Membranes and Cell Signalling Lecture 1 PDF

Membranes and Cell Signalling - Lecture 1 Introduction to Biomembranes Julian Pakay Intended Learning Outcomes Discuss how biomembranes are composed of different classes of lipids and how this influences their physical properties. Explain the basic bio-membrane structure a...

Membranes and Cell Signalling - Lecture 1 Introduction to Biomembranes Julian Pakay Intended Learning Outcomes Discuss how biomembranes are composed of different classes of lipids and how this influences their physical properties. Explain the basic bio-membrane structure and how it was determined. Compare the original fluid-mosaic model with our current understanding of biomembranes. Discuss the experiments which have led to the new paradigm of the fluid-mosaic model. Where are membranes found? A > eX0 Y cytosoliz  Function of membranes organised. effective-cells ame - & Compartmentation/ Barrier - enables compartmentalisation of enzymes, metabolites, pathways Specific “transport” - allows specific compounds to cross the barrier due to the barrier - may be passive or active (pump) - membrane potential) Maintenance of electrical potential > - - FOuV /resting - many membranes can generate and store an electrical potential difference across the two sides plants/eyes Trapping light energy > - - some membranes can trap light energy and convert it to electrical or chemical energy ↓ Provision of a [special environmentJ (eg for synthesis) compartmentalizat of > - into resice a “Signal” Recognition/ Transduction ligand binds receptors> - to eg packing energies. signaltransductt ↳ - receptors for hormones, neurotransmitters, viruses - generation of 2nd messengers - cell-cell interactions (eg. recognition, antigen presentation) for cells Shape Maintenance B-activ (house keeping game) maintain particular shape > -. - submembranous [cytoskeleton1 controls shape and movement Endocytosis/ exocytosis - membranes are constantly trafficked Phospholipids Are the Main Lipid Constituents of Most Biomembranes Polar hydrophilic head group Non Polar hydrophobic fatty acyl tails Camphipathic structure ↳ combinat of both polar + non polar characteristics  DClasses of lipids ① ② (a) Phosphoglycerides eg phosphatidylcholine (PC), phosphatidylethanolamine (PE) , phosphatidylserine (PS), phosphatidylinositol (PI) ③ ④ (b) Sphingolipids – derivatives of sphingosine eg sphingomyelin (SM) distinct structure > - fused ring structure (c) Sterols – eg cholesterol.. Lipidomics reveals a remarkable diversity of lipids in human plasma Quehenberger et al. 2010, J Lipid Res Over 500 lipid species identified and quantified in human plasma  is different > - across the lipids retrains quite similar Most derivatives of glycerol-3-phosphate - Fatty acids can vary in length, contain no double bonds (saturated) or 1,2 or 3 hydrophobic double bonds (unsaturated) e - Plasmalogen – one fatty acyl chain linked by ester and one by ether to glycerol  ] > - important tail hydrophobic Derivatives of sphingosine (in red) – amino alcohol with long hydrocarbon tail Other fatty acyl chains linked by amide bond Other sphingolipids have sphingosine linked to a sugar residue or branched oligosaccharide  Sebring structure oo O Basic structure is a 4-ring hydrocarbon Amphipathic – single hydroxyl group is equivalent to the polar head group in other lipids TWhat would happen to phospholipids if they were dispersed in an aqueous solution? ↳ 3 possible ways  Amphipathic molecules in solution layer phospholipid double of ② - spherical shape O -. - single layer mhospholipid of & structure. - Spherical · ③ more ,move stable layer) flat/sheet double laged/bilayer = The type of structure formed①by a pure phospholipid or a mixture of phospholipids depends on the length ② ③ of the fatty acyl chains and their degree of saturation, on the temperature, on the ionic composition of ④ > - how you lipids release the in the water. the aqueous medium, and on the mode of dispersal of the phospholipids in the solution. In all three forms, hydrophobic interactions cause the fatty acyl chains to aggregate and exclude water molecules from the “core.” NB: micelles are rarely formed from natural phosphoglycerides, whose fatty acyl chains generally are too bulky to fit into the interior of a micelle. Developing the model for biomembrane structure Ett In the 1950's Robertson noted the structure of membranes seen in the above electron micrographs. He hypothesized that the [railroad track appearance J came from the binding of osmium tetroxide to proteins and polar groups of lipids. Proteins channels etc. -> Receptors) What is missing from this model?  Freeze-etching confirmed the structure of End & the bilayer bilager ! used to determine what technique is Is the exposed surface polar or non-polar? ① cryo biological sample[frozen at -100°CJ - S this uncrolmanometer ② > - surface cut with a liquid nitrogen cooled microtome (knife) in a vacuum bilayer I > Protocol Or ③ 2 layer sample splits along interior membrane plane – revealing two fracture faces ⑭ - ↓ the two surfaces are coated with a heavy metal and a replica is made of each half – amenable to viewing by an electron microscope Empt for exams  Fend The result of freeze etching? Smooth n Rough more proteins the bumps Frotins alot & Inner What are all these lumps on Brother & campy - the inside of the lipid bilayer? proteinsouter more proteins Richattin less a ↑ Take away point] & [Conclus from freeze etching] nembrane the plasma layers in confirms that there are I compared to the > - proteins present as -Inner leaflet has a lot more outer leaflet. By 1970 beginning to appreciate how the bilayer looks because of the freeze etching technique > - Gi & O proteins mobile/have movement. Given that these membranes areO fluid (rather than crystalline) under physiological conditions – are the lipids and protein mobile? - 4)  Are proteins and lipids mobile in the bilayer? - Yes. > - I diff mobility ① lateral diffusion the same plane) ↳ more and left (on right ② transverse diffusion ↳ flipping ↓ ATP involved difficult to happen. Which is easier (energetically) – for lipids and for proteins embedded in the bilayer? - ↳ lateral diffusu /no ATP involved)  zarliest that introduced to How was lateral diffusion demonstrated? > - This was technique was explain lateral diffust. Human – mouse heterokaryon experiment - mixing not affected by: inhibition of protein synthesis depletion of ATP but lowering temperature to What are spherocytes? - smaller rounder red blood cells less surface area than normal erythrocytes less flexible and less durable caused by a defect in cytoskeletal proteins (spectrin, ankyrin, band 3 or protein 4.2 S · - has proteins. ↓ inside nothing.  How do RBCs get their shape? network of integral and peripheral membrane proteins anchored to the cytoplasmic face of the RBC membrane. What do we mean by - integral and peripheral? ↓ transmembrane ↓ side of the both layers either - across inner/outer > pans membrane bilayer of plasma periphe eripheral Integral Sheetz et al. compared lateral mobility of membrane proteins in normal vs spherocytic mice (lacking spectrin) Sodium dodecyl sulfate poly-acrylamide electrophoresis (SDS-PAGE) of erythrocyte “ghosts” indeed does not have spectiv RB2 ↑ confirm that spheresitic Labelled glycoproteins with fluorescent DTAF or fluorescent concanavalin A (Con A) DTAF - Dichlorotriazinyl aminofluorescein Con A - a lectin (carbohydrate-binding protein) /T↓ t spectives / spectineseet Il spheros itic RBG a) coomassie stain b) fluorescent image of gel FRAP used following labelling to look at diffusion rates RBL spheresitic I 3 lateraldiffust I fast) RBC -original - lateraldits an shad 3 # cytoskeleton preservan affects diffuse lateral 50x! Protein interactions and cytoskeletal structure can lead to a non-random distribution of membrane proteins More mosaic than fluid 1972 current  Membranes and Cell Signalling - Lecture 2 The Structure of the Bilayer Julian Pakay What you need to know… Micro-domains within the bilayer Asymmetry between the leaflets in a bilayer Transverse mobility of lipids – key experiments How transverse movement is catalysed Apoptosis and lipid asymmetry Our current model of the membrane (more protein than we originally thought)  "parts" We know that proteins can form micro-domains in membranes [microdomains) fast ⑭ spectrin ”corrals” in erythrocytes # What about the lipids themselves? Are they distributed randomly throughout each leaflet?  Eg Receptors initzin. > - Evidence comes from Glycophosphatidylinositol (GPI) I anchors for proteins - lipid attached to in - protein > protein to sit helps membrane i - ↓ membrane. on to The plasma - plasma functionality ranges from enzymatic to antigenic and adhesion. also play a critical role in a variety of receptor-mediated signal transduction pathways. Synthesis and trafficking of GPI-linked proteins 7.Est luk protina suc membrane  (Brown and Rose, 1992) anchor breakdown proteins and lipids Kid interin - - 1 GPI-linked proteins- solubilise poorly in non-ionic detergents (TX100) - cannot breakdown harsh ↳ ↳ less /harsher) compared to ioniz Surprising since you would expect hydrophobic phosphatidylinositol to partition in detergent micelles GPI-linked proteins solubilise poorly in non-ionic detergents (TX100) ⑮ ER golgi cell surface O PLAP - Human placental alkaline phosphatase (GPI-anchored) Insolubility of PLAP not due to protein-protein interactions Still insoluble in high salt or alkaline carbonate buffer Insoluble fraction contains lipid – detergent resistant membrane there were lipid vesicles in the TX100 insoluble HP-TLC fractions Neutral lipids Acidic lipids I S I S * Sphingolipids – cerebrosides (CB), lactosyl ceramide (LacCer), Forssman antigen (Gb5) , Sulfatides (sulf), sphingomyelin (SPM). A much higher fraction of sphingolipids in the insoluble fraction cannot be broken stable - down I > A - ny acted neuous Cholesterol and sphingolipid also shown to aggregate in artificial membranes  Lipid Raft Model 'liquid-ordered' phase extracellular 'liquid-disordered' phase cytosol Sphingolipids only in the outer leaflet of the plasma membrane bilayer. Fatty acids of sphingolipids are very different from those of glycerolipids, consisting of very-long-chain (up to C26) largely saturated acyl chains - largely saturated acyl chains Sphingolipids tend to have more free hydroxyl groups, both in the long-chain bases and fatty acid components than glycerolipids, and these enter into hydrogen bonding and contribute to the stability of rafts. Packing of cholesterol with the saturated acyl chains of sphingolipids is thermodynamically favoured over that with unsaturated acyl chains, and cholesterol is essential to the process of raft formation. Polarity in Epithelial Cells Receptors - GPI-linked proteins targeted here - to lumen Cholesterol and sphingolipid rich exposed - I Viral budding shows different membrane composition Different membrane composition cell-cell ~ communicate advest 99 molecules Atomic force microscopy Lipid rafts – feeling is believing atomic force microscopy reveals sphyingomyelin rafts (orange) protruding from a dioleoylphosphatidylcholine background (black) in a mica-supported lipid bilayer. Placental alkaline phosphatase (yellow peaks), a glycosylphosphatidylinositol-anchored protein, is shown to be almost exclusively raft-associated. Properties of rafts 50% of the plasma membrane may consist of rafts. (the apical membrane of epithelial cells may behave like a large raft). rafts are sphingolipid-rich regions ('liquid-ordered' phase) surrounded by glycerophospholipid-rich domains ('liquid-disordered' phase). ordering is responsible for the resistance to attack by detergents. rafts are relatively small (approximately 50 nm diameter and containing roughly 3000 sphingomyelin molecules) and mobile. of proteins due presence. to > - ⑧ they are thicker than normal membranes (46 versus 40 angstroms). sphingolipids tend to have more free hydroxyl groups, both in the long-chain bases and fatty acid components than glycerolipids, and these enter into hydrogen bonding and contribute to the stability of rafts.  of How can rafts influence function? movement help in > certain proteins Craft > - trafficking 4/t within www proteins > proteins "islands' dynamic structures – mobile, coalesce andO grow The aid in trafficking certain proteins segregation of signalling components -micro-domains (eg a protein activated by phosphorylation within the raft and might be prevented from interacting with an inactivating phosphatase in another Raff region of the membrane) T · i downstream signalling molecules) 8 8 concentration or sequestration of signalling molecules T in response to receptor activation or other stimuli, sphingolipid compositions in rafts may be altered with effects on membrane architecture or morphology producing further downstream events.  bilayer-fluid-mosaic-rafts = The updated fluid – mosaic model - a new paradigm Current data does not fit the Singer-Nicholson “fluid-mosaic” model: i. non-random co-distribution patterns of receptors in notrandomised the plasma membrane Cytoskeleton holds rafts place in > - ii. quasi-permanent molecular contacts to cytoskeletal elements and signal-transducing molecules iii. much shorter barrier-free path than expected for unrestricted diffusion domain structure of the lipid components of membranes has the capacity to segregate or co-localize membrane proteins iv. participation of integral membrane proteins in the maintenance of membrane domains suggests that proteins are as important as structural elements as lipids *v. dynamic reorganization of protein elements in membrane domains allows for streamlined cellular responses and is restricted by protein–lipid and protein–protein interactions.  Movement within bilayer ① lateral diffusion ② I flip transverse diffusion ( difficult-ATP involved) Which is easier (energetically) – for lipids for proteins embedded in the bilayer? not ATP involved lateral diffuse > - Movement within bilayer lateral diffusion transverse diffusion For lipids: 1-2 hours for a phospholipid to “flip” transversely movement is 109 times faster laterally For proteins: tranverse “flipping” has not been observed  AHowever when we examine membranes... - Examgus m Outer leaf – extracellular tipids Inner leaf - cytosolic total % % ] 20 importat 20 e To to cut the to different very s % colo 20 % 15 % - total Data from human erythrocytes Can you think how this data was obtained? I  I Protocol for lipid distribute Determining lipid asymmetry know st stop the flipping ① use phospholipases A2 to “clip” the - RBC outer leaflet PLs PL → lysoPL + fatty acid -inner leaflet PLs are protected 1. dissolve lipids in organic solvent ⑳ 2. separate by 2D-TLC (thin layer ③ chromatography) identify different lipid - classes 3. determine asymmetric distribution of phospholipids ⑭ Determining lipid asymmetry cont...  Alternative method for determining surface-exposed PS. (Note: works better for cells other than RBCs) Annexin V – a human intracellular protein, binds PS (function not clear) recombinant fluorescent annexin V is commercially available (as experimental tool) binds to cells with exposed PS => annexin V trimerisation and internalisation => highly fluorescent cells used as a probe to measure apoptosis in vitro and in vivo -measure amount of fluorescent-annexin V bound using “microscopy” or flow “cytometry”  Mechanism for maintaining asymmetry Two hypotheses: a) that the rate of "flip-flop" is very slow (i.e. RBC membranes are synthesised in an asymmetric fashion and remain so for the life of the RBC) b) that a specific mechanism operates to maintain amino PLs at inner leaflet How to test these hypotheses? You can measure “flip-flop” rates incubate erythrocytes with phospholipid vesicles containing “fluorescent or radiolabelled” phospholipids (*PS, *PE and *PC). incubate the RBCs at 37°C (in presence of an energy source) remove samples at different times. Assess PL asymmetry using PLase treatment or back extraction of phospholipids. min What does this imply?  - > to indicate apoptosis s Flip-flop of PS and PE is fast therefore “facilitated” transport? ATP flipping requires = *PS and *PE are “translocated” very rapidly to the inner leaflet (in ATP-replete erythrocytes) whilst *PC remains mainly at the outer leaflet. (i.e. mimics endogenous PL distribution) Conclusion: must be facilitated or “active ” transport Protein-mediated, ATP-dependent Involves members of the P-type ATPase family (ATP8 translocase) or "flippase" There are flippases, floppases and scramblases in the plasma membrane ⑮ # apoptosis- important ATP dependent ATP dependent ATP independent Activation of a “scramblase ” causes loss of PL asymmetry “eat me” signal => senescence or cell death/apoptosis  Apoptosis – pathway of programmed cell death in multi-cellular organisms. Normal (in detail later in semester!) PS 0%o I - involves a series of biochemical events leading to a El altered cell morphology, blebbing, cell shrinkage, loss of PS PL asymmetry and chromosomal DNA fragmentation Apoptosis is % cell ⑮=> PS at the outer leaflet is an indicator of apoptosis. 11/(//I 0% / Is this cell a healthy/viable well Differentiation of fingers and toes in a developing human embryo occurs ↓is 9 outer because cells between the fingers Ans : no > - on leaflet. undergoing apoptose. apoptosis , Membranes and Cell Signalling - Lecture 3 Membrane Proteins Julian Pakay What you need to know… focus on PS - indicator of apoptosis > - Phospholipase - + A2 enzyme ?? ) ↳ in outer leaflet high > - X good Methods to measure lipid asymmetry/translocation How proteins associate with the membrane Protein asymmetry is absolute Integral vs peripheral membrane proteins Types of membrane proteins Differentiating membrane proteins Predicting transmembrane domains Mechanism for maintaining asymmetry ATP requires Two hypotheses: & lateral diffust transverse diffusa/ flipping compared to ↑ X a) that the rate of "flip-flop" is very slow (i.e. RBC membranes - are synthesised in an asymmetric fashion and remain so for the life of the RBC) phospholipids. b) that a specific mechanism operates to maintain amino PLs - at inner leaflet > - importance ofPS How to test these hypotheses? ↑ FRAP lateral diffuse -> V  diffust il transverse You can measure “flip-flop” rates - > - incubate erythrocytes with phospholipid vesicles containing “fluorescent or radiolabelled” phospholipids (*PS, *PE and *PC). incubate the RBCs at 37°C (in presence of an energy source) remove samples at different times. Assess PL asymmetry using PLase treatment or back extraction of phospholipids. · min > - less ↓ than 60 fastest sea less are efficient at removing damage via - within a minute apoptosis > - What does this imply?  diffust ? > & How to measure transverse Using fluorescent phospholipid analogues to determine translocation rates prepare membranes (erythrocyte ghosts) !! Protocol to RMS membrane * - - * X into plasma add fluorescent analogue (nitrobenzoxadiazole (NBD) and incubate – readily - incorporates into the outer leaflet andfourescent analogisa > washalay - wash membranes with buffer containing BSA – back extraction of analogue recover membranes by centrifugation and solubilise in detergent determine fluorescence of extract  BSA can be used to back-extract the analogue Preferentially binds to the longer acyl chain of the analogue wash buffer. a X -Bindstounbound Shutt itself into O NDT incopatea e 9  before Jes out quit Same in a Joptions J optione PS - A student is designing an experiment to use fluorescently-tagged phosphatidylserine to measure the activity of ATP-dependent - amino-phospholipid translocase in mammalian erythrocytes. The structure of unmodified 18:1 phosphatidylserine is represented by (A). However there are two possible fluorescently-tagged analogues commercially available, (B) and (C). needs be similar organic structure phospholipid in as chosing analogue to -> when ↑ ↳ sit " nicely" beside the phospholipid > - has to incoporate with phospholipid Which analogue would you recommend the student to purchase and why? head of the stree a ofthestructureinteracts B the more than the ↳ Option - Are B and C more or less polar than A? Explain how this could affect the interpretation of the experiment. C polar > - more than A > ↳ B -  for incoporate incoporates better !?) betor looking which Option is if a are > > you one - - at the looking eneyme would ? Option - flowerse enzyme recognise -> which avalogue recognition an ↓ head enzyent recognise fail better than the ? why > - exposed recognise a the -  very clean > - not > - wash buffer /remove any excess clean , and que does not birdto so but birds to pSy Bar, 20 μm A. initial fraction of analogs in outer leaflet. B. In fibroblasts, disappearance of NBD-PS from the cell surface is due to fast translocation across the plasma membrane (and endosomal membranes), resulting in a labeling of various intracellular membranes. C. NBD-SM is internalized via endocytic vesicles resulting in the appearance of intracellular fluorescent spots (C).  Is the translocation protein dependent? > - => Yes NEM (n-ethylmaleimide reactive toward thiols) & Thigher I S > - doe than green/yellow binds to to Biotin streptavidia porty · binds to specifically Exotic Must be protein specific (pustNanslationas - - ↓ recognises. it binding to protein 3 ↓ How much flourescence there is activity of proteins. Active vs inactive flippase - ↓ Quit - concept is same flips PS peak 1 peakz activity higher Y low y activity ↳ tells you activity of protein Why study membrane proteins? Constitute more than 30% of cellular proteins. more than half of all FDA approved drugs bind to membrane proteins yet they remain the most challenging targets for structure determination! Protein content of biomembranes How are these proteins associated with the membrane? A lot of our information about membranes has come from RBCs – why?  integral us peripheral Types of membrane proteins ↓ ↓ either side both side (1) (7) (P) (7) (2) (P) (P) Anatomy of an integral membrane protein 1 Transmembrane protein) - Disulphide bridges (non-reducing) Oligosaccarides non-cytosolic (added in lumen of golgi and ER) hydrophilic Jhyb Reducing conditions hydrophilia How to solubilise membrane proteins Detergent disrupts the lipid bilayer Protein enters solution as a protein-lipid detergent complex Lipid is also solubilised Reconstitute a membrane protein  How about if we want just the peripheral proteins? membrane > - Integral protein Y Peripheral Jeasier to remove holds the protein to ↳ target bonds that membrane the What forces/types of bonds hold peripheral proteins to the membrane? These noncovalent interactions may include ionic bonds, hydrophobic interactions, hydrogen bonds and van der Waals forces (dispersion attractions, dipole-dipole and dipole-induced dipole interactions). Integral > Peripheral > Cytosolia chardest) I easiest)  Extracting just the peripheral proteins Protocol Josied  A sample problem Yeast cells in culture were harvested by centrifugation and then resuspended in a physiological Hepes buffer. Cells were lysed by the addition of 0.45µm glass beads and vortexing six times for 20 seconds with 1 min incubations on ice between each burst. To remove unlysed cells, the crude yeast lysate was centrifuged at 1,000 g for 3 mins at 4°C. The crude yeast lysate was then incubated with a physiological buffer, 1% triton X-100, 1M NaCl or 100mM Na2CO3, pH 11. The lysate was then centrifuged at 175,000 g, and separated into supernatant (S) and pellet (P) fractions. Aliquots of each fraction were separated by SDS-PAGE and immunoblotted with affinity-purified antibodies to specifically detect the proteins Sec35p, Sed5p and PGK. Refer to figure below (next slide) Which of these proteins (Sec35p, Sed5p and PGK) is likely to be cytosolic and which is likely to be membrane associated? If you think any of these proteins are membrane associated, decide if they are peripheral or integral to the membrane. Explain your answer describing the purpose of each of the different treatments Refer to figure below Which of these proteins (Sec35p, Sed5p and PGK) is likely to be cytosolic and which is likely to be membrane associated? If you think any of these proteins are membrane associated, decide if they are peripheral or integral to the membrane. Explain your answer describing the purpose of each of the different treatments to look at last step of protocol = most accurate place > - supernatant. - cytosolic Peripheral vs Intergral Membrane Proteins Number of intergral membrane protein structures solved  Solving membrane protein structure Soluble proteins can be studied by “X-ray” crystallography => structure at 2-3Å resolution (see Membrane Protein Lectures -Megan Maher) Membrane proteins are difficult to study for a number of reasons. e 0 , Their surface is relatively hydrophobic and they can only be extracted from the cell membrane with detergents. They are also often flexible and unstable. This leads to challenges at all levels, including expression, solubilisation, purification, crystallisation, data collection and structure solution.  Can we predict if a protein is an integral or peripheral membrane protein based on sequence? Does this sequence code for an integral membrane protein? [ I LSTTGVAMHTSTSSSVTKSYISSQTNDTHKRDTYAA hydropathy plot. TPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEI TLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSP DTDVPLSSVEIENPETSDQ  Performing hydropathy plots to predict transmembrane regions The polarity (hydropathy) scale describes the change in free energy associated with transfer from a non-polar environment to an aqueous environment (kcal/mol). Phe +3.7 a negative change in free energy implies that the transfer will be favourable. Met +3.4 Empirically, it has been found that a cumulative (additive) value of Ile +3.1 +20kcal/mol for 20 consecutive residues indicates that a polypeptide Leu +2.8 segment is likely to be membrane spanning. Right are the polarity scale Val +2.6 values for each of the amino acids, where hydrophobic residues have Cys +2.0 positive values and hydrophilic residues have negative values. Trp +1.9 Ala +1.6 Thr +1.2 Gly +1.0 Ser +0.6 http://www.bioinformatics.org/sms2/one_to_three.html Pro -0.2 (converts 1 letter code to 3 letter code) Tyr -0.7 His -3.0 Gln -4.1 Asn -4.8 Glu -8.2 Lys -8.8 Asp -9.2 Arg -12.3 Glycophorin - single trans-membrane domain protein Yuydrophila 7 hydrophobic hydrophilia myscopic Integral attern Japropos- Juydrophi  Prediction of multi-transmembrane domain proteins Hydropathy plot predictions work well for single transmembrane segment proteins. Can they be used to predict multi-transmembrane spanning proteins? Bacteriorhodopsin – a known 7-transmembrane domain protein. It has some charged amino acids in the polar pore that acts as a H+ channel. CR protein &  Potential problems with hydropathy plots GPCR > - alternate peaks * transmembrane domains 000000 empty > - limitate Multi-transmembrane segment proteins are usually recognised as IMPs but the number of TM segments may not be correctly predicted. A Problems: a) some transmembrane segments may not be predicted (eg. amphipathic helices in ion channels) - common problem b) a peak in a hydropathy plot does not prove the existence of a transmembrane helix – need “experiments” to confirm What about the other class of membrane spanning proteins? hydropathy plot? Beta-barrel primary sequences What would the hydropathy plot look like? Hydropathy plot for porin Membranes and Cell Signalling - Lecture 4 Introduction to Cell Signalling https://www.youtube.com/watch?v=zme5 VcSYpCg Adrian Elcock  The crowded cytoplasm A cell is not a bag filled with water! Cellular interiors are 30–40% volume occupied by macromolecules of specific volume close to 1 ml g−. Some of the pathways controlling cell homeostasis Juxtapose complexity with crowding!  Major Concepts to Understand Signalling allows cells to perceive and respond to their environment. Main events in signalling. Types of signalling. What makes a good signal? Plasticity in signalling. What makes a good receptor? Chapter 15 Lodish – Signal Transduction and G-Protein-Coupled Receptors  There are diverse signalling events in living organisms Organisms interact with their environment They sense the extracellular environment via: Molecules dissolved in water or air that bind to cell surface receptors or enter through pores or transporters Extracellular solid substrates Mechanical interactions Monitoring light, temperature, pressure, movement etc They sense neighbouring cells via: Direct cell to cell junctions through which molecules are exchanged Exchange of diffusible molecules which bind to cell surface receptors or enter through pores or transporters Mechanical interactions  What happens when a signal arrives? Nucleus Gene Cytoplasm expression Receives a signal 1. Perception of a signal – usually dedicated proteins called receptors. 2. Transmission of the signal by the receptor into the cell. 3. Passing on the message to a series of Initial New proteins cellular components (transduction or (primary) Signal transduction signal cascade). signal 4. Arrival of signal at final destination in ↓ Receptor the cell. 5. Response of the cell. is nucleus usually Metabolism/cytoskeleton How do cells respond to environmental signals? membrane transduct/cascade signal J response  Types of signalling which can take place in an organism Class of signalling Range Comment Electrical Long Propagation of an electrical potential along a cell Endocrine Long Release and perception of hormones Paracrine Short Release and perception of extracellular signals Cell-cell contact Neighbouring cells Either by receptor mediated signalling or gap junction/plasmodesmata Autocrine Same cell Use of diffusible signals The types of cellular signalling  Intercellular communication What makes a good signal? ~ bloodstream 1. Be able to travel from site of manufacture to the target site relatively easily > - move/released 2. Made, mobilized and altered (turned off) relatively quickly 3. Specificity > to - target all Adrenaline and the “fight or flight” response After a threat is detected a sympathetic response is relayed from the hypothalamus to the adrenal glands. Acetylcholine released from pre-ganglionic fibres increases sodium conductance of the chromaffin cell membrane. Chromaffin cells are modified post-ganglionic neurons. The depolarization of the cell membrane leads to an influx of calcium through voltage-sensitive channels. Hormones (adrenaline and noradrenaline) stored in granules are released by the regulated secretory pathway into the blood stream import to transport honeome Jigand Release of Adrenaline to release adrenalive Exocytosis of granules from chromaffin cells Principle secretory products of chromaffin cells are the catecholamines adrenaline and noradrenaline (epinephrine and norepinephrine). About 5-6mg of catecholamines are stored in the adrenal medulla. Adrenaline is normally less than 10ng/l in healthy adults. Can increase 50-300 fold under stress. This increase occurs within seconds and effects occur within seconds..  how long it stays in the body Removal- of Adrenaline - Half-life of circulating adrenaline is less turned off within seconds than- 10 seconds In neuronal cytosol principally degraded by monoamine oxidase In endothelium, liver, heart and other tissues degraded by catecholamine-O-methyl -transferase The concentration of a molecule can be adjusted quickly if the half-life is short For example, two intracellular signalling molecules X and Y, both of which are normally maintained at a concentration of 1000 molecules per cell. Molecule Y is synthesized and degraded at a rate of 100 molecules per second, with each molecule having an average lifetime of 10 seconds. Molecule X has a turnover rate that is 10 times slower than that of Y: it is both synthesized and degraded at a rate of 10 molecules per second, so that each molecule has an average lifetime in the cell of 100 seconds. If a signal acting on the cell boosts the rates of synthesis of both X and Y tenfold without any change in the molecular lifetimes: at the end of 1 second the concentration of Y will have increased by nearly 900 molecules per cell (10 × 100 - 100), while the concentration of X will have increased by only 90 molecules per cell.  The concentration of a molecule can be adjusted quickly if the half-life is short - pland exte Jinhom Advenaline I effectsalived Numbers in blue = half-life The effects of adrenaline * * How can the same molecule cause different effects in different tissues? Samrealmolecule Adrenaline causes smooth muscle relaxation in the O airways but causes of Type contraction of the smooth receptor muscle that lines most arterioles present signal ↓neculies of mupter / same. to effect different downstream Specificity – the correct response in the correct cell – receptor dependent Diges What makes a good receptor? There are three crucial criteria: 1. The receptor has to have specificity – detecting only the molecule (or range of molecules) that the cell wishes to perceive. 2. The binding affinity must be such that it can detect the signalling molecule at the concentrations at which it is likely to be found in the vicinity of the cell. 3. The receptor must be able to transmit the message that the signalling molecule conveys to the cell – usually by modulation of the further components in a signalling cascade. ①Receptor-Ligand Specificity Example The structure of the β1 adrenergic receptor with agonist bound has been solved All four agonists bind in the catecholamine pocket in a virtually identical fashion dobutamine isoprenaline salbutamol carmoterol Warne et al. (2011) Nature 469; pp241-245 The secondary amine and β-hydroxyl groups shared by all the agonists (except for dobutamine, which lacks the β-hydroxyl); form potential hydrogen bonds with Asp 121 and Asn 329, whereas the hydrogen bond donor/acceptor group equivalent to the catecholamine meta-hydroxyl (m-OH) generally forms a hydrogen bond with Asn 310. In addition, all the agonists can form a hydrogen bond with Ser 211. [Polar and non-polar interactions 3 involved in agonist binding to β1-adrenergic receptor. - > between receptor and ligand Amino acid residues within 3.9A˚ of the ligands are depicted, with residues highlighted in blue making van der Waals contacts (blue rays) and residues highlighted in red making potential hydrogen bondswith favourable geometry (red dashed lines) or hydrogen bonds with unfavourable geometry (blue dashed lines). Examining Receptor Binding Sites 3) structure of receptor/binding site) must Small patches of amino acids are important for be complimentary to 3D structure of ligand specific binding between growth hormone and its receptor 3D structure was determined for the growth hormone – growth hormone receptor complex. 28aa in the hormone were in the binding interface with the receptor. These were analysed by alanine scanning – each one in turn was mutated to alanine. Substitution by alanine deletes all interactions made by atoms beyond the β carbon and should reveal the contribution to binding energy made by the removed portion of the side chain Clackson and Wells (1995) Science 267 8aa on the hormone contributed to 85% of binding energy. These 8 were far apart on the primary see Lodish 15-3 sequence but help form a hydrophobic pocket in the folded protein. Membranes and Cell Signalling - Lecture 5 Receptors – Basic Principles and Types What you need to know… How Receptor – ligand affinity relates to the magnitude of the physiological response The classes of receptors How conformational changes in receptors mediate signal transduction – learn how the R and R* structures of the adrenaline receptor were determined. How G-protein coupled receptors work How tyrosine kinase receptors work How intracellular receptors work  for quiz Receptor-Signal Binding Affinity > - Will come up You can view ligand (signal molecule) binding to a receptor as a simple reversible reaction koff > - X & kon ↳ koff is the rate constant for dissociation of the ligand from its receptor > forward reacts - kon is the rate constant for formation of the receptor-ligand complex from free ligand and receptor  At Equilibrium… Koff Kon Jchanistry = The rate of dissociation is equal to the rate of formation and can be described as a simple equilibrium-binding equation * or Kd = koff/kon O Kd is the dissociation constant and is a measure of the affinity of the receptor for its ligand. binds to - how well receptor ligand The lower koff is to kon the more stable the receptor-ligand complex is and the tighter the binding. The lower Kd is the tighter the binding. the binding higher kd , the looser the How can we experimentally determine the Kd?  LECSO) 7 i Performing Binding Assays we then look at 50% binding th th Specific binding = -B See Lodish et al 15.2, 6 7 Edition Mouse cell line incubated with increasing concentrations of 125I Epo – 4°C, 1 hr..7 3 Cells centrifuged to remove unbound Epo and amount of radioactivity bound to the cells is I ⑪ - measured. platen of specific binding Curve A represents specific high affinity binding to total no · of. receptors Epo receptors as well as low affinity binding to other 1. 75 molecules on the cell surface. Curve B is non-specific bining – control cell line not expressing Epo receptor 0. 11 nm Curve C is calculated difference between A and B. Aut Number of Epo binding sites per cell = 2200 - 37. fuoles per 100 cells 106 cells (3.7x10-15 moles x6.02x1023 molecules/mole/106 cells ) 3 7x10-. per ↳ 7x10-13) (6 02X102) receptors per 13.. 10% cells ↳> 2227400000 : 105 => in 1 2227 4 receptors cell > - 2200 =. receptors The Kd is the concentration required to bind 50% of the - receptors = 1050 => receptors/cell. 200 + 2 2 1100= 1100 Therefore Kd = 1.1 x 10-10M or 0.1nM -  A direct binding assay works well for ↑ high affinity receptors but… not use - Some ligands bind to their receptors with much lower affinity – adrenaline and other catecholamines If the Kd is greater than 10-7M ie. where koff is relatively large compared to kon then during the measurement some of the ligand will dissociate from the receptor. need toknowitaffinition t - In this case you need to use a competition assay  - low affinity Competition Assays To detect weak binding of a ligand to its receptor you can use a competition assay with another ligand which binds with high affinity (lower Kd). 100 * Inhibition of alprenolol binding (%) 80 isoprenaline adrenaline alprenolol IP A 60 use constant amount of radio-labelled alprenolol – Kd = 3 x 10-9 40 add increasing amount of competitor to displace alprenolol. 20 the concentration of competitor which inhibits binding by 50% approximates the Kd 0 10-8 10-6 10-4 * antagonist Competitor concentration (M)  More on Kd => ⑪ accept very important If basal levels response - > bad -> ↑ thanked , cells are in constant so % of response occurs : basal levels of lingand must be lowerand Signalling systems have evolved so that a rise in the level of signalling : cells sensitive to molecule gives a proportional physiological response. are any chances in the system Therefore the binding affinity (Kd) of a receptor for a ligand must be higher than the normal circulating levels of the ligand. (Basal levels) kd 1.4 x 10 M] Example the Kd for the hepatocyte insulin receptor for insulin is 2 -10 * And suppose the normal insulin concentration in blood is 5 x 10-12M. We can - S lower than calculate the fraction of insulin receptors with bound insulin at equilibrium. this of ? fractional get > - how to > - occupancy 1 4x10-0. Around 3.5% of receptors will be bound to insulin - " in body our response And a 5-fold increase in insulin will lead to a 5-fold increase in bound receptor => ↑ response in body What if the normal circulating insulin concentration was 1.4 x 10-10M? - what percentage of receptors are bound to insulin? - what if you increased insulin 5-fold again?  bound to how many receptors are tells > - you Determining Fractional Occupancy ligand. Fractional Occupancy (F.O.) = [RL]/[RT] = [RL]/[R] + [RL] Kd = [R] [L]/[RL], therefore [RL] = [R] [L]/Kd  What if the normal circulating insulin concentration was 1.4 x 10 -10M? a) - what percentage of receptors are bound to insulin? b) - what if you increased insulin 5-fold again? (Kd for the hepatocyte insulin receptor for insulin is 1.4 x 10-10M) 11 4x10-10) * Important to know. 50 % a) F 0 = = 0) 11 4x10-10) X = (1 4x10.. -. +. 4x10-10) -(1 5.