2023_L11.pdf

1 Lecture 11 Lipids, Membranes and Cellular Transport 2 The Molecular Structure and Behavior of Lipids Lipid molecules tend to be insoluble in water, but they can associate to form water-soluble structures such as: o Micelles...

1 Lecture 11 Lipids, Membranes and Cellular Transport 2 The Molecular Structure and Behavior of Lipids Lipid molecules tend to be insoluble in water, but they can associate to form water-soluble structures such as: o Micelles o Vesicles o Bilayers 3 The Molecular Structure and Behavior of Lipids The simplest lipids are the fatty acids. Their basic structure is a hydrophilic carboxylate group attached to one end of a hydrocarbon chain, which contains typically 12 to 24 carbons. An example is stearic acid, a C-18 fatty acid. Stearic acid is an example of a saturated fatty acid, one in which the carbons of the tail are all saturated with hydrogen atoms Many important naturally occurring fatty acids are unsaturated, that is, they contain one or more double bonds. An example is oleic acid, a C-18 D9 fatty acid. 4 The Molecular Structure and Behavior of Lipids Structures of the ionized forms of some representative fatty acids: 5 The Molecular Structure and Behavior of Lipids Membrane lipids are amphipathic. They tend to form surface monolayers, bilayers, micelles, or vesicles when in contact with water. Most naturally occurring fatty acids contain an even number of carbon atoms. If double bonds are present (unsaturation), they are usually cis.  The Molecular Structure and Behavior of Lipids 6  The Molecular Structure and Behavior of Lipids 7 Fats, or triacylglycerols, are triesters of fatty acids and glycerol. They are the major long-term energy storage molecules in many organisms. 8 The Molecular Structure and Behavior of Lipids Fat storage in animals serves three distinct functions: 1. Energy production - most fat in most animals is oxidized for the generation of ATP, to drive metabolic processes. 2. Heat production - specialized cells (in “brown fat” of warm- blooded animals, for example) oxidize triacylglycerols for heat production, rather than to make ATP. 3. Insulation - in animals that live in a cold environment, layers of fat cells under the skin serve as thermal insulation. The blubber of whales is one obvious example. 9 The Molecular Structure and Behavior of Lipids 10 The Molecular Structure and Behavior of Lipids Adipocytes (animal fat storage cells)make up a large part of adipose tissue. The designations MFC and VSFC correspond to “mature fat cell” and “very small fat cell,” respectively. 11 The Molecular Structure and Behavior of Lipids When fats are hydrolyzed with strong bases such as NaOH or KOH (in earlier times, wood ashes were used), a soap is produced. This process is called saponification. The fatty acids are released as either sodium or potassium salts, which are fully ionized. However, as cleansers, soaps have the disadvantage that the fatty acids are precipitated by the calcium or magnesium ions present in “hard” water, forming a scum and destroying the emulsifying action. 12 The Molecular Structure and Behavior of Lipids Synthetic detergents have been devised that do not have this defect. One class is exemplified by sodium dodecyl sulfate (SDS): NaO3SO(CH2)11CH3 The salts of dodecyl sulfate with divalent cations (i.e., Ca2+ and Mg2+) are more soluble. Recall that SDS is widely used in forming micelles about proteins for gel electrophoresis. There are also synthetic nonionic detergents, like Triton X-100:  The Molecular Structure and Behavior of Lipids 13 Structure of a typical wax: Waxes are formed by esterification of fatty acids and long-chain alcohols. The small head group can contribute little hydrophilicity, in contrast to the significant hydrophobic contribution of the two long tails. Thus, the waxes are completely water insoluble. Often serve as water repellents, as in the feathers of some birds and the leaves of some plants. In some marine microorganisms, waxes are used instead of other lipids for energy storage. In beeswax, they serve a structural function. As with the triacylglycerols, the firmness of waxes increases with chain length and degree of hydrocarbon saturation.  The Structure and Properties of 14 Membranes and Membrane Proteins There are four major classes of membrane-forming lipids: o Glycerophospholipids o Sphingolipids, o Glycosphingolipids, o Glycoglycerolipids Their structure is that of a large head group attached to a double tail yielding a roughly cylindrical molecule. Such cylindrical molecules can easily pack in parallel to form extended sheets of bilayer membranes with the hydrophilic head groups facing outward into the aqueous regions on either side. They differ principally in the nature of the head group. The bilayer is roughly 6 nm thick, with ~1.5 nm of interface on either side of the ~3 nm hydrophobic core.  The Structure and Properties of 15 Membranes and Membrane Proteins Phospholipids and membrane structure: a)Fatty acids are wedge-shaped and tend to form spherical micelles. b)Phospholipids are more cylindrical and pack together to form a bilayer structure. c)A computer simulation of a phospholipid bilayer.  The Structure and Properties of 16 Membranes and Membrane Proteins Stereochemistry of glycerophospholipids: a)Glycerol is a prochiral molecule. Phosphorylation of one CH2OH group or the other gives the R- or S- enantiomer of glycerol phosphate. a)The same molecule can be called L- glycerol-3-phosphate or D-glycerol-1- phosphate depending on the carbon numbering scheme.  The Structure and Properties of 17 Membranes and Membrane Proteins c) The sn (stereochemical numbering) system assigns the pro-S carbon as C1. All glycerophospholipids are derivatives of sn-glycerol-3- phosphate.  The Structure and Properties of 18 Membranes and Membrane Proteins Glycerophospholipid structure: a)Stereochemical view of a generalized glycerophospholipid. a)The same structure represented in the convention used in this text, with hydrophobic groups to the right, hydrophilic to the left. R3 is a hydrophilic group  The Structure and Properties of 19 Membranes and Membrane Proteins  The Structure and Properties of 20 Membranes and Membrane Proteins  The Structure and Properties of 21 Membranes and Membrane Proteins A second major class of membrane constituents is built on the amino alcohol sphingosine, rather than on glycerol. The structure of sphingosine includes a long-chain hydrophobic tail, so it requires the addition of only one fatty acid to make it suitable as a membrane lipid. If a fatty acid is linked via an amide bond to the NH2 group, the class of sphingolipids referred to as ceramides is obtained:  The Structure and Properties of 22 Membranes and Membrane Proteins An especially important example is sphingomyelin (part of myelin sheath that surrounds some nerve cellaxons), in which a phosphocholine group is attached to the C-3 hydroxyl.  The Structure and Properties of 23 Membranes and Membrane Proteins  The Structure and Properties of 24 Membranes and Membrane Proteins Examples of glycosphingolipids: a)A cerebroside - an important constituent of brain cell membranes. A ganglioside - This particular ganglioside, called GM2 or the Tay-Sachs ganglioside, accumulates in neural tissue of infants with Tay-Sachs disease. The defect responsible for enzyme that normally cleaves the terminal N-Acetylgalactosamine.  The Structure and Properties of 25 Membranes and Membrane Proteins Another class of lipids, less common in animal membranes but widespread in plant and bacterial membranes, are the glycoglycerolipids, exemplified by monogalactosyl diglyceride: Copyright © 2013 Pearson Canada Inc. 10 - 25  The Structure and Properties of 26 Membranes and Membrane Proteins Cholesterol, is a component of many animal membranes. It influences membrane fluidity by its bulky structure.  The Structure and Properties of 27 Membranes and Membrane Proteins Much of our current understanding concerning biological membranes is based upon the fluid mosaic model proposed by S. J. Singer and G. L. Nicolson in 1972. A membrane is a fluid mixture of lipids and proteins. Peripheral membrane proteins are associated with one side of the bilayer and can be separated from the membrane without disrupting the bilayer. Integral membrane proteins are more deeply embedded in the bilayer and can only be extracted under conditions that disrupt membrane structure. Many integral membrane proteins extend through the bilayer.  The Structure and Properties of 28 Membranes and Membrane Proteins Structure of a typical cell membrane: Proteins are embedded in and on the phospholipid bilayer; Some of them are glycoproteins, carrying oligosaccharide chains. The membrane is about 6 nm thick. Most membranes are more densely packed with proteins than is shown here.  The Structure and Properties of 29 Membranes and Membrane Proteins Experimental demonstration of membrane fluidity: When cells with surface membrane protein marked by fluorescent tags are induced to fuse, the proteins gradually mix over the fused surface. 30 The Structure and Properties of Membranes and Membrane Proteins  The Structure and Properties of 31 Membranes and Membrane Proteins The gel–liquid crystalline phase transition in a synthetic lipid bilayer: a)A schematic view of the change at the transition temperature. Below this temperature the hydrocarbon tails are packed together in a nearly crystalline gel state (left). Above this temperature the movement of the chains becomes more dynamic, and the interior of the membrane resembles a liquid hydrocarbon (right). a)Detection of the transition by calorimetry. Measurement of the heat absorbed by a membrane as the temperature is raised each degree shows a sharp spike at the transition temperature (Tm) for a pure dipalmitoylphosphatidyl choline bilayer. This well-defined transition from gel to liquid is called melting of the membrane. When 20 mol % cholesterol is mixed into the bilayer, the Tm is not changed, but the transition is broadened.  The Structure and Properties of 32 Membranes and Membrane Proteins  The Structure and Properties of 33 Membranes and Membrane Proteins The transition temperature (Tm) for a membrane depends on its lipid composition. Lipids with longer, saturated tails tend to increase the Tm. Those with more cis double bonds and/or shorter tails will reduce the Tm. Under physiological conditions, biological membranes exist in a semi-fluid liquid crystalline state.  The Structure and Properties of 34 Membranes and Membrane Proteins The two leaflets of a membrane usually differ in lipid composition. Lipid composition in the outer leaflet (green) and inner leaflet (gold) of the plasma membrane is graphed for three cell types. o PC phosphatidylcholine o PE phosphatidylethanolamine o PS phosphatidylserine o PI = phosphatidylinositol o SP = sphingomyelin  The Structure and Properties of 35 Membranes and Membrane Proteins Examples of structures for several integral membrane proteins:  The Structure and Properties of 36 Membranes and Membrane Proteins Bacteriorhodopsin - an integral membrane protein: Functions as a lightdriven proton pump in certain bacteria. Seven helices span the membrane and hold a molecule of the lightabsorbing pigment retinal (convert light into metabolic energy).  The Structure and Properties of 37 Membranes and Membrane Proteins The sequence and postulated structure of glycophorin A (MNS blood group protein in human erythrocyte): This protein was the first integral membrane protein to be sequenced. The external (N-terminal) domain carries 15 O-linked and one N-linked oligosaccharides; together these constitute ~60% of the total protein mass. o The single transmembrane helix is highly hydrophobic. o The cytosolic C-terminal domain is quite hydrophilic.  The Structure and Properties of 38 Membranes and Membrane Proteins Co-translational insertion and folding of transmembrane helices in an integral membrane protein: Some parts of the protein (in this case, the loops) are conducted across the bilayer through the translocon; transmembrane portions exit the translocon and remain embedded in the bilayer. The transmembrane helices fold after they are inserted.  The Structure and Properties of 39 Membranes and Membrane Proteins A protein channel called the “translocon” facilitates the insertion of integral membrane proteins into the membrane bilayer. Model for translocon function: a) The “resting” translocon. b) The “active” translocon. c) If a segment of the nascent peptide is sufficiently hydrophobic it will partition into the lipid bilayer. Hydrophilic sequences (loops) partition to either side of the bilayer, depending on the orientation of the transmembrane segments.  The Structure and Properties of 40 Membranes and Membrane Proteins The “inside positive” rule: Wild-type leader peptidase (Lep) from E. coli orients its two transmembrane helices with the termini in the periplasm and the loop between the helices in the cytoplasm. Addition of four Lys to the N-terminus and removal of positive charge from the loop yield a mutant Lep that has the opposite membrane topology. The membrane potential is indicated by greater negative charge on the cyotplasmic side compared to the periplasmic side.  The Structure and Properties of 41 Membranes and Membrane Proteins Membrane rafts are rich in cholesterol, sphingolipids, and GPI-linked proteins. The bilayer is thicker in rafts than in the surrounding membrane. The proteins coalesce and form nanometer-sized dynamic raft domains, which may be stabilized by interactions with actin fibers. Rafts can associate to form larger structures (“platforms”). Certain proteins interact preferentially with rafts (orange shading), while others do not (brown shading) or are excluded. 42 Transport Across Membranes Adaptation to hydrophobic mismatch in a membrane: If the thickness of the bilayer core and the hydrophobic surface area of an embedded protein do not match, either the protein will undergo conformational change or the bilayer will change composition until the dimensions of these hydrophobic regions match. Copyright © 2013 Pearson Canada Inc. 10 - 42 43 Transport Across Membranes 44 Transport Across Membranes For a substance that can pass through a membrane, the normal state of equilibrium is achieved when the concentrations of the substance are equal on both sides of the membrane. Equalization of the concentrations of some substance across a membrane can be circumvented by: (1) binding of the substance to macromolecules (2) maintaining a membrane potential (if the substance is ionic) (3) coupling transport to an exergonic process The rate of nonmediated transport, as measured by membrane permeability, is proportional to the diffusion and partition coefficients and inversely proportional to membrane thickness. Facilitated transport, via pores, permeases, or carriers, can increase the rate of diffusion across a membrane by many orders of magnitude. 45 Transport Across Membranes The three major mechanisms for facilitated transport: 46 Transport Across Membranes Valinomycin is an antibiotic that acts as an ion carrier. The outside of this roughly spherical cyclic polypeptide is hydrophobic. The central cavity surrounded by oxygens complexes a K+ ion. The surface is covered with CH3 groups (not shown). 47 Transport Across Membranes The channel-forming hemolysin from S. aureus: Ribbon drawings of the a-hemolysin heptamer, viewed (a) looking down the sevenfold axis (b) perpendicular to the sevenfold axis (c) one protomer extracted from the heptamer structure The heptamer is 10 nm in diameter and 10 nm in length, as measured along the sevenfold axis. The b-barrel stem, which penetrates the membrane, is about 6 nm long. 48 Transport Across Membranes Gramicidin A is an antibiotic that acts as an ion pore. Two molecules of gramicidin A form a pore through the membrane by adopting a helical conformation, with their hydrophobic side chains in contact with the lipid. Note the N-termini are inside and the C-termini are outside the bilayer core. The inside of the helix forms the hydrophilic pore. The hydrogen bonding in this open helical structure resembles that in b-sheet polypeptides. This is possible because of the alternating D and L residues.  Transport Across Membranes 49 Many types of eukaryotic cells must move large amounts of water rapidly across their membranes as part of their physiological function. o Erythrocytes (which experience a wide range of solution osmolarity as they transit through lungs, capillaries, and kidneys) o Secretory cells in salivary glands o Epithelial cells in the kidney Although water can cross membranes, it does so relatively slowly; thus, the inherent permeability of membranes toward water is not sufficient to support the rapid transport observed in many cell types. Such rapid transport is achieved by water-specific channels called aquaporins. The aquaporins function as tetramers of identical monomers. Each monomer contains six membrane-spanning helices and two shorter helices that contain a conserved N-terminal Asn-Pro-Ala (NPA) motif.  Transport Across Membranes 50 The aquaporin water channel: a)Cartoon rendering of human aquaporin-5 tetramer looking along the four water channels. The two short helices containing the NPA sequence are shown in blue. b)A cutaway view of the water channel in one of the monomers. The narrowest part of the channel is where the two short helices meet. Note the location of Asn76 and Asn192 at this restriction. The two helical macrodipoles and Arg195 provide an electrostatic barrier to H3O+ passage. c)Schematic view of the aquaporin channel, showing the electrostatic repulsion of H3O+ and the reorientation of the water molecules as they pass through the central restriction.  Transport Across Membranes 51 Ion selectivity is achieved by optimal geometry of chelating groups in ion channels. The structure of the potassium channel pore: Copyright © 2013 Pearson Canada Inc. 10 - 51 52 Transport Across Membranes Selective binding of Na+ and K+ in ion channels: a)Two binding sites make up the selectivity filter in the transmembrane region of LeuT. a)Four are bound in the filter of the KcsA channel.  Transport Across Membranes 53 A model for voltage-gating in the channel: The channel portion of the voltage-gated channel is structurally homologous to the KcsA channel. The Arg- and Lys-rich S4 helices are highlighted in blue. The depth of these helices in the bilayer changes as a function of the membrane potential. 54 Transport Across Membranes The rate of facilitated diffusion approaches a maximum value when all available transporters are saturated with substrate, whereas nonmediated diffusion shows a linear increase in rate as substrate concentration Increases. 55 Transport Across Membranes In active transport, substances are moved across a membrane against a concentration gradient. Direct or indirect coupling of transport to ATP hydrolysis provides the required free energy. The Na+-K+ pump acts in all cells to maintain higher concentrations of K+ inside and Na+ outside. 56 Transport Across Membranes The structure of the Na+-K+-ATPase with K+ bound: 57 Transport Across Membranes A schematic diagram of the functional cycle of the Na+–K+ pump: The subunit is believed to have two states: o one open only to the outside (brown o one open only to the inside (blue) A dot between two symbols indicates noncovalent binding, a line indicates covalent attachment (as in phosphorylation). Cardiotonic steroids (e.g., digoxin) inhibit the Na+–K+ pump, resulting in increased Ca2+ ion concentration in heart muscle, which, in turn, leads to stronger contractions of the heart muscle. 58 Transport Across Membranes In cotransport, the unfavorable movement of a substance through the membrane is coupled to the favorable transport of another substance. The sodium–glucose cotransport channel is also presumed to have two possible states o one open only to the outside o one open only to the inside of the cell Transition to the inside open state is stimulated by glucose binding to E.Na+ Return to the outside-open state occurs upon Na+ release to the inside of the cell. The sodium gradient from inside to outside provides the driving force for the unfavorable transport of glucose. 59 Transport Across Membranes 60 Transport Across Membranes In transport by modification, a substance that has diffused through a membrane is modified so that it cannot return. This method is used by many bacteria for the uptake of sugars. The sugars are phosphorylated, either during their diffusion through the membrane or as soon as they emerge into the cytosol.  Transport Across Membranes 61 Specific transport processes: This composite plant–animal cell illustrates some of the most important specific transport processes. All of the substances shown here, and many more, are transported in specific directions across cellular membranes. 62 Transport Across Membranes Bulk transport across membranes involves the formation of clathrin-coated vesicles and/or caveolae. (a)Formation of a clathrin-coated vesicle initiates with formation of a coated pit (top two panels), which then buds (third panel from top) forming a free coated vesicle (bottom panel). (a)Caveola formation occurs at sites rich in cholesterol and sphingolipids due to insertion of caveolin into one leaflet of the membrane. Budding of the caveola yields a free vesicle.  Excitable Membranes, Action Potentials, 63 and Neurotransmission Neurons conduct electrical impulses by membrane potential changes in regions of the plasma membrane cell. The cell body contains the nucleus and most of the cellular machinery. The dendrites receive signals from the axons of other neurons The axon transmits signals via the synaptic termini, which communicate to the dendrites of other neurons or to muscle cells. Along the axon are Schwann cells, which envelop the axon in layers of an insulating myelin membrane. The Schwann cells are separated by nonmyelinated regions called the nodes of Ranvier.  Excitable Membranes, Action Potentials, 64 and Neurotransmission Use of squid giant axons for studies of neural transmission: Electrodes attached to a voltmeter record the potential across the axonal membrane. At the resting axon ion concentrations shown here, the voltmeter would read about 60 mV. If the axon is stimulated at point A by a depolarizing pulse, the traveling membrane potential will shortly pass point B, where it can be recorded.  Excitable Membranes, Action Potentials, 65 and Neurotransmission The resting potential of a nerve fiber is determined by the permeabilities of the membrane to different ions, particularly K+, which has a high permeability due to K+ leak channels. The action potential is generated and propagated because a small depolarization of the nerve cell membrane opens voltage gated channels, allowing ions to flow through.  Excitable Membranes, Action Potentials, 66 and Neurotransmission The voltage-gated channel cycles during an action potential:  Excitable Membranes, Action Potentials, 67 and Neurotransmission The action potential: a)Changes in membrane conductance at a point on an axon as a neural impulse passes. The membrane first becomes permeable to sodium ions, allowing a large inward rush of Na+. A decrease in the Na+ permeability results and is in turn followed by an outward flow of K+ b)Changes in membrane potential accompanying the permeability changes shown in (a). As Na+ rushes in, the potential increases and becomes positive. As K+ influx increases, the potential decreases, undershooting the resting potential, before returning to the resting potential.  Excitable Membranes, Action Potentials, 68 and Neurotransmission Transmission of the action potential:  Excitable Membranes, Action Potentials, 69 and Neurotransmission Neurotoxins can act by blocking gates in the axonal membrane in closed or open states. Tetrodotoxin is found in some organs of the puffer fish. TTX binds specifically to the Na+ channel, blocking all ion movement. Saxitoxin is contained in the marine dinoflagellates responsible for “red tide” also block Na+ channels. Veratridine is found in the seeds of a plant of the lily family, Schoenocaulon officinale. This toxin also binds to the Na+ channels but blocks them in the “open” configuration. These toxins have proven to be useful in studies of axonal structure and conduction, for their tight binding makes them excellent affinity labels for the channel. 70 Techniques for the Study of Membranes Examination of membrane structure as it exists within cells depends heavily on electron microscopy (EM). An especially useful variant of this method is the freeze fracture technique. If a membrane is frozen quickly and then broken by a sharp blow from a microtome knife, it frequently splits along the plane between the bilayer leaflets Membranes from individual types of cells or purified organelles can usually be obtained by lysis of the cell or organelle, followed by differential centrifugation.