according to the Nernst equation.

* The molecular structure of the protein is shown in Figure 11-38. It is a pentamer. The five subunits form a channel that is lined with negative charges. The gate is deep within the channel. The binding of two acetylcholine molecules causes a conformational change that alters the positioning of subunits, and thereby opens the gate.

* fig 11-38

How neuromuscular junctions create muscle contractions

(Neuromuscular Transmission Involves the Sequential Activation of Five Different Sets of Ion Channels)

An action potential travelling down to the presynapse causes voltage-gated Ca++ channels to open, causing an influx of Ca++, which results in the release of

neurotransmitters. The neurotransmitters bind the receptor-channels on the postsynapse. Activation of acetylcholine receptors results the influx of Na+, which causes local membrane depolarization, which activates local voltage-gated Na+ channels. The resulting self-propagating membrane depolarization of the plasma membrane activates voltage-gated Ca++ channels that lead to the opening of Ca++-gated Ca++ channels on the

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adjacent sarcoplasmic reticulum that acts as a

Ch. 10 Membrane Structure

Define plasma membrane

* separates the cytoplasm from the external environment

* encloses th cell, defines its boundaries, and maintains the essential difference between the cytosol and the extracellular environment

* its a thin film of lipid and protein molecules held by noncovalent bonds.

* its a dynamic, fluid structure

* eukaryotic cells have internal membranes that create organelles such as the cell nucleus

lipid bilayer

a fluid, impermeable barrier to water-soluble molecules

* membrane proteins may be embedded in the bilayer (which mediate all functions of the membrane such as xport of molecules across, atp synthesis catalysis, receptors, etc)

* provides the basic structure of cell membranes and is attributable to the properties of lipids which assemble spontaneously into bilayer when exposed to water

the 3 most abundant membrane lipids Phosphoglyceride, sphingolipids, and sterols

* phospholipids, the most abundant of which are phosphoglycerides

* phosphoglycerides are amphiphilic bc they have a polar head and 2 nonpolar tail regions (bc they have a hydrophilic end and a hydrophobic end). the backbone of it is the 3-carbon compound glycerol whereas the backbone of the phospholipid sphingomyelin is sphingosine. Many membranes contain cholesterol, a sterol that contains a rigid ring structure attached to a single polar hydroxyl group (Figure 10-4).

* fig 10-4

Phospholipids Spontaneously Form Bilayers

The nonpolar tails of phospholipids do not favorably interact with water (Figure 10-7). This unfavorable interaction decreases entropy, because water forms an ordered structure around the nonpolar tail in an attempt to minimize interactions. To avoid a decrease in entropy, which is energetically unfavorable, phospholipids in water will spontaneously form a bilayer in which the nonpolar tails interact with each other and polar heads are exposed to water.

(they spontaneously aggregate in the presence of water to bury their hydrophobic tails int heir interior where they are shielded from water and expose their hydrophilic heads to water, done in 2 ways: by forming spherical micelles with tails inward or double layered sheets (bilayers) w/ hydrophobic tails sandwiched bt hydrophilic head grps.. ths purpose of this is to minimize entropy/free ennergy by producing as many energetically favorable hydrogen bonds as possible.)

* fig 10-7

How The Lipid Bilayer Is a Two-Dimensional Fluid

The bilayer is semi-fluid, allowing certain types of movements and preventing others (Figure 10-11). Lipids can diffuse laterally (e.g., from left to right) and rotationally (rotate 360o). However, it is energetically unfavorable for them to flip from one leaflet of the bilayer to the other.

* fig 10-11

How The Fluidity of a Lipid Bilayer Depends on Its Composition (lipid tails double bonds, shorter/long lipids, cholesterol, high temps)

An optimal fluidity of a biological membrane is needed to ensure the proper integrity of the membrane and to promote the function of membrane proteins. Fluidity can be altered by altering the lipid composition.

* structural features of lipids make the bilayer more or less fluid. These include the following:

* Double bonds in the lipid tails make the bilayer more fluid. This is because double bonds diminish packing between the tails.

* Shorter lipids tails make the bilayer more fluid. Shorter tails are freer to diffuse laterally and rotationally.

* At high concentrations, cholesterol modulates membrane fluidity.

* High temperature makes the bilayer more fluid.

How Despite their Fluidity, Lipid Bilayers can Form Domains of Different Compositions (Lipid Rafts)

In addition to asymmetry with regards to the leaflet, the membrane bilayer is not uniform in that there are thicker regions of the membrane, known as lipid rafts that are composed of a grouping of lipid and protein molecules that are somewhat stuck together (Figure 10-13). They diffuse within a lipid bilayer as a single unit. Researchers speculate that lipid rafts may promote the function of certain membrane proteins.

* fig 10-13

How The Asymmetry of the Lipid Bilayer Is Functionally Important

With regard to the two leaflets of a membrane, asymmetry exists in the following ways (Figures 10-15):

The composition of different types of lipids varies in the two leaflets.

The orientations of membrane proteins are asymmetrical.

As we will learn later in the course, this asymmetry is very important with regard to membrane function.

* fig 10-15

Define glycolipids

Glycolipids are lipids that have carbohydrates attached to the polar head group (Figure 10-16). When found in the plasma membrane, the carbohydrate portion projects to the outside of the cell. When found in organellar membranes, the carbohydrate projects away from the cytosol; it projects into the lumen of organelles. As we will learn later in the course, glycolipids play a role in cell recognition and help to protect the outer surface of cells.

* fig 10-16

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membrane proteins

are embedded within the lipid bilayer or attached to its surface

The various ways membrane proteins can be associated with the lipid bilayer (integral, peripheral)

The structure of a portion of a protein can be embedded in the bilayer. This is shown for the first, second, and third proteins in Figure 10-17. These are integral membrane proteins.

* Peripheral membrane proteins are associated with the cell membrane but do not go through both lipid leaflets. They can be associated with the cell membrane by having an amphiphilic helix that partitions into the inner leaflet of the lipid bilayer (protein #4), are anchored to the membrane by a covalently attached lipid or hydrophobic moiety (protein #5 & 6), or by simply binding to a membrane- associated protein.

* fig 10-17

How lipid anchors control the membrane localization of some signaling proteins

Some membrane proteins are covalently attached to a fatty acid chain or a prenyl group (Figure 10-18). This anchors the protein to one side of the membrane where it carries out its biological activity.

* fig 10-18

~(how a MP attaches to bilayer reflectsi ts function

* transmembrane Ps function on both sides of bilayer and transport molecules across (ex: receptors)

* proteins that function on 1 side of bilayer are bound only to the lipid monlayer on that side

* ex: some intracellular signaling Ps that relay EC signals to interior of cell are bound to cytosolic monlayer by 1+ covalenty attached lipid groups which can be fatty acid chains or a prenyl group. usually >1 fatty acids attach the protein firmly to bilayer.)

In most transmembrane proteins, the polypeptide chain crosses the lipid bilayer in which helical conformation?

alpha-helical conformation - is (by far) the most common (Figure 10-19). When a protein contains a sequence of 20-25 amino acids, most of which have nonpolar side chains, such a sequence may fold into an helix and completely cross the membrane. This is termed a transmembrane segment of a protein.

(this conf. satisfies the hydrogen bonding requirements of the protein. Ps can also fold into a beta-sheet that is rolled into a cylinder (beta-barrel)

How can computer programs predict the likelihood that a protein contains an alpha-helix that is a transmembrane segment?

By creating a hydropathy plot to illustrate the relative polarity of protein regions. When a segment is very positive in this plot, it means that it contains a high proportion of nonpolar amino acids. If a very positive region is long enough (i.e., contains 20 amino acids or more), it is likely to be a transmembrane segment.

(segments with 20-30 hydrophobic amino acids can span bilayer as an alpha-helix and can be identified in hydropathy plots which localize potentiala lpha-helical membrane spanning segments of a P)

How Transmembrane Alpha-Helices Often Interact with One Another

As shown in Figure 10-21, transmembrane alpha-helices in multipass membrane proteins often interact, and these interactions contribute to protein structure. Interactions between helices are particularly important for the function of many membrane channels and transporters.

~( alpha-helices form dimers help together by noncovalent bonds between 2 helices...)

How Some Beta-Barrels Form Large Transmembrane Channels

Alternatively, and less common, some proteins have beta-strands that cross the membrane. These proteins have multiple beta-strands that form a cylindrical structure called a beta-barrel (Figure 10-23).

*Such proteins are frequently found in the outer membrane of certain bacteria and in the outer membrane of mitochondria and chloroplasts. They function as transmembrane channels.

~(beta-barrels are more rigid than alpha-helices, many are channels/pores that allow small hydrophilic mols across, polar aa's line the channel on the inside and nonpolar aa's side chains form outside of barrel which interact w/ hydrophobic region of bilayer, loops of protein protrude into lumen of channel to narrow it so only certain solutes can pass, making porins highly selective)

Many Membrane Proteins Are Glycosylated

Like lipids, the structure of a protein can have carbohydrates that are covalently attached (Figure 10-25).

~~(* the extracellular parts of most proteins are glycosylated (have an oligosaccharide added to them), the cell coat/glycoalyx protects the cell from mechanical/chemical damage and prevents unwanted cell-cell interactions)

Membrane Proteins Can Be Solubilized and Purified in Detergents

If a researcher wants to purify and study a particular type of membrane protein, it must be removed from the membrane. This can be accomplished via detergents, which are lipid-like molecules that naturally form micelles (Figure 10-27).

Lipids remove proteins from a membrane such that a single protein is found within a micelle (Figure 10-28). Protein/lipid micelles can be purified by chromatography techniques, described in Chapter 8.

(the detergent disrupts the bilayer by binding to hydrophobic regions of MPs where they displace lipid mols w/ a collar of detergent mols. since one end of detergent molds is polar, this binding brings MPs into soln as detergent-protein complexes.)

The 2 methods used to see how many membrane proteins diffuse in the plane (surface) of the membrane: heterokaryons and FRAP

(MPs dont flip flop across bilayer but they rotate perpendicular to bilayer [rotational diffusion] and move laterally within membrane [lateral diffusion])

The diffusion of membrane proteins can be studied by a variety of methods. In the experiment of Figure 10-32, mouse and human cells were fused to create a heterokaryon. The cells were then exposed to an antibody that specifically recognized a membrane protein in the mouse cell and to a different antibody that recognized a human membrane protein. Each antibody was labeled with a different fluorescent molecule. Immediately after fusion, the two sides of the heterokaryon were different colors. Over time, however, the different colored fluorescent molecules became intermingled due to lateral diffusion of the membrane proteins in the plasma membrane. (2 separately labeled antibodies that distinguishes mouse and human PR proteins were created. at first, the mouse and human proteins were confined to their own halves of the heterokaryon, then the 2 sets of proteins diffused and mixed over entire cell surface in 1/2 hr)

A second method to monitor lateral diffusion is FRAP (fluorescence recovery after photobleaching) (Figure 10-33). In this method, the cell surface is labeled with a fluorescent molecule and then a region is bleached with a laser beam. The bleaching permanently destroys the fluorescence. it is measured how long it takes for the fluorescent molecules in the adjacent, unbleached region to invade the bleached region. As in the previous experiment, this is due to lateral diffusion. (allows estimation of the diffusion coefficient for diff. MPs in diff cells which is highly variable b/c interactions w/ other proteins impedes movement)

What is the function of the cortical cytoskeleton?

To give Membranes Mechanical Strength and Restricts Membrane Protein Diffusion

Underlying the plasma membrane is the cortical actin-enriched cytoskeletal network that gives the plasma membrane mechanical strength as well as helps restricts the lateral movement of membrane proteins. This is most obvious in red blood cells (RBC's), which have to deform their cell shape to go through narrow capillaries. RBC's that have impaired cortical cytoskeletons cannot withstand the forces as they flow through the capillaries, and as such lyse, leading to spherocytosis.

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structure of a polypeptide chain (protein aa sequence)

(amino/N terminus) NH3+-CH(R)-C(=O)-[peptide bond]-NH-CH(R)-C(=O)(O-) (Carboxyl/C terminus)

enzymes

proteins that catalyze chemical reactions

~ the shape of a protein is determined by?

its amino acid sequence. which has side chains that will determine which bonds hold the protein together. this may include hydrogen bonds, electrostatic (ionic) bonds, and dispersion forces [all noncovalent bonds] (as well as hydrophobic clustering where hydrophobic side chains of certain aa's are forced together in an aqueous env to maximize h-bonding of water mols whereas polar side chains arrange themselves ont he outside of the protein so they can h-bond with water and other polar mols)

what are 2 common protein folding patterns

alpha-helix and beta-sheet

* protein domains

The structures of proteins are organized into functional modules called domains.

Each domain has a unique 3D structure.

* (protein domain - a substructure produced by any part of a polypeptide chain that can fold independently of the rest of the protein into a compact, stable structure. it is the modular unit from which many larger proteins are made. different domains usually have different functions.

(protein domains are modular units from which larger proteins are built. the structures of proteins are organized into functional modules called domains. each domain has a unique 3D shape)

the 4 levels of organization of a protein

Primary structure - the amino acid sequence

Secondary structure - stretches of polypeptide chain that form alpha-helices and beta-sheets

tertiary structure - the full 3D organization of a polypeptide chain

quaternary structure - a complex of more than 1 polypeptide chain's structure

homologous genes

genes derived from the same ancestral gene

how gene duplications create them

Gene duplications can create two or more homologous genes within a single species. Over time, duplicated genes accumulate mutations that cause them to be specialized in their function.

how gene families arise/define them

A gene family is a group of homologous genes within a single species in which the members have related but specialized functions. For example, a gene family may encode proteins involved with cell contraction. The members of the family may have slightly different contractile properties and may be expressed in different types of cells.

how computer technology can identify homologous genes

why is this useful?

Using computer technology, a gene sequence can be used to search a database to determine if that database contains genes that are homologous.

This is useful because

homologous genes often have similar functions. Therefore, if a researcher identifies a new gene, the identification of homologous genes in a database can be a useful clue regarding the new gene's function if the function of any of the homologues is already known.

* exon shuffling and its evolutionary role in creating proteins

Eukaryotic proteins are often the result of exon shuffling in which a sequence encoding a particular domain has been inserted into many different genes. This creates proteins with a modular structure, which means that a protein has different regions that carry out specific types of function. Examples are shown in Figures 3-10 and 3-15. You don't need to memorize these examples, but be familiar with the general concept.

(protein module - a subset of domains that were especially mobile during evolution)

Many proteins are composed of two or more subunits. The subunits may be encoded by the same gene or by different genes.

how structures in cells are capable of self-assembly, what this means

The surfaces of two different proteins often are attracted to each other, and will assemble in an energetically favorable way. We will consider examples of this when we discuss certain proteins later in the course, such as cytoskeletal proteins.

(the info for forming the complex assembles of macromolecules in cells is contained in the subunits themselves as shown by purified subunits` ability to spontaneously assemble into the final structure under appropriate conditions/ when individual components (macromols) are mixed in a test tude they spontaneously reform the original structure

Cell culture

A cell culture refers to a population of cells that have been grown in the laboratory.

the purpose of cell culturing methods for biologists

Cell culturing methods enable cell biologists to obtain large numbers of cells for experimentation. In addition, the components that are added to a cell culture can be controlled, which is another experimental advantage. In this section, we will consider some of the issues associated with cell culturing methods, and how cells can be manipulated experimentally.

how/why cells can be isolated from tissues

(cells are physically disrupted to gain access to their components. cells are dissociated from tissues and separated according to cell type resulting in a homogenous population of cells which can be analyzed before or after the cells proliferate in a culture)

If the cells are derived from unicellular species, such as bacteria or yeast, such cells are typically obtained from colleagues or scientific stock centers at various places around the world. If the cells are from a multicellular species, such as animals and plants, the situation is a bit more complex. In some cases, a researcher may begin with a tissue sample from an organism that contains more than one type of cell. For example, a small tissue sample from the tail of a mouse would contain skin cells, blood vessel cells, etc. To start a cell culture, researchers often times want that culture to be composed of a single type of cell. Therefore, the different cells types within a tissue must be separated from each other. Figure 8-2 describes one method for separating individual cells, fluorescence activated cell sorting.

~(intact tissues provide the most realistic source of material for cells. you must isolate individual cells by disrupting extracellular matrix and cell-cell junctions which hold cells together)

** Fluorescence activated cell sorting - individual cells travel single file in a fine stream and pass through a laser beam. the fluorescence of each cell is rapidly measured. a vibrating nozzle generates tiny droplets containing one of no cells. the droplets with one cell are given a positive or negative charge at the moment of formation depending on whether the cell they contain is fluorescent. then they are deflected by a strong electric field into an appropriate container

how/why its useful that cells can be grown in culture

Many types of cells will grow on solid growth media. In the case of animal cells, the solid media is often coated with a protein, such as collagen, that enables the cells to adhere. In addition, liquid media of a defined composition is required to maintain cell growth. Such media contains amino acids, vitamins, salts, sugar, and various growth factors, among others. The composition of the growth media can be altered to study its effects on cell division, cell function, etc. For example, a hormone can be added to a liquid culture of cells, and then a researcher can study the effects of that hormone on cell structure and/or function. One advantage of solid growth media is that it enables researchers to visualize the cells as they are growing. We will consider methods of cell visualization in Chapter 9

~(cells grown in culture provide a more homogenous population of cells from which to extract material. cultured cells may be examined under microscopem and analyzed to see the effects of adding/removing specific molecules like hormones and growth factors.

in vivo

"in the living organism"

in vitro

"in glass"

~primary cultures

cell cultures prepared directly from intact tissues of an organism. Such primary cultures take on the characteristics of their origin. For example, cells derived from nerves will extend processes that can form chemical synapses with other neurons.

cell line

Primary cultures can be used to form a cell line, which is a population of cells that have been derived from a single cell via many, many cell divisions.

~secondary cultures

when cells are removed from primary cultures and recultured repeatedly

~ replicative cell senescence/senescent

that cells stop dividing after a certain # of dell divisions in culture

primary cultures vs cell cultures

primary cultures can be passaged over weeks and months, they cannot be passaged indefinitely; eventually they die. For example, fibroblasts can divide 25-40 times before dying due to progressive shortening of the cells' telomeres. In contrast, many cell cultures used by cell biologists are immortal, which means that they will continue to divide without becoming senescent. An immortal cell line has an experimental advantage because researchers can continue to propagate more cells as long as they want. Some immortal

cell lines were originally derived from tumors. An example is the HeLa cell line.

Alternatively, other immortal cell lines were derived from normal tissues, but later incurred mutations during growth in cell culture that made them immortal.

Some examples of cell lines are shown in Table 8-1. Please do not memorize this table. Cell lines are shared among researchers. When scientists use the same cell lines in their experiments, it is easier to directly compare their results.

heterokaryon, hybrid cells, hybridoma definition and its purpose

In certain types of experiments described later in this course, different cells are made to fuse with one another. This initially creates a heterokaryon, which is a combined cell with two nuclei.

Later, hybrid cells will be produced that have a single nucleus containing genetic material from both of the original cells. If one of the parent cells was from a tumor cell line, the hybrid cell is called a hybridoma. Hybridoma cell lines are used extensively to produce monoclonal antibodies which recognize specific proteins.

(~ derived from fusion of antibody-producing b lymphocytes with cells derived from a transformed beta lymphocyte cell line, producing a hybrid that can make a particular antibody and multiply indefinitely)

(the point of producing monoclonal antibodies for a protein is to localize the protein inc ells and tissues, track its movement, and purify it to study its structure and function)

fractionation

fractionation refers to methods that involve the breaking open of cells, and the subsequent separation and isolation of particular cellular components.

For example, cell fractionation could involve the isolation of mitochondria from cells.

first step of fractionation

The first step involves the breakage of cells, a phenomenon called cell lysis.

the various types of cell lysis (osmotic lysis, sonication, french press, cell wall digestion)

Homogenization: involves a glass tube and a pestle that spins rapidly. The cells are squeezed between the pestle and glass, and the shearing force that occurs causes them to lyse.

Osmotic lysis: only used to break open animal cells, which lack a cell wall. Cells are placed in a hypotonic media, which causes them to swell and eventually burst.

Sonication: exposure of cells to high-energy sound waves, which causes them to burst.

French press: cells are forced through a small orifice under extremely high pressure, causing them to lyse.

Cell wall digestion: for plant and yeast cells, the cell wall can be removed via enzymatic or mechanical treatments to create protoplasts. These can them be broken open via osmotic lysis, etc.

how cells are separated into their component fractions (cell extract/cell homogenate, then:

centrifugation to form a supernatant/pellet (in differential centrifugation)

OR

velocity centrifugation

After a cell is lysed, the components are released, thereby generating a cell extract. If the cells were lysed by homogenization, the cell extract may be called a cell homogenate. Individual components have their own characteristic densities. For example, mitochondria are a relatively heavy component. Depending on what component a researcher wishes to study, the cell extract can be centrifuged at various speeds, causing different components to be found in the supernatant or pellet. You should understand this approach, termed differential centrifugation, which is described in Figure 8-6.

Rather than separating supernatants and pellets, an alternative approach is to

3

centrifuge a cell extract until certain components have separated, but a pellet has not yet formed (Figure 8-7). The bottom of the tube can then be punctured, and portions of the fluid in the tube, called fractions, can be collected and analyzed. In velocity centrifugation, components would eventually form a pellet, but the centrifugation is done only a short period of time to avoid this from happening.

equilibrium centriguation

In equilibrium centrifugation, the tube contains a salt or sugar gradient, and components migrate until their densities match the density of the gradient. At this point, a component has reached equilibrium and it will remain where it is.

chromatography, what it is and how it works

Chromatography is the most popular method to purify proteins. The rationale is that each protein has a unique set of features that enable it to be separated from other proteins. As shown in Figure 8-8, a mixture of proteins is loaded on a column that contains beads. The proteins flow in between the beads and eventually are eluted from the bottom. Two different proteins may flow through the column at different rates, enabling them to be separated from each other.

But why do proteins flow through a column at different rates? The answer lies in their chemistry. The surfaces of the beads are designed to interact with proteins in some particular way (Figure 8-9). For example, in ion-exchange chromatography, the beads interact with charges on the surfaces of proteins (i.e., with charged amino acids). Gel- filtration affects the flow of proteins based on their size. In affinity chromatography, the beads have some molecule on their surface that the protein of interest binds tightly to. In all three cases, the properties of the beads influence the flow rate of proteins through the column, and thereby promote a separation of different types of proteins. In some cases, more than one column is needed to get a very pure preparation of a protein.

(Proteins are separated based on their chemical properties

a sample of solution containing a mixture of diff. proteins is loaded into a column containing beads/a permeable solid matrix

the surface of the beads interact with the proteins in a way that depends on the properties of both the beads and proteins

proteins may be separated by:

charge in (ion-exchange chrom)

hydrophobicity (hydrophobic chrom)

size (gel-filtration chrom)

ability to bind to certain small mols/other macromols (affinity chrom)

all these chrom types differ in their matrices

solvent is then passed slowly through column and collected in separate tubes. the components of the sample travel at diff rated thru column and are fractionated into diff tubes)

Immunoprecipitation

Immunoprecipitation is a rapid Affinity Purification Method

Antibodies that specifically bind to a protein of interest are conjugated to small agarose beads that can be used to precipitate the desired protein by centrifugation.

(Specific antibodies that recognize the protein to be purified are attached to small agarose beads

*doesnt use a column. Ab-coated beads are added to a protein extract in a test tube and mixed for a short amount of time allowing Abs to bind to proteins

* beads collected w/ low speed centrifugation

* it purifies small amounts of enzymes from cell extracts for analysis of the proteins enzymatic activity)

How Genetically-Engineered Tags provide an easy way to purify proteins

In some cases, researchers produce fusion proteins between the protein of interest and a protein or amino acid sequence that will bind tightly to an antibody. This is accomplished via cloning methods. Then, the fusion protein can be purified as above using antibodies on agarose beads.

(any gene can be modified to make its protein with a special recognition tag attach to it in order to make protein purification rapid. the recognition tag is an epitope which is recognized by an antibody. the Ab purifies the protein by affinity chrom or immunoprecip.)

cell-free systems

A cell-free system is a mixture of cytoplasmic and/or nuclear components purified from the cells that are needed to catalyze a certain biological process, for example in vitro protein synthesis or DNA replication. Cell-free systems are used to study molecular details of complex cell processes.

(it allows you to study biological processes free from all the complex side rxns that occur in a living cell. cell homogenates are fractionated to purify the macromols needed to catalyyze a biological process of interest. pure componses once isolated are added separately to define its role in the overall process. it determines the molecular details of complex cell processes.

read summary p451

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SDS Polyacrylamide Gel Electrophoresis

please study Figure 8-13,14. Keep in mind that SDS-PAGE denatures a protein, so it's not usually used to purify a protein in a functional state. Rather, it's used mostly to monitor protein purity and to identify a protein in a cell extract.

~(proteins migrate thru a highly crosslinked gel of polyacrylamide. proteins migrate at a rate depending on their net charge, shape, and size. proteins of interest dissolved in soln of negatively charged detergent - sodium dodecyl sulfate (SDS) which binds hydrophobic regions of proteins, unfolding them and rendered freely soluble. beta-mercaptoethanol added to break S-S linkages in proteins. when proteins are SDS solubilized and run thru polyacrylamide gel, the proteins bind detergent which makes its charge and migrates towards the pos. electrode when voltage is applied. proteins of same size move thru gel with similar speeds bc they bind same amounts of SDS and have same neg. charge. larger proteins bind more SDS and have more neg charge but also more drag force so they move slower. thus, a mixture of proteins is fractionated into protein bands arranged in order of mol weight.

Two-Dimensional Gel Electrophoresis

Two-Dimensional Gel Electrophoresis provides greater protein separation

This method is an extension of SDS page (Figures 8-16, 8-17). First, proteins are separated in a tube gel according to their isoelectric points. The tube gel has a pH gradient, and proteins migrate until their net charge is zero. The tube gel is then laid horizontally onto an SDS gel, which is the second dimension. The SDS gel separates proteins according to their masses. Because 2D-gel electrophoresis separates proteins based on two different properties, it is quite effective at resolving each protein within a mixture of many different proteins.

Western blotting/immunoblotting

The method of detecting proteins is called Western blotting or immunoblotting. A specific protein or proteins that share a particular feature can be identified on a gel by using a specific antibody that is coupled to a radioactive isotope, an enzyme, or a fluorescent dye. First, the proteins on a gel have to be transferred by "blotting" onto a nitrocellulose or nylon membrane. The membrane is placed over a gel and a strong electric field is applied, which transfers proteins onto the membrane. The membrane is then soaked in a solution with a labeled antibody to detect the protein/s of interest.

Mass Spectrometry

Mass Spectrometry provides a highly sensitive method for identifying unknown proteins

*mass spectrometry is used to determine short peptide sequences. If the genome of the organism has already been sequenced, the peptide sequence can be converted to the encoded DNA sequence, and the entire gene sequence can then be identified from within the genome sequence. For example, one can identify the different proteins resolved by 2D-gel electrophoresis via mass spectrometry. Each spot on the 2D gel, which technically represents one different protein, can be extracted from the gel, trypsinized into peptides, and the identity determined by MALDI- TOF mass spectrometry.

cell doctrine

The cell doctrine states that all living things are composed of cells. Much of the early work supporting this doctrine came from microscopy studies.