Lecture Slides Membranes PDF

Biochemistry Yves Bollen Overview Lecture 1: thermodyanamics (Energy, entropy, enthalpy, work) Lecture 2: Interactions (covalent bond, non-covalent interactions, affinity of ligand binding, ATP) Lecture 3: Protein structure (amino acids, peptide bond, 1st, 2nd, 3rd, 4ht structure) Lectur...

Biochemistry Yves Bollen Overview Lecture 1: thermodyanamics (Energy, entropy, enthalpy, work) Lecture 2: Interactions (covalent bond, non-covalent interactions, affinity of ligand binding, ATP) Lecture 3: Protein structure (amino acids, peptide bond, 1st, 2nd, 3rd, 4ht structure) Lecture 4: Protein dynamics (protein folding, dynamics of folded proteins, misfolding, aggregation and disease) Lecture 5: Enzyme kinetics (Enzymes are catalysts, Mechanisms, Michaelis-Menten model for enzyme reactions, Inhibition of enzymes) Lecture 6: Cofactors, regulation of enzyme activity, cooperativity, allostery Lecture 7: Biological membranes Membranes make compartments in cells Lipids in membranes Membrane proteins Biochemistry 2 Structure of a cell All cells are surrounded by a plasma membrane. The membrane keeps wanted molecules inside and unwanted molecules outside. It forms the barrier between the living cell and the environment. Eukaryotic cells also contain many intracellular compartments, that are also all surrounded by membranes. Biochemistry 3 Plasma membrane The plasma membrane is composed of lipids and proteins. Due to the hydrophobic core, most molecules that dissolve in water cannot pass the membrane. This is the essence of the barrier function. Biochemistry 4 Fatty acids The hydrophobic core of membranes is formed by fatty acids. A fatty acid is a linear chain of carbons that only have hydrogen atoms bound, with a carboxylic acid at the end. The chain of fatty acids is a-polar because hydrogen and carbon have similar electronegativity. Fatty acids can vary in their length (number of carbon atoms) and in the carbon-carbon bonds: they can be all single bonds (“saturated”, because the carbons are saturated with hydrogen), or they can contain one or more double bonds (“unsaturated” because there is still room for some hydrogen on the carbons). Biochemistry 5 Fatty acids Saturated Unsaturated The single carbon-carbon bonds allow free rotation. The chain can thus be very flexible, but it can also stretch out completely to allow for a very compact packing. The double c=c bond in unsaturated fatty acids is cis, meaning that both carbons point to the same side of the c=c bond. The bond is rigid, which induces a kink in the chain of carbons. The kink prevents compact packing. Trans double bonds in fatty acids are rare naturally, but they are formed in the production of some food products due to hardening of fats or during heating. Their effect on human health is unknown. Biochemistry 6 Names of fatty acids Fatty acids have historical names, such as linoleic acid or palmitic acid. They are also identified using a systematic nomenclature. The name depends on the number of carbon atoms and the number and position of double bonds. The fatty acid shown below has 18 carbon atoms and 4 double bonds. This is indicated as 18:4. The acidic end is called the α (alpha) end, the other end is called ω (omega) (first and last letter of the Greek alphabet). The first double bond is at position 3 when counting from the ω-end. Hence the systematic name of this fatty acid is 18:4ω3. Biochemistry 7 Omega fatty acids Humans do not have the enzymes to create ω-3 and ω-6 double bonds, whereas we do need these fatty acids. They are therefore considered essential fatty acids: they need to be present in our diet. They are found e.g. in fat fish, and are now also added to other food products. Biochemistry 8 Fats Plants and animals store energy in the form of fat. Fats consist of three fatty acids coupled to the three -OH groups of glycerol, thus forming triacylglycerol. The carboxylic acid group of the fatty acid forms an ester bond with glycerol. Fluidity of the fat depends on the composition of the fatty acids. Saturated fatty acids pack well and are solid at room temperature. Unsaturated fatty acids do not pack well, leading to a fluid fat: oil. Biochemistry 9 Phospholipids Most lipids in cellular membranes are diacylglycerols. Only two out of three -OH groups of glycerol are occupied by a fatty acid. The third -OH of glycerol is coupled to a polar group called the head. As a result, lipids have a polar head and a hydrophobic tail. Many different head groups are found. The majority are coupled to glycerol via a phosphate. These lipids are called phospholipids: phosphatidylcholine, phosphatidylethanolamide, phosphatidylserine and phosphatidylinositol. Phosphocholine and phosphoethanolamine are neutral (zwitterionic) head groups. Phosphoinositol and phosphoserine are negatively charged. Membranes thus have a negatively charged surface. Biochemistry 10 Other lipids in the membrane Glycolipids have a sugar in the polar head group, like inositol or galactose. Sphingomyelin has an amino-alcohol core instead of glycerol, and the choline head group. Cerebroside has both: it resembles sphingomyelin (amino-alcohol core) but it has a sugar as head group. Biochemistry 11 Cholesterol Cholesterol is also present in mammalian membranes. It has a sterol core: a hydrophobic carbon structure of 4 rings and a short branched hydrocarbon tail. Sterols also function as hormones. It has a very small hydrophilic head: only an OH group. It is smaller than phospholipids. It can fill the gaps in the membrane created by unsaturated (kinked) fatty acids, and thereby modify the membrane fluidity. Biochemistry 12 Hydrophobic effect The hydrophobic effect forces the hydrophobic tails of lipids to stick together, with the polar heads facing the water. The smallest possible structure is a micelle. Micelles of phospholipids are however not stable, there is not enough room for two tails per head: two tails have the same diameter as one headgroup. That is why phospholipids rather form liposomes or bilayer sheets: two layers of lipids with the tails facing each other. Sheets are 4-5 nm thick, with the hydrophobic core spanning around 3 nm. Biochemistry 13 Membranes are fluid yet stable Although at first glance they appear rather fragile, membranes are surprisingly sturdy. And at the same time very fluid. Remember: membranes are held together by electrostatic interactions of headgroups with water and ions, and by the hydrophobic effect. As long as the bilayer is maintained, membranes can be deformed easily. Biochemistry 14 Controlling fluidity Lipid bilayers have a melting temperature (Tm). Below this temperature they are gel-like, above the temperature they are more fluid. The actual lipid composition determines the Tm. Long and saturated fatty acids make the membrane more stiff, the Tm shifts to higher temperature. Short and unsaturated fatty acids make the membrane more fluid. They shift the Tm to lower temperature. Membranes are typically in the fluid phase. Organisms that cannot control their body temperature tend to adjust the lipid composition of their membrane to the prevalent temperature. At higher temperature they make longer and more saturated lipids, and vise versa. Biochemistry 15 Fluidity of the membrane Lipids are free to move around in the plane of the membrane. They diffuse rapidly, making a random walk. Lipids cannot flip from one layer to the other. Flippases and scramblases are enzymes that catalyze the flipping of lipids from one leaflet to the other. Flippases use ATP and are selective: not all lipids are flipped. As a result, the two leaflets do not have identical lipid composition: they are asymmetric. Scramblases do the same, but they are not selective. Biochemistry 16 Flippases and scramblases Scramblases randomly exchange one lipid from one leaflet against one lipid from the other leaflet. Flippases actively (ATP required) and selectively flip lipids to one leaflet, thus maintaining asymmetry. The polar headgroup of the lipids is guided through the hydrophobic core of the membrane via a hydrophilic cleft on the inside of a flippase. Phosphatidylcholine (PC) and sphingomyelin (SM) occur mostly in the outer leaflet of the membrane, whereas phosphatidylethanolamine (PE) and -serine (PS) are found mostly on the inside. Biochemistry 17 Fluid mosaic model The fluid mosaic model states that the lipids diffuse randomly in the plane of the membrane, which thus forms a two-dimensional fluid. Proteins diffuse randomly in this 2D lipid. However, since the 1980’s it has become evident that membranes do contain micro- domains with distinct composition and function. Biochemistry 18  Lipid rafts in the plasma membrane Lipid rafts are thicker and more rigid that the rest of the membrane. Lipid rafts are enriched in cholesterol, glycosphingolipids and receptor proteins. They are 20-200 nm in diameter. They most likely form spontaneously, because separating long stiff lipids into domains is energetically more favorable than having them mixed with short fluid lipids. A Intracellular space B Extracellular space 1. Non-raft membrane 2. Lipid raft 3. Lipid raft associated protein 4. Non-raft membrane protein 5. Glycosylation modifications 6. GPI-anchored protein 7. Cholesterol 8. Glycolipid Biochemistry 19 Fluid microdomains in bacterial membrane For a long time bacteria were considered too small and simple to have domains in their membranes. A few years ago it was shown that bacteria also have raft-like domains. Recently, we were able to show that the membrane of E. coli also contains domains that are more fluid than the bulk membrane. Oswald et al (2016) MreB-dependent organization of the E. coli cytoplasmic membrane controls membrane protein DiI-C12 – Preference for disordered regions General lipid dye - BODIPY FL-C12 diffusion. Biophys J. 110(5):1139-49 A22 inhibits MreB. MreB is an actin-like cytoskeletal protein. Biochemistry 20  Domain stability depends on cytoskeleton Epi illumination 32 ms exposure. No A22. Epi illumination 32 ms exposure. With A22. Domains form spontaneously but fall apart again without the Lipid dye - DiI-C12 cytoskeleton of MreB, which is the bacterial homologue of actin. Preference of disordered regions A22 is an inhibitor of MreB, it makes the cytoskeleton disassemble. Biochemistry 21 Individual lipids move in and out of domain Lipid dye - DiI-C12 TIRF illumination 12 ms exposure. No A22. Preference of disordered regions Biochemistry 22 Proteins move randomly in membrane plane We genetically fused E. coli membrane proteins to GFP and recorded video’s of single fluorescently labelled membrane proteins diffusing in the membrane. Their diffusion coefficient scales with their diameter. CstA-eGFP 32 ms exp. Bar = 1 µm Biochemistry 23 Proteins in the membrane Biochemistry 24 Proteins that span the membrane Most membrane-spanning proteins consist of one or more α-helices connected by shorter or longer loops on both sides of the membrane. Particularly on the cytoplasmic side of the membrane, these proteins are in contact with other proteins. The latter are referred to as “membrane associated proteins”. Biochemistry 25 Membrane-spanning helices Hydrophobic side chains are shown in green. Which of these two proteins is a membrane protein? The one on the right. Membrane proteins have many hydrophobic side chains facing out. They are in contact with the hydrophobic tails of lipids (hydrophobic effect). Biochemistry 26 Amphipathic helix Many helices of membrane proteins are amphipathic. This means that they have a hydrophilic and a hydrophobic side. The are found either on the surface of the membrane, or they form a hydrophilic channel through the membrane. Biochemistry 27 Lipid-anchored proteins Some proteins are bound to the membrane via a covalently bound lipid molecule. The c-terminus of the protein is bound to phospho- ethanolamide (a common lipid headgroup), which is bound to a branched oligosaccharide that is in turn attached to a phosphatidylinositol lipid containing two fatty acid tails. The entire lipid anchor is called glycosylphosphatidyl- inositol (GPI) anchor. The proteins can be released from the membrane in a controlled manner by phospholipase enzymes. Biochemistry 28 Membrane transport The membrane forms a hydrophobic barrier around the cell. It keeps wanted molecules inside and unwanted molecules outside. However, some molecules need to pass the membranes. For example, the cell needs to take up fuel and building blocks (sugars, amino acids, metal ions, oxygen) and get rid of waste products. Several mechanisms exist to transport small molecules across the membrane. We discriminate four categories: simple diffusion, facilitative diffusion, gated channels and active transport pumps. Transport system are called passive if they do not require an energy source, and active if they require hydrolysis of ATP to achieve transport. Biochemistry 29  Membrane transport In the following slides we assume an inward-facing electrochemical gradient. For now we interpret this as: high concentration outside, low concentration inside. More details will be provided later. Biochemistry 30 Simple diffusion Gases such as oxygen and CO2 and lipid-soluble substances (e.g. steroid hormones) can cross the membrane by simple diffusion. Simple diffusion is a random process. It does not require energy. It occurs in both directions. Due to chance (entropy!) there will be net transport from the side with a higher concentration to the side with lower concentration. For example, consider an active cell that consumes O2 and produces CO2. As a result it has lower O2 and higher CO2 concentrations than its environment. This leads to net import of O2 and net export of CO2. Biochemistry 31 Facilitative transport Molecules that are larger, more polar or charged cannot pass the lipid bilayer spontaneously. They require a carrier protein that guides them over the membrane. Carriers have substrate specificity, but no directional specificity. Transport occurs in both directions, just like in diffusion. There is net transport in the direction of the gradient. At equal concentrations inside and outside there will be no net transport: equilibrium. Carriers do not require ATP and are considered as passive transporters. Most membranes contain selective pores that allow water to cross the membrane. Biochemistry 32 Structure of a carrier protein Lactose permease is a typical carrier; binds substrate very selectively switches conformation between inside- open and outside-open. quite fast transfer (102 – 104 molecules/s) Abramson, J. et al. (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301: 610-615. Biochemistry 33 Transport kinetics Whether a molecule crosses the membrane by simple diffusion or via a carrier protein can be investigated experimentally, by measuring transport rates at various substrate concentrations. For simple diffusion the rate will keep increasing with concentration. For carrier-mediated diffusion, the rate will level off due to saturation of the carrier. At high substrate concentration the carrier capacity becomes limiting. Carrier- mediated diffusion thus follows the Michaelis-Menten model for enzyme kinetics. Biochemistry 34 Gated channels Gated channels form a selective pore for certain ions, but only upon stimulation by some trigger. The trigger can be a voltage change, binding of a ligand or phosphorylation. For example, a nerve impulse consist of a voltage change leading to a passive flux of Na+ ions through a voltage-gated channel. At rest, Na+ concentration is high outside the cell and low inside. Upon opening of the voltage-gated Na+-channel, the Na+ ions diffuse rapidly into the cell. This step does not require energy. Energy is required however to restore the original situation with high Na+ concentration outside. Biochemistry 35 Channel selectivity How can an ion channel be selective? Why does a K+ channel not transport Na+? This is controlled by so-called selectivity filters inside the channel. The ion must loose its hydration (water) shell: it enters the channel as a naked ion. Dehydrating an ion is energetically very costly. The favorable water-ion interactions need to be replaced with even stronger ion-protein interactions. The proteins make use of the fact that different ions have different radii. For example, K+ is 1.33Å, Na+ is only 0.95Å (1Å is 0.1 nm or 10-10 m). Na+ thus has weaker interactions inside the channel, and can therefore not be dehydrated by a K+-channel and will not enter it. And vice versa, a K+ ion does not fit inside a Na+ channel because it is too large. Biochemistry 36 Structure of a potassium channel KcsA is a bacterial potassium channel. It is tetrameric. Shown here are only two of the four subunits. Selectivity filter is shown in the middle. Sequence of the filter amino acids is TVGYG (bottom to top). Shown are backbone oxygen atoms and threonine sidechain oxygen (red sticks). These oxygens interact with the K+ ions (purple). The smaller Na+ ions do not bind so well and will therefore not loose their water shell. Biochemistry 37 CFTR: a ligand- and phosphorylation-gated ion channel Cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand- gated Cl- channel that also requires phosphorylation. First, the regulatory domain (R) is phosphorylated by Protein Kinase A (PKA). A subsequent conformational change exposes the ATP binding domain (ABD). Hydrolysis of ATP leads to opening of the channel, allowing chloride ions to diffuse out of the cell, following the gradient. Biochemistry 38 Active transport Diffusion - either simple or facilitated by a pore or carrier protein - can only lead to net transport from high to low concentration. In fact, we should talk about an electrochemical gradient, taking also the membrane electrical potential into account. But we will leave that until a bit later. For now, we will simply consider concentration differences. If a compound needs tot be transported against the gradient, energy is required. We speak of primary active transport if the energy is taken from ATP hydrolysis. Secondary active transport means that the flow of one compound down its gradient is used to transport something else against its gradient. Biochemistry 39 Primary active transport A sodium-potassium antiporter pumps Na+ out of the cell and K+ into the cell, while Na+ is already high outside and K+ high inside the cell. It is primary active transport because it hydrolyzes ATP to change conformation after binding of 3 Na+ ions. Once it is open towards the outside, Na+ is released and K+ is bound, followed by dephosphorylation and a return to the original conformation. An antiporter transports one compound in one direction and another compound in opposite direction. A symporter transports two compounds in the same direction. Biochemistry 40 Secondary active transport Secondary active transport of glucose by the Na+-glucose cotransporter. Small intestines The sodium-potassium antiporter actively maintains low Na+ inside the cell, as seen on the previous slide. In the intestinal lumen, Na+ binds to the glucose transporter and takes a glucose molecule along into the cell, even when intracellular glucose concentration is high due to the large concentration difference of sodium: cotransport. Glucose can then diffuse down its concentration gradient into the extracellular fluid. Biochemistry 41 Secondary active transport is common Biochemistry 42 Multi-drug resistance Antibiotic resistance is becoming a severe problem for global health. An increasing number of bacteria have become resistant to nearly all available antibiotics. In many cases, antibiotic resistance is caused by a protein that pumps a large variety of compounds out of the cell. Also in cancer therapy drug resistance occurs due to the production of protein pumps. These drug-resistance proteins perform primary active transport: they hydrolyze ATP to facilitate the pumping process. Biochemistry 43 24-01-2008: Military hospital installed at medical center Enschede (NL) to replace infected ICU Cause of the infection in this case was MRSA: Methicillin Resistant Staphylococcus aureus Multi - Resistant Staphylococcus aureus Biochemistry 44 Not an incident, but a structural problem Biochemistry 45 Structure of multi-drug resistance protein The bacterial multi-drug transporter SAV 1866 from Staphylococcus aureus constitutes a typical ABC (ATP binding cassette) transporter, harboring its ATP binding cassettes on two domains. It is yet unclear how this one pump can transport many different drugs out of the cell, and how it recognizes drugs from its own metabolites. Usually, pumps have a high substrate specificity and only transport one or a few structurally similar compounds. Dawson, R.J.P., and Locher, K.P. (2006) Structure of a bacterial multidrug ABC transporter Nature 443: 180-185 Biochemistry 46 Carriers and pores can be antibiotics For cells it is essential to maintain certain gradients of ions, for example Na+ and K+, as we have just seen. In mammals Na+ is 5-15 mM inside cells and 145 mM outside, and the opposite for K+: 140 mM inside and only 5 mM outside. Compounds that can make the plasma membrane permeable for ions kill the cell. Several antibiotics are known to work according to this mechanism. They are called ionophores and they act either as a channel or as a carrier. Biochemistry 47 Valinomycin Valinomycin is a carrier for K+ transport. This cyclic ionophore is produced by Streptomyces and transports up to 104 K+ ions/s out of the cell, following its concentration gradient. For sterical reasons, its affinity for K+ is 10000 times higher than for Na+. Valinomycin consists of twelve alternating amino acids and esters: three repeats of [L-valine, D-α- hydroxyisoveleric acid, D-valine and L-lactic acid]. Note that one of these amino acids is a D-amino acid. Biochemistry 48 Gramicidin A, B and C Gramicidins A, B and C are peptide antibiotics produced by the soil bacterium Brevibacillus brevis. Gramicidins A, B and C are nonribosomal peptides, thus they have no genes and they are not synthesized on ribosomes. They consist of 15 amino acids of which 6 are D-amino acids. Their amino acid sequence is: formyl-L-X-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp- D-Leu-L-Y-D-Leu-L-Trp-D-Leu-L-Trp-ethanolamine A gramicidin head-to-tail dimer makes ion-conduction channels in membranes. They are applied in e.g. eye droplets, to combat infections. Biochemistry 49 Mitochondria Within this course, mitochondria are the only organelles to be discussed. Mitochondria are dedicated for oxidative phosphorylation: generating ATP from respiration products. Mitochondria are surrounded by two membranes, an outer membrane and a highly folded, highly impermeable inner membrane. The invaginations of the inner membrane are called cristae. This is where ATP generation takes place. The volume between the membranes is called intermembrane space. The volume inside the inner membrane is called the matrix. Most soluble enzymes involved in oxidation are located here. Mitochondria have their own DNA, encoding for 13 mitochondrial proteins. All the other proteins in mitochondria are encoded in the nucleus and transported from the cytoplasm into the mitochondria via a dedicated protein translocation mechanism. Biochemistry 50 Mitochondrial metabolism at a glance Pyruvate, the primary product of glycolysis is actively transported into the mitochondrial matrix, where it is oxidized, decarboxylated and coupled to CoA. It then enters the Krebs/citric acid cycle. The electrons that are taken out of pyruvate are used to pump protons across the inner membrane into the intermembrane space. The pH difference across the inner membrane is around 1 pH unit. This pH difference is then used to generate ATP using the ATP synthase machinery discussed previously (lecture 4 protein dynamics - motor proteins). Biochemistry 51 Summary Membranes make compartments in cells Lipids in membranes saturated, unsaturated fatty acids phospholipids with various head groups cholesterol domains, rafts Membrane proteins amphipathic pores, channels, carriers active and passive transport antibiotics: ionophores, multidrug resistance proteins Biochemistry 52