Biological Membrane Transport - Crossing the Berlin Wall of the Cell

Biological Membrane Transport “Crossing the Berlin Wall of the cell - Membrane Transport” 1 Physical Properties of Membranes • Not permeable to large polar solutes and ions • Permeable to nonpolar compounds and some small polar compounds (e.g., water) • Permeability can be artificially increased by chemical treatment. – when we want to get DNA into the cell • Very stable yet dynamic and flexible structures • Can exist in various phases and undergo phase transitions • Can fuse • Functionality provided by embedded proteins Three Types of Membrane Proteins Peripheral • Associate with the polar head groups of membranes • Relatively loosely associated with membrane – through ionic interactions with the lipids or aqueous domains of integral membrane proteins • Removed by disrupting ionic interactions either with high salt or change in pH • Purified peripheral membrane proteins are no longer associated with any lipids. Amphitrophic Integral • Span the entire membrane • Have asymmetry like the membrane • Amphitrophic proteins can be conditionally attached to the • membrane by covalent interaction with lipids or carbohydrates attached to lipids. • • Biological regulation results in attachment • to, or cleavage from, lipids. – different domains in different compartments Tightly associated with membrane – Hydrophobic stretches in the protein interact with the hydrophobic regions of the membrane. Removed by detergents that disrupt the membrane Purified integral membrane proteins still have phospholipids associated with them. 2 Membrane Transport Dr. Kornberg: “The Berlin Wall of the Cell”-part 1 Membrane Transport Dr. Kornberg: “The Berlin Wall of the Cell” -part 2 3 Membrane Transport Dr. Kornberg: “The Berlin Wall of the Cell” • Cell membranes are permeable to small nonpolar molecules that passively diffuse through the membrane. • Passive diffusion of polar molecules involves desolvation and thus has a high activation barrier, unless desolvation energy is lowered. • Transport across the membrane can be facilitated by proteins that provide an alternative diffusion path. • Such proteins are called transporters or permeases. Lecture 01.23.17 (21:04-23:56 & 35:27-38:29)-Berlin Wall Types of Membrane Transport Non-mediated [S]out >> [S]in [S]out<< [S]in All others are Mediated X–Sin A–X A Sout Group translocation Ion channel (down (against electrochemical gradient; may be gated concentration gradient) by a ligand or ion) 4 Membrane Transport Three Classes of Transport Systems Membrane Transport What is this electrochemical gradient? • Transport across a membrane must be energetically favorable. There are two types of energies at play: – Concentration dependence: The solute moves toward chemical equilibrium across the membrane. – Electrical dependence: The solute moves toward charge equilibrium across the membrane. This is why we say “down” or “against” the electro-chemical gradient. How is this gradient quantified? 5 Membrane Transport Energetics [Sout] Transport can be considered like a chemical reaction with regard to mass action of Sout to Sin Sout ⇌ Sin Outside [Sin ] DG’ = DG°’ + RTln _______ [Sout ] But, DG°’ = 0 because there is no chemical reaction: [Sin] Inside [Sin ] DG’ = RTln ____ [Sout ] If [Sout] > [Sin], DG’ is ⊝ If [Sout] < [Sin], DG’ is ⊕ But, if S is charged, we must account for this: [Sin] DG’ = RTln _____ + zF D$ [Sout] Where “z” is the charge on S, and D$ is the membrane electrical potential in volts Membrane Transport Energetics [S⊕ out] ⊕ ⊕ ⊕ ⊕ Outside ⊕ Sout ⇌ Sin Sin DG’ = RTln ____ + zF D" Sout Where “z” is the charge on S, and D" is the membrane electrical potential in volts; simply called the “membrane potential” ⊝ ⊝ ⊝ ⊝ Inside ⊝ [S⊕ ] in D" = charge difference “in” versus “out” So, if its more negative in than out, D" is ⊝ (as depicted) And, if its more positive in than out, D" is ⊕ Now, if D" is negative, and S has a positive charge (z is +1, as depicted), then zF D" makes a negative contribution to DG’ making it even more favorable. As a further consequence, if D" is maintained, then at equilibrium [Sin] > [Sout]. 6 Membrane Transport How do you experimentally determine the kind of transport? Non-mediated Mediated Simple diffusion Facilitated diffusion, ionophore mediated, active transport Diffusion is governed by Fick’s law of diffusion: Q = rate of diffusion. D = diffusion coefficient A = area across which diffusion occurs [Sout](P1) and [Sin](P2)= concentrations on each side of membrane. Q L = thickness of the membrane. Sout DA/L is the permeability coefficient. Thus, “diffusion” is Non-mediated Mediated behaves like saturation kinetics C + Sout ⇌ CS ⇌ C + Sin Q Q= Sout Qmax [Sout] Thus, “diffusion” Kd + [Sout] is Mediated Membrane Transport Examples: Facilitative Diffusion Ionophore Maltoporins GLUT1 transporter Aquaporin Selective ion channel for potassium (K-channels) Active Transport Primary (1°) Na/K ABC Secondary (2°) Na/Glc Bicarb/Cl Group Translocation Bacterial phosphotransferase system (PTS) 7 Membrane Transport Facilitative Diffusion Membrane Transport Examples of Facilitative Diffusion (including ionophore mediated) Ionophore Valinomycin • Proteins of the E. coli outer membrane • Maltoporin (derived from PDB ID 1MAL) is a maltose transporter (a trimer; each monomer consists of 16 β strands). 8 Membrane Transport Examples of Facilitative Diffusion Membrane Transport Examples of Facilitative Diffusion 9 Membrane Transport Examples of Facilitative Diffusion Prevention of proton (+H3O) transport: Gates the size of pore at 2.8 Å role of His180 – role of Arg195 – Repels hydronium ions role of NPA – Positive poles at ends of helices repels hydronium ions, but also breaks up network of water molecules Membrane Transport Examples: Facilitative Diffusion Ionophore Maltoporins GLUT1 transprter Aquaporin Selective ion channel for potassium (K-channels) Active Transport Primary (1°) Na/K ABC Secondary (2°) Na/Glc Bicarb/Cl Group Translocation Bacterial phosphotransferase system (PTS) 10 Membrane Transport Examples of Facilitative Diffusion a4 Membrane Transport K channel https://www.youtube.com/watch?v=IXPmgprE8rg&feature=youtu.be KcsA K+ channel in complex with a monoclonal Fab antibody fragment 11 Membrane Transport Examples of Facilitative Diffusion How is the >100X specificity for K+ achieved? Ion Radius Energy of (Å) dehydration (kcal/mole) 0.6 – 98 Na 1.0 – 72 K+ Li+ + This picture is not quite correct as K+ makes eight coordination bonds; this was revealed by the high resolution structure 1.3 – 55 + Rb 1.5 – 51 Cs+ 1.7 – 47 Membrane Transport Examples of Facilitative Diffusion Ion Radius Energy of (Å) dehydration (kcal/mole) Li+ 0.6 – 98 Na+ 1.0 – 72 K+ 1.3 – 55 Rb+ 1.5 – 51 + 1.7 – 47 Cs How is the >100X specificity for K+ achieved? 12 Membrane Transport Examples of Facilitative Diffusion Ion Radius Energy of (Å) dehydration (kcal/mole) Li+ 0.6 – 98 Na+ 1.0 – 72 K+ 1.3 – 55 Rb+ 1.5 – 51 Cs+ 1.7 – 47 While specificity for Na+ over K+ can be achieved by the size of the binding site, that won’t work the other way around. To get specificity of K+ over Na+, the protein takes advantage of the higher (30%) energy of dehydration. It takes more energy to dehydrate Na+ than K+ and the protein doesn’t make as good bonds to Na+ ion as does water shell (see off-set oxygens in polygon). In addition, there is no room for the axial waters in the pore, which are not needed for K+ 13