Structure Membrane and Transport Proteins
OBJECTIVES Outline the structural features of membranes that are generally shared by all membranes: a. lipid bilayer b. fluid structure c. asymmetry d. transmembrane proteins e. receptors f. channels
To know the basic structures of the phospholipids that are found in membranes and the importance of their amphipathic properties. To understand the fluid-mosaic concept of membranes. How are transporters similar to enzymes? How are they different? Describe the differences between, and give examples of Active (both primary and secondary) and passive transport Uniporters, Symporters and Antiporters Be able to describe the structure and function of: -the GluT1 transporter -the Na+K+ ATPase -the Na+ Glucose transporter
OUTLINE I. Membrane functions A. Define the external boundary of cells B. Regulate transport in and out C. Divide internal space of cells into discrete compartments to segregate processes (example: fatty acid oxidation and synthesis segregated by mitochondrial membrane) D. Contain components responsible for energy generation (oxidative phosphorylation) E. Intra- and inter-cell communication (receptors, second messengers) F. Site of cellular component assembly (Ex: on ER)
II. Membrane structure Necessary attributes: All these above functions require certain properties: tough but flexible;
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selectively permeable; asymmetric; self-sealing A. Membranes are lipid bilayers... 1. lipids =~50% of mass of most animal cell membranes 2. Made of amphipathic lipids Forces responsible for the lipid aggregation are hydrophobic and van der waals interactions; and also some electrostatic ints, and H-bonds between the polar heads, and with water in surrounding solution. The Point: NO covalent bonds responsible for stability of lipid bilayer aggregate 3. Consequences of this lipid bilayer; It forms a permeability barrier; most ions and polar molecules are unable to broach that hydrophobic barrier due to energetic considerations Water is an exception; it is a very polar molecule, but small enough to cross the bilayer 4. Lipid composition in lipid bilayers vary with different types of cells
B. Not simply lipid bilayers... have proteins associated with them -Membrane proteins are responsible for most membrane functions; receptors, enzymes, transport proteins C. Fluid Mosaic model Membranes are solutions of oriented lipids--as lipid bilayers, with polar heads out, nonpolar tails in; and proteins are embedded within or associated upon this bilayer in a mosaic fashion Membrane is fluid; lipids within the bilayer, and proteins, can move laterally within the plane of the bilayer, but do not (usually) rotate from one side of the bilayer to the other; that is, no "flip-flop";
D. Membranes are asymmetric So, the structure of the membrane maintains the direction of the lipids and proteins within it; the sidedness of the membrane is maintained
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Many of the functions of the membrane proteins are dependent upon this asymmetry; energy generation, transport, recognition and signal effectors
E. Membranes are fluid Membranes must be fluid to work properly; if membranes solidify, a decrease in permeability may constrain protein conformational changes necessary for functions, etc. Membranes are not rigid static structures. What factors are important in the fluidity (melting point) of a membrane? a. length of Fatty acids (Fas) in the membrane lipids; longer = higher melting temps (longer chains interact more, more vdWaals ints to disrupt) b. Degree of saturation in the membrane lipid Fas; Membranes with more saturated Fas will have higher melting temps; sat. FAs fit together tighter, more vdWaals; unsaturated FAs have kinks due to cis carbon-carbon bonds, fewer vdWaals, and therefore lower melting temps. c. Percentage of steroid molecules (cholesterol in many eukaryotes, but not all; sitosterol in plants, ergosterol in fungi); Basic structure of sterols: -stiff, bulky steroid nucleus -more flexible hydrocarbon tail -polar hydroxyl group "head"
Overall role of cholesterol is to moderate extremes of both solid and fluid states; stabilizes membrane fluidity at varying environmental temps
Final point: Fluidity depends on the temperature surrounding the membrane Certain cell types have a different membrane lipid composition due to their position in an organism; (example; the membrane lipid Fas in the cells in reindeer feet are more unsaturated and shorter, in order to stay fluid in cold temperatures) Both bacteria and laboratory-cultured cells can internally regulate the lipid composition of their membranes under varying growth temps and so regulate, and stabilize the fluidity of their own cell membranes
F. Classifications of proteins based on their physical POSITION in bilayer as opposed to their function or chemistry
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Membrane proteins can be associated with lipid bilayer in various ways; 1. Peripheral proteins -do not extend into lipid bilayer, are bound to either face of the membrane by noncovalent interactions only--usually to the integral membrane lipids
2. Integral ; either a. transmembrane proteins; extend through the lipid bilayer, have hydrophobic and hydrophilic portions b. others that can be considered integral are attached to the lipid bilayer by covalent bonds of some type: such as addition of a fatty acid chain covalently attached by insertion into cytoplasmic side of bilayer In keeping with the asymmetry of membranes, transmembrane proteins show "sidedness" These integral membrane proteins also show themes in secondary structure; either alphahelix, or sometimes beta-barrels (beta-sheet in a closed barrel). Both these secondary structures maximize H-bonding between the participants in the (polar) peptide bonds of the protein; there is no water in the inside of the bilayer sandwich
III Overview of membrane transport Membranes act as permeability barriers, but the plain truth is that cells live and grow by exchanging molecules with their environment, so nutrients such as sugars and amino acids must be able to get across somehow...waste products must be removed (urea, CO2) , and intracellular concentrations of ions have to be continually readjusted. Ion concentrations outside a cell are very different from those inside; maintenance of these differences is crucial for cells survival and function. Inorganic ions plentiful in cell environment that we need to consider: Na+, K+, Ca++, Cl-, H+
A. Transport across membranes without transporters Simple diffusion across bilayer: allowed = lipid-soluble molecules and -Small nonpolar molecules like O2, CO2, N2 can rapidly diffuse across bilayer, -Small uncharged polar molecules can if small enough: water, urea
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Water is also allowed across membrane through special channel proteins called Aquaporins--these let water through much faster
B. Transport across membranes with transporters For larger uncharged polar molecules (such as glucose), and all ions, transfer depends upon transport proteins that span the bilayer and specifically provide passage. -carrier proteins are highly selective, -many found in each membrane; different dependent on type of cell. -all membrane transport proteins so far studied have been found to be multipass transmembrane proteins
IV. Transporters in detail A. Overview -have moving parts (ie., show conformational changes) to shift specific molecules across the membrane -can be coupled to a source of energy to catalyze active transport or not (but there are both active and passive carrier proteins) Process is reminiscent of enzyme-substrate reactions: -Binding is stereospecific and through multiple weak non-covalent interactions -carrier protein has a binding site (analogous to active site on an enzyme) -when binding sites are full, carrier is saturated, and rate of transport is maximal = Vmax; specific to that carrier protein -also, a Kt, = equal to concentration of transported molecule when the transport rate = 1/2 Vmax -binding of transported molecule can be blocked by competitive inhibitors, (compete for same binding site, may or may not be actually transported) or by mixed inhibitors, which bind elsewhere besides binding site, but alter the behavior of the binding site. -NOT like enzymes in that transported molecule is not altered as in substrate to product Look at the kinetics; -"Substrate " = glucose out -"Product" = glucose in -Vmax -Kt = concentration necessary to get to Vmax/2 (lower is better)
-note that the carrier mediated diffusion process is saturatable, while simple diffusion is not -mediated diffusion is faster than simple diffusion -it is very specific
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Structurally, transporters span the lipid bilayer at least once, and usually several times, forming a transmembrane channel lined with hydrophilic amino acids
Can describe transporters in several ways; first by direction B. Uniport, symport, antiport -uniporters transfer single molecule, one way Other, coupled transport processes cannot be described so easily: -transfer of one solute depends on simultaneous or sequential transport of another solute; -either in same direction: = symport -or opposite direction: = antiport
Transport can also be broken down according to whether or not the process requires energy. C. Passive transport; (Facilitated diffusion) -Movement of molecules "downhill" from a region of high concentration to a region of low concentration, occurs spontaneously as long as a path exists -Requires no expenditure of energy by cell (Electrical forces as well as concentration gradients can drive this type of transport:)
examples Ex: glucose carrier found in plasma membrane of erythrocytes GluT1 (several different types) ; -consists of protein that spans bilayer several times (12) times; forms a hydrophobic cylinder with a hydrophilic channel through which the glucose is transported. -thought to be able to adopt at least two conformations and switches reversibly and randomly between them -In one conformation, the carrier exposes binding sites for glucose to the exterior of cells; in the other it exposes this site to the interior of the cell. The flow of glucose is regulated by concentration gradient across the membrane; -Luckily, glucose is almost immediately used (converted into G-6-P) so that the [glucose] is always lower inside than out, maintains conc. gradient for glucose Transport proteins of this type do NOT determine the direction, just permit the flux of solute based on gradient of concentration Though passive, highly selective. The hallmarks of passive transporters: -high rates of transfer down concentration gradient
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-Saturatable -specific -Fully reversible (according to [gradient])
D. Active transport To move a solute against its concentration gradient, to drive the "uphill" flow , transport has to be coupled to some other process carried out only by certain types of carrier proteins that can harness some energy source -either ATP hydrolysis (primary) or an ion gradient (secondary)
1. Primary; -Directly use energy from hydrolysis of ATP examples -Na+K+ ATPase -an antiporter; pumping Na+ out of cell against its concentration gradient, pumping K+ in against its gradient mechanism 1. Binding of Na+ on cytosolic (inside of cell) side 2. phosphorylation of aspartic acid residue on cytosolic side coupled to ATP hydrolysis 3. 1, 2 cause conformational change that transfers the Na+ across membrane, releases it 4. K+ binds from outside 5. dephosphorylation 6. causes return to initial conformation, releases K+ Really two K+ binding sites, 3 Na+ binding sites; for every molecule of ATP hydrolyzed, 3 Na+ pumped out, 2 K+ pumped in Probably more complex conformational changes actually occur, but this is a working model. Function of this? maintenance of concentration differences across cell membrane; ([K+] is typically 10-20X higher inside cells than out; [Na+] reversed) Ouabain, a poison used in Africa to increase the efficacy of hunting arrows, blocks this transporter and causes death.
2. Secondary active transport -active driven transport by ion gradients; free energy from movement of an inorganic ion down its electrochemical gradient can pump other solutes uphill against their gradient.
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The gradient that drives this has to have been established by primary active transport -these all function as coupled transporter; either symporters or antiporters -in animal cells, Na+ is the usual co-transported ion whose electrochemical gradient provides the energy for the active transport of the other molecule -since Na+ gradient maintained by Na+K+ ATPase, this primary active transport indirectly drives the secondary active transport examples -Intestinal epithelial cells -contain a variety of symport systems that use the Na+ gradient to import sugars, amino acids (each transport protein specific, of course) -Solute and Na+ bind to different sites on the carrier protein -symports: Na+ moves into the cell down its concentration gradient, DRAGS the specific other molecule into cell with it AGAINST its [gradient] Antiports can work this way too; two molecules moving in opposite direction, but one must be moving down its gradient while other against) Na+ / Glucose transporter moves Na+ down its concentration gradient at the same time that it moves glucose into cells against the glucose concentration gradient. In this case both solutes are moving in the same direction and is a SYMPORTER. In this instance an energetically unfavorable reaction is driven by an energetically favorable one.
IV. Channel proteins A.Porins -relatively large, permissive channels found in outer membranes of bacteria, mitos and chloroplasts B. Ion channels -responsible for electrical excitability of muscle cells; nervous cells -form narrow hydrophilic pore, allowing the passive movement of small inorganic ions -always passive transport -very selective; permit some ions to pass, but not others; implies that the pores are narrow enough to force contact between inside of pore and the ion being allowed through--must be correct size and charge.
Differences from transporters? -Rate of flux through ion channels is much higher than for transporters; much more efficient than carrier proteins at the transport: rates ~1000X greater than with carrier proteins
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-NOT saturatable -canNOT be connected to energy source --always passive transport here -this doesn't mean the ion channels are not regulated, though...ion channels are NOT continuously open. They have "gates" which open and close in response to a specific stimulus: by some cellular event which forces an allosteric transition on the channel , open closed
Summary
Problems 1. Ruptured biological membranes are self sealing due to all of the following except: A. Covalent interactions between lipids B. Hydrophobic interactions between lipid tails C. An increase in entropy of the system upon sealing D. Hydrogen bonds between the head groups of the lipids and H2O E. All of the above are important in membrane self-sealing 2. Which of the following is a function only of the lipids, and not the proteins in a membrane? Explain. A. To act as an entry point for facilitated diffusion for specific sugars B. To determine the fluidity of the membrane C. To determine the asymmetry of the membrane D. To act as pumps and/or gates for ions E. None of the above 3. Proteins 1, 2, and 3 are somehow associated with the intact biological membranes of a culture of cells. After the cells containing these proteins are exposed to a severe drop in pH, only protein 1 remains associated with the membrane. You can conclude which of the following? Why? A. 2 and 3 are peripheral B. 2 and 3 are integral C. 1 may be peripheral with a covalent attachment or may be integral D. A and C are correct
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4.Two different species of bacteria have been isolated: one from a hot spring with an average water temp. of 400 C, the other from a glacial lake with an average water temp. of 40C. a) Which of the two bacterial species would be expected to have more unsaturated fatty acids in its membrane lipids? b) Which would have longer chain fatty acids? 5. Compare and contrast symport and antiport. Describe the transport system mediated by the Na+K+ ATPase; is it a symport or antiport system? 6. Compare and contrast passive and active transport. Describe the transport system mediated by the Na+K+ ATPase; is it an active or passive system? 7. Compare and contrast primary and secondary active transport. Describe the transport system mediated by the Na+K+ ATPase; is it a primary or secondary active system? 8. The GluT1 transporter of erythrocytes is a classic example of a passive transport system. Passive transporters are often compared to enzymes; how is this transport system similar to, and different from, enzymes? Answers 1. Ruptured biological membranes are self sealing due to all of the following except: A. Covalent interactions between lipids B. Hydrophobic interactions between lipid tails C. An increase in entropy of the system upon sealing D. Hydrogen bonds between the head groups of the lipids and H2O E. All of the above are important in membrane self-sealing A is the correct answer. There are NO covalent bonds between membrane lipid molecules in a membrane bilayer 2. Which of the following is a function only of the lipids, and not the proteins in a membrane? Explain. A. To act as an entry point for facilitated diffusion for specific sugars B. To determine the fluidity of the membrane C. To determine the asymmetry of the membrane D. To act as pumps and/or gates for ions E. None of the above B Proteins do not influence the fluidity of a membrane. A useful analogy is as follows: imagine sticks and rocks scattered in a stream. The fluidity of the water flowing around these obstructions (analogous to proteins in the bilayer) is NOT affected by the obstructions. The fluidity of the membrane is determined by the temperature of the environment, and by the type of lipid molecules (saturated, unsaturated, long or short tails, amount of sterols) in the bilayer.
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3. Proteins 1, 2, and 3 are somehow associated with the intact biological membranes of a culture of cells. After the cells containing these proteins are exposed to a severe drop in pH, only protein 1 remains associated with the membrane. You can conclude which of the following? Why? A. 2 and 3 are peripheral B. 2 and 3 are integral C. 1 may be peripheral with a covalent attachment or may be integral D. A and C are correct D A change in pH would not destroy any covalent bonds nor would it disrupt the hydrophobic interactions that hold the bilayer together and the integral proteins within. It would only affect proteins held by weak noncovalent interactions such as ionic or H-bonds, i.e. it would release peripheral proteins with no covalent attachment. 4.Two different species of bacteria have been isolated: one from a hot spring with an average water temp. of 400 C, the other from a glacial lake with an average water temp. of 40C. a) Which of the two bacterial species would be expected to have more unsaturated fatty acids in its membrane lipids? Glacial lake species. The unsaturated fatty acids would keep their membranes fluid at low temperatures. b) Which would have longer chain fatty acids? Hot spring species. The longer fatty acids would decrease fluidity of their membranes at high temperatures. 5. Compare and contrast symport and antiport. Describe the transport system mediated by the Na+K+ ATPase; is it a symport or antiport system? In both symport and antiport, two solutes move through the membrane simultaneously or sequentially . In symport, both move in the same direction; in antiport, one solute goes in one direction, the other in the opposite direction. The Na+K+ ATPase of the plasma membrane is an antiport system. It moves K+ into the cell and Na+ out in a ratio of 2K+ per 3Na+. Neither of the ions can be transported unless the other is present, which is characteristic of cotransport systems. 6. Compare and contrast passive and active transport. Describe the transport system mediated by the Na+K+ ATPase; is it an active or passive system?
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In passive transport, also called facilitated diffusion, the transported species always moves down its concentration gradient and no net accumulation above the equilibrium point occurs. It requires no direct energy input. Active transport, by contrast, results in the accumulation of a solute on one side of the membrane. Active transport is thermodynamically unfavorable, and occurs only when coupled (directly or indirectly) to an exergonic process such as the absorption of sunlight, an oxidation reaction, the breakdown of ATP, or the concommitant flow of some other chemical species down its concentration gradient. The Na+K+ ATPase of the plasma membrane is an active transport system. This couples breakdown of ATP to the simultaneous movement of both Na+ and K+ against their concentration gradients. For each molecule of ATP converted to ADP and Pi, this transporter moves K+ in and Na+ out in a ratio of 2K+ per 3Na+. 7. Compare and contrast primary and secondary active transport. Describe the transport system mediated by the Na+K+ ATPase; is it a primary or secondary active system? In primary active transport, solute accumulation is coupled directly to an exergonic chemical reaction (e.g., conversion of ATP to ADP + Pi). Secondary active transport occurs when endergonic (uphill) transport of one solute is coupled to the exergonic (downhill) flow of a different solute that was originally pumped uphill by primary active transport. The Na+K+ ATPase of the plasma membrane is a primary active transport system.
8. The GluT1 transporter of erythrocytes is a classic example of a passive transport system. Passive transporters are often compared to enzymes; how is this transport system similar to, and different from, enzymes? Similarities: The process of glucose transport can be described by analogy with an enzymatic reaction in which the "substrate" is glucose outside the cell (S out), the "product" is glucose inside (Sin), and the "enzyme" is the transporter, T. When the rate of glucose uptake is measured as a function of external glucose concentration, the resulting plot is hyperbolic; at high external glucose concentrations the rate of uptake approaches Vmax. The rate equations for this process can be derived exactly as for enzyme-catalyzed reactions, yielding an expression analogous to the Michaelis-Menten equation: Vo = Vmax [S]out Kt + [S]out in which V0 is the initial velocity of accumulation of glucose inside the cell when its concentration in the surrounding medium is [S] out, and Kt (Ktransport) is a constant analogous to the Michaelis constant, a combination of rate constants characteristic
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of each transport system. This equation describes the initial velocity--the rate observed when [S]in = 0. Kt, like Km, is a measure of the affinity of transporter for substrate; the lower the Kt, the higher the affinity. Because no chemical bonds are made or broken in the conversion of S out to Sin, neither "substrate" nor "product" is intrinsically more stable, and the process of entry is therefore fully reversible. As [S] in approaches [S]out, the rates of entry and exit become equal. Such a system is therefore incapable of accumulating the substrate (glucose) within cells at concentrations above that in the surrounding medium; it simply achieves equilibration of glucose on the two sides of the membrane at a much higher rate than would occur in the absence of a specific transporter. In other words, the process is saturatable. Transporters bind their substrates with stereochemical specificity through many weak, noncovalent interactions. The negative free-energy associated with these weak interactions counterbalances the positive free-energy change that accompanies loss of the water of hydration from the substrate, lowering the energy of activation for transmembrane passage. Differences: Substrates are moved from one compartment to another, but are not chemically altered.
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