BCH3033 Biochemistry 1 Chapter 11 B PDF

BCH3033: Biochemistry 1 Chapter 11 B 03.21.2024 Donella Beckwith, Ph.D. [email protected] 1 Integral –vs- Peripheral Proteins Peripheral proteins (membrane protein): can vary in shape PPs do not have a hydrophobic region of amino acids but they have many hydrophilic amino acids exposed on their surf...

BCH3033: Biochemistry 1 Chapter 11 B 03.21.2024 Donella Beckwith, Ph.D. [email protected] 1 Integral –vs- Peripheral Proteins Peripheral proteins (membrane protein): can vary in shape PPs do not have a hydrophobic region of amino acids but they have many hydrophilic amino acids exposed on their surface Can attach to integral membrane proteins or interact with the phosphate head of the lipids Ex: cytochrome C, adrenodoxin reductase, flavoproteins (e’ transport proteins) Transporters Remove by shifting the pH Enzymes, anchorage Recognition, receptors Enzymes, receptors intrinsic extrinsic 2 Covalently Attached Lipids Anchor or Direct Some Membrane Proteins GPI-anchored protein (glycosylphosphatidylinositol): exclusively on the outer face and are clustered in certain regions glycosylphosphatidylinositol associate reversibly with membranes (found in both membranes and the cytosol) Extracellular face 3 Question Which statement is true? A. Hydropathy analysis provides a measure of H-bonding within the transmembrane portion of a membrane-spanning protein. B. For all glycoproteins of a plasma membrane, the glycosylated domains are always found on the extracellular face of the bilayer. C. Proteins may be embedded in, but never span, a biological membrane. D. Once a membrane is synthesized, the lipid composition of each monolayer does not change. 4 Acyl Groups in the Bilayer Interior Are Ordered to Varying Degrees liquid-ordered (Lo) state = gellike state in which all types of motion of individual molecules are strongly constrained liquid-disordered (Ld) state = state in which individual hydrocarbon chains are in constant motion (lateral and rotational) 5 Fatty Acid Composition Affects Membrane Fluidity at physiological temperatures (~37 ˚C): – long-chain saturated fatty acids tend to pack into an Lo phase – kinks in unsaturated fatty acids interfere with packing, favoring the Ld state – shorter-chain fatty acyl groups favor the Ld state 6 Sterol Content Affects Membrane Fluidity sterols have paradoxical effects on bilayer fluidity: – they interact with phospholipids containing unsaturated fatty acyl chains, compacting them and constraining their motion – they associate with sphingolipids and phospholipids having long, saturated fatty acyl chains, making the bilayer fluid, otherwise w/o sterols adopt the LO state At lower temperatures, cholesterol increases fluidity Cholesterol acts as a BUFFER At higher temperatures, it reduces fluidity Constrained motion Fluid but more ordered Constrained motion 7 Fatty Acid Composition Affects Membrane Fluidity at physiological temperatures (~37 ˚C): – long-chain saturated fatty acids tend to pack into an Lo phase but upon adding cholesterol favors Ld – kinks in unsaturated fatty acids interfere with packing, favoring the Ld state but upon adding cholesterol favors LO – shorter-chain fatty acyl groups favor the Ld state 8 Content Affects Membrane Fluidity At lower temperatures: – Increase the concentration of unsaturated fatty acids = increase fluidity – Increase the sterol concentration = increases fluidity – Decrease the concentration of saturated fatty acids = increases fluidity 9 Question Membrane fluidity: A. B. C. D. is maximal when the membrane lipids are in the Lo state. is a function of temperature if composition is held constant. increases with saturated fatty acid content. is not affected by sterol content. 10 Question Liposomes were constructed for an experiment with liver cells in culture. The experiment will be at 25 °C and not 37 °C. Which change could NOT increase liposome fluidity at the lower temperature? A. increasing the concentration of sterols B. increasing the concentration of polyunsaturated fatty acids in phospholipids C. decreasing the concentration of saturated fatty acids in sphingolipids D. increasing the concentration of triacylglycerol in liposomes 11 **Question** A cell was growing happily at 37 °C, but the temperature was then changed to 40 °C. What modifications to the plasma membrane could a cell make to counteract the increase in temperature? A. Add more cholesterol into the plasma membrane. B. Introduce more saturated fatty acids into the phospholipids. C. Decrease the number of double bonds in fatty acids in the phospholipids. D. All of the answers are correct. 12 Sphingolipids and Cholesterol Cluster Together in Membrane Rafts microdomains (rafts) = clusters of cholesterol and sphingolipids that make the bilayer slightly thicker and more ordered than neighboring, phospholipid-rich regions – can be up to 50% of the cell surface 13 Proteins and Lipid Rafts proteins must have hydrophobic helical sections long enough to segregate into the thicker bilayer regions of rafts lipid rafts are enriched in: 1. proteins that have two long-chain saturated fatty acids covalently attached through Cys residues 2. GPI-anchored proteins 14 Transbilayer Movement of Lipids Requires Catalysis transbilayer (“flip-flop”) movement has a large, positive free-energy change membrane proteins facilitate the translocation of individual lipid molecules 15 Flippases flippases = catalyze translocation of the amino- phospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) from the extracellular to the cytoplasmic leaflet of the plasma membrane – consume ~1 ATP per molecule of phospholipid translocated – related to the P-type ATPases (active transporters) 16 Floppases floppases = move plasma membrane phospholipids and sterols from the cytoplasmic leaflet to the extracellular leaflet – are ATP-dependent – members of the ABC transporter family – each specializes in movement of specific lipids 17 Scramblases scramblases = move any membrane phospholipid across the bilayer down its concentration gradient – not dependent on ATP; some require Ca2+ – lead to controlled randomization of the head-group composition on the two faces of the bilayer 18 Lipids and Proteins Diffuse Laterally in the Bilayer individual lipid molecules undergo Brownian movement FRAP = fluorescence recovery after photobleaching – rate is a measure of the rate of lateral diffusion of the lipids 19 Hop Diffusion of Individual Lipid Molecules single particle tracking confirms lipid molecules diffuse laterally within small regions movement from one region to another (“hop diffusion”) is rarer 20 Some Membrane Proteins Are Free to Diffuse, Whereas Others Are Not membrane proteins are limited in movement by: – associating to form large aggregates (“patches”) – anchoring to internal structures 21 Caveolae and Caveolin caveolae (“little caves”) = specialized rafts caveolin = integral protein that binds to the cytoplasmic leaflet of the plasma membrane – forms dimers – associates with cholesterolrich membrane regions – forces the bilayer to curve inward to form caveolae 22 Question Which statement regarding caveolae is false? A. B. C. D. They are a means of expanding the cell surface. Their extracellular leaflets are lipid rafts. They tend to be cholesterol poor. They result from the formation of caveolin dimers. 23 Question What can be learned from a hydropathy plot of caveolin? A. The protein may have several α helices and at least one β strand. B. There is a calcium-binding domain. C. There are three polypeptides in the functional protein. D. It provides a prediction of two membrane-spanning regions. 24 Membrane Curvature and Fusion Are Central to Many Biological Processes curvature changes are central to the ability of biological membranes to undergo fusion with other membranes without losing continuity 25 Cardiolipin cardiolipin = can create or recognize membrane curvature – Phospholipid – Unique structure: 2 phosphate residues and 4 fatty acyl chains – located in mitochondrial and bacterial membranes 26 Fusion Proteins fusion proteins = mediate specific fusion of two membranes by bringing about specific recognition and a transient local distortion of the bilayer structure 27 SNARE Proteins SNAREs (snap receptors) = family of proteins v-SNAREs = SNAREs in the cytoplasmic face of the intracellular vesicle t-SNAREs = SNAREs in the target membrane with which the vesicle fuses Membrane fusion during neurotransmitter release, a v-SNARE and a t-SNARE bind to each other and undergo a structural change. 28 Integral Proteins of the Plasma Membrane Are Involved in Surface Adhesion, Signaling, and Other Cellular Processes integrins = surface adhesion proteins that mediate a cell’s interaction with the extracellular matrix and with other cells – carry signals in both directions across the plasma membrane – heterodimeric proteins composed of two unlike subunits, α and β 29 Cadherins and Selectins cadherins = involved in surface adhesion – undergo homophilic interactions with identical cadherins in an adjacent cell selectins = have extracellular domains that bind specific polysaccharides on the surface of an adjacent cell – require Ca2+ – essential part of the bloodclotting process 30 Question Integrins, cadherins, and selectins: A. are never all found in the same cell. B. are only found in caveolae. C. are sometimes integral and sometimes peripheral membrane proteins. D. allow for attachments between cells or between a cell and the extracellular matrix. 31 Summary of Transport Types nonpolar compounds can dissolve in the lipid bilayer and cross a membrane unassisted polar compounds and ions require a specific membrane protein carrier 32 Three General Classes of Transport Systems cotransport systems = simultaneously transport two solutes across a membrane – antiport = moves in opposite directions – symport = moves in the same direction uniport systems = carry only one substrate 33 Membrane Potential, Vm membrane potential, Vm = a transmembrane electrical gradient that occurs when ions of opposite charge are separated by a permeable membrane – produces a force that: opposes ion movements that increase Vm drives movements that reduce Vm 34 Electrochemical Gradient electrochemical gradient (electrochemical potential) = determines the direction in which a charged solute moves across a membrane – composed of: the chemical gradient the electrical gradient (Vm) 35 Active Transport Results in Solute Movement against a Concentration or Electrochemical Gradient results in the accumulation of a solute above the equilibrium point thermodynamically unfavorable (endergonic) – must be directly or indirectly coupled to an exergonic process 36 Two Types of Active Transport primary active transport = solute accumulation is coupled directly to an exergonic chemical reaction secondary active transport = endergonic transport of one solute is coupled to the exergonic flow of a different solute that was originally pumped uphill by primary active transport 37 Free-Energy Change for the Conversion of a Substrate to a Product general equation for the free-energy change: ∆G = ∆G′° + RT ln ([P]/[S]) (11-2) where ∆G′° is the standard free-energy change, R is the gas constant (8.315 J/mol·K), and T is the absolute temperature 38 Free-Energy Change for Transport of an Uncharged Solute Transport a solute from a region where its concentration is C1 to a region where its concentration is C2 and no bonds are broken, then ∆G′ is zero free-energy change for transport, ∆Gt, of a solute from a region where its concentration is C1 to a region where its concentration is C2 is: ∆Gt = RT ln(C2/C1) (11-3) 39 Free-Energy Change for Transport of an Ion without movement of an accompanying counterion, the process is electrogenic (produces an electrical potential) free-energy change for transport, ∆Gt, of an ion is the sum of the chemical and electrical gradients: ∆Gt = RT ln(C2/C1) + ZF ∆ψ (11-4) where Z is the charge on the ion, F is the Faraday constant, and ∆ψ is the transmembrane electrical potential (in volts) 40 Question Calculate the energy cost (free-energy change) of pumping an uncharged solute against a 106-fold concentration gradient at 25 °C. Recall that R is the gas constant (8.315 J/mol K). A. B. C. D. 34,233 kJ/mol 2.87 kJ/mol −34 kJ/mol 34 kJ/mol ∆Gt = RT ln(C2/C1) 106-fold concentration gradient 106 1 41 Model of Glucose Transport into Erythrocytes cycles between two extreme conformations: – T1 form: glucosebinding site is exposed on the outer membrane surface – T2 form: glucosebinding site is exposed on the inner surface 42 The Glucose Transporter of Erythrocytes Mediates Passive Transport glucose enters the erythrocyte by passive transport via GLUT1 analogous with an enzymatic reaction where: – glucose (“substrate”) outside is Sout – glucose (“product”) inside is Sin – transporter (“enzyme”) is T 43 Rate Equations for Glucose Transport analogous to the Michaelis-Menten equation: Vmax [S]out V0 = Kt + [S]out (11-1) where V0 is the initial velocity of accumulation of glucose inside the cell, [S]out is the concentration of glucose in the surrounding medium, and Kt (Ktransport) is a constant analogous to the Michaelis constant 44 Kinetics of Glucose Transport into Erythrocytes Double-reciprocal plot 45 Glucose Transporters in Humans Table 11-1 Glucose Transporters in Humans Transporter Tissue(s) where expressed Kt (mᴍ) Role/characteristics GLUT1 Erythrocytes, blood-brain barrier, placenta, most tissues at a low level 3 Basal glucose uptake; defective in De Vivo disease GLUT2 Liver, pancreatic islets, intestine, kidney 17 In liver and kidney, removal of excess glucose from blood; in pancreas, regulation of insulin release GLUT3 Brain (neuron), tests (sperm) 1.4 Badal glucose uptake; high turnover number GLUT4 Muscle, fat, heart 5 Activity increased by insulin GLUT5 Intestine (primarily), testis, kidney 6 Primarily fructose transport GLUT6 Spleen, leukocytes, brain >5 Possibly no transporter function GLUT7 Small intestine, colon, testis, kidney 0.3 ⸻ GLUT8 Testis, sperm acrosome −2 ⸻ GLUT9 Liver, kidney, intestine, lung, placenta 0.6 Urate and glucose transporter in liver, kidney GLUT10 Heart, lung, brain, liver, muscle, pancreas, placenta, kidney 0.3 Glucose and galactose transporter GLUT11 Heart, skeletal muscle 0.16 Glucose and fructose transporter GLUT12 Skeletal muscle, heart, prostate, placenta ⸻ ⸻ 46 Question Another GLUT transporter was discovered in vertebrates. Which observation would NOT be expected if this protein is similar to other GLUT transporters? A. Transport kinetics yield a hyperbolic curve. B. The hydropathy plot suggests many transmembrane domains. C. Transporters decrease in concentration on the plasma membrane as glucose levels drop outside the cell. D. The ATP/ADP ratio decreases when the transporter is maximally active. 47 Response Another GLUT transporter was discovered in vertebrates. Which observation would NOT be expected if this protein is similar to other GLUT transporters? D. The ATP/ADP ratio decreases when the transporter is maximally active. GLUT transporters mediate passive transport. A decreasing ATP/ADP ratio would indicate active transport. 48 Question P-type ATPases: A. B. C. D. undergo reversible phosphorylation on an Asp residue. transport anions. transport phosphate up (against) its concentration gradient. transport phosphate down its concentration gradient. 49 Response P-type ATPases: A. undergo reversible phosphorylation on an Asp residue. P-type ATPases couple phosphorylation and dephosphorylation of the critical Asp residue in the P (phosphorylation) domain of the transporter to alternate exposure of solute-binding sites on the inside and the outside of the membrane. All P-type pumps have similar and similar mechanisms. 50 Question Which consequence of the action of a Na+K+ ATPase in the plasma membrane is useful? A. B. C. D. depleting the cells of Na+, which can be harmful drawing K+ into cells, contributing to buffering capacity maintaining charge equilibrium across the membrane membrane repolarization 51 Response Which consequence of the action of a Na+/K+ ATPase in the plasma membrane is useful? D. membrane repolarization For each molecule of ATP converted to ADP and Pi, the transporter moves two K+ ions inward and three Na+ ions outward across the plasma membrane. Cotransport is therefore electrogenic, creating a net separation of charge across the membrane. In animals, this produces a negative membrane potential, which is essential to the conduction of action potentials in neurons. 52 ABC Transporters Use ATP to Drive the Active Transport of a Wide Variety of Substrates ABC transporters = family of ATP-driven transporters that pump substrates across a membrane against a concentration gradient – have two ATP-binding domains (“cassettes”) and two transmembrane domains – example substrates: amino acids, peptides, proteins, metal ions, various lipids, bile salts, and hydrophobic compounds, including drugs 53 Mechanism of ABC Transporters substrates move across the membrane when two forms of the transporter interconvert – driven by ATP hydrolysis 54 Some ABC Transporters in Humans Table 11-2 Some ABC Transporters in Humans Gene(s) Role/characteristics Text reference ABCA1 Reverse cholesterol transport; defect causes Tangier disease Fig. 21-47 ABCA4 Only in visual receptors, recycling of all-trans-retinal Fig. 12-19 ABCB1 Multidrug resistance P-glycoprotein 1; transport across blood-brain barrier ⸻ ABCB4 Multidrug resistance; transport of phosphatidylcholine ⸻ ABCB11 Transports bile salts out of hepatocytes Fig. 17-1 ABCC6 Sulfonylurea receptor; targeted by the drug glipizide in type 2 diabetes Fig. 23-27 ABCG2 Breast cancer resistance protein (BCRP); major exporter of anticancer drugs p. 396 ABCC7 CFTR (Cl₋ channel); defect causes cystic fibrosis Box 11-2 multidrug transporter (MDR1) = human ABC transporter with very broad substrate specificity – encoded by the ABCB1 gene – removes toxic compounds Placenta Blood brain barrier – responsible for resistance of tumors to drugs 55 Question Which statement is false? A. The mechanism of ATP transporters is believed to involve two forms of the transporter. B. The stoichiometry of ATP transporters is about one ATP hydrolyzed per molecule of substrate transported. C. Inhibition of MDR1 is often associated with treatment failure in cancers of the liver, kidney, and colon. D. ABC transporters are also present in simpler animals and in plants and microorganisms. 56 Response Which statement is false? C. Inhibition of MDR1 is often associated with treatment failure in cancers of the liver, kidney, and colon. MDR1 pumps chemotherapeutic drugs out of cells, thus preventing their accumulation within a tumor and blocking their therapeutic effects. Overexpression of MDR1 is often associated with treatment failure in cancers of the liver, kidney, and colon. Highly selective inhibitors of these multidrug transporters are expected to enhance the effectiveness of antitumor drugs. 57