Liposomes, micelles, membranes

Transcription

1 Liposomes, micelles, membranes Architectures of phospholipids Membrane proteins 1

2 NB Queste diapositive sono state preparate per il corso di Biofisica tenuto dal Dr. Attilio V. Vargiu presso il Dipartimento di Fisica nell A.A. 2014/2015 Non sostituiscono il materiale didattico consigliato a piè del programma. 2

3 References Books and other sources Physical Biology of the Cell, R. Phillips et al., 2th ed., Chap. 11 Biochemistry (5 th ed.), Berg et al., Chap. 12 Movies Exercise 3

4 Architectures of amphipathic lipids Amphipathic nature of phospholipids leads to two major architectures in polar (aqueous) solvents: Micelles (one layer exposed to solvent) Membranes (including vesicles/liposomes, two faces leaflets exposed to solvent) 4

5 Micelles or membranes? Most phospho-/glyco-lipids prefer bilayer than micelle architectures. Reason is the bulkiness of two fatty acid chains, which does not allow fit of hydrophobic tails to the interior of micelle. Fatty acids salts (e.g. soaps) containing only one chain readily form micelles. Other phases, such as hexagonal (H II ), cubic, and more complex also found. 5

6 Micelles or membranes? Most phospho-/glyco-lipids prefer bilayer than micelle architectures. Reason is the bulkiness of two fatty acid chains, which does not allow fit of hydrophobic tails to the interior of micelle. Fatty acids salts (e.g. soaps) containing only one chain readily form micelles. Other Micelles phases, are such limited as hexagonal in size, (Hcannot II ), cubic, extend and more beyond complex ~20 also nm. found. Bilayers can have macroscopic dimensions, up to ~10 6 nm. One reason for the preference of the latter in organisms. 6

7 Micelles or membranes? All of these phases are formed by different natural lipids under variable conditions 7

8 Self-assembly of amphipathic lipids Lipid bilayers (as well as micelles) form spontaneously in polar solvents (self-assembly). Process mainly driven by optimization of hydrophobic interactions among lipid tails. Electrostatic and H-bonds interactions between polar heads and solvents contribute to stabilization. Membranes are cooperative structures: Tend to be extensive. Tend to close on themselves (forming vesicles/compartments) as to avoid hydrophobic edges exposed to solvent. Are self-sealing (holes thermodynamically unstable). 8

9 Self-assembly of micelles 9

10 Self-assembly of bilayers 10

11 Key features of membranes Set of common features. All membranes: Are sheet-like structures of 6-10 nm in thickness (3-4 nm hydrophobic core). Contain lipids (phospho-, glyco-, sphingo-lipids, sterols), proteins and carbohydrates. Have amphipathic lipids that self-assemble in aqueous environment, are held together by cooperative non-covalent interactions, and function as a barrier to the flux of polar molecules. Are asymmetric: inner and outer leaflet (faces) always differ. Are 2D solutions of oriented proteins and lipids: in general they can translate into the membrane plane, but rotations across the membrane are infrequent. Most are electrically polarized, with negative inside (typically ~ -60 mv). Different membranes have their peculiarities (e.g. the plasma membrane is peculiar with respect to the membranes of organelles) and associated proteins can be also highly specific of that membrane. 11

12 Composition of membranes Asymmetry is a key feature of biological membranes (e.g. plasma) 12

13 Composition of membranes Highly variable lipids mixtures, depending on function. 13

14 Composition of membranes Highly variable protein/lipid mass ratio (from 1:5 to 4:1) depending on the role of specific membranes: - Myelin (membrane insulating certain nervous fibers) has low content of proteins (15-30%) because pure lipids are good insulators. - Plasma membranes contain ~50% of proteins (pumps, channels, receptors, enzymes). - Mitochondrial and chloroplasts internal membranes transduce energy, need proteins (~75% are proteins). Type of proteins also vary with specific function of membrane. 14

15 Functions of biological membranes Biological membranes define the inside and outside of cell and its organelles in eukaryotic cells Prevent leaking of intracellular material and penetration of unwanted compounds. Selective permeability through membrane proteins (pumps and channels) floating in lipids sea. Additional functions associated to other membrane proteins: energy storage, information transduction, receptor functions, enzymatic activity, etc 15

16 Plasma membranes in some cells Plasma membranes are present in all kinds of cells. Differ in Gram- and Gram+ bacteria. 16

17 Plasma membranes in some cells Gram- have two membranes separated by a cell wall (made of proteins, peptides, and carbohydrates) lying in between. Outer membrane quite permeable to small molecules owing to the presence of porins. Gram+ and archaea have only a single membrane surrounded by cell wall. 17

18 Plasma membranes in some cells Differ in prokaryotic and eukaryotic cells: Eukaryotic cells (with exception of plant cells) do not have cell walls, and their cell membranes consist of a single lipid bilayer. In plant cells, the cell wall is on the outside of the plasma membrane. 18

19 Plasma membrane FM model Fluid mosaic model: Fluid because membranes are 2D solutions of oriented lipids and proteins Mosaic because they are a composition of different lipids and proteins 19

20 Plasma membrane FM model Fluid mosaic model: Fluid because membranes are 2D solutions of oriented lipids and proteins Mosaic because they are a composition of different lipids and proteins 20

21 Membrane proteins classification Most processes carried out by membranes are mediated by proteins. Classification based on the strength of interaction with lipid bilayer, reflecting degree of burial into the membrane 21

22 Membrane proteins classification Integral membrane proteins (yellow) interact strongly with hydrocarbon chains of lipids: Can be removed by agents competing with hydrophobic interactions, such as detergents or organic solvents (e.g. hexane, toluene). Most integral proteins span the entire lipid bilayer. 22

23 Membrane proteins classification Peripheral membrane proteins (azur) bound to hydrophilic heads of lipids by electrostatic interactions and/or H-bonds: Can be dissociated by mild means, such as highly concentrated ionic solvents (e.g. 1 M NaCl) Some further stabilized by covalent anchor to hydrophobic chains 23

24 Membrane proteins topologies α-helices and β-barrels (β-sheet curled to form a hollow cylinder) preferred structures assumed by integral membrane proteins (embedded in lipid bilayer). Intra-chain H-bonds in α-helices and β-sheets thermodynamically favored in hydrophobic environment, where no polar competitor molecules are present. Porin Bacteriorhodopsin Prostaglandin synthase Biochemistry, J. Berg et al., 5 th ed.,

25 Membrane proteins topologies α-helices usually span the entire bilayer perpendicularly to its plane (~20 aa in length). Most residues non-polar, distribution optimized to enhance stability: hydrophobic/apolar aa points towards lipid chains Polar/charged residues create functional networks Bacteriorhodopsin Biochemistry, J. Berg et al., 5 th ed.,

26 Membrane proteins topologies Propensity of α-helices to insert into hydrophobic bilayers can be estimated from hydropathy (HP) index, i.e. the (per-residue) free energy of transfer of mono-aa α-helices from hydrophobic environment to water: Empirically, HP 20 kcal/mol for 20 aa sequence to be found as TM α-helix. In a HP plot, peak should correspond to TM helix. Biochemistry, J. Berg et al., 5 th ed.,

27 Membrane proteins topologies PHE, MET, ILE highest value of transfer free energy favourable TM helix. ARG, ASP, LYS, GLU most negative values of transfer free energy unfavourable TM helix. However Peak in HP index does not imply a sequence will give a trans-membrane (TM) helix! Only valid for α-helices, not applicable to β-barrel! HP index of a porin (β-barrel) Biochemistry, J. Berg et al., 5 th ed.,

28 Membrane proteins topologies β-barrels formed by antiparallel curled β-sheets. Outside residues mostly nonpolar, inside (water-filled) hydrophilic, realized through alternation of hydrophobic and hydrophilic aa along each strand. H-bond Biochemistry, J. Berg et al., 5 th ed.,

29 Membrane proteins topologies Some proteins are attached to bilayer surface and have hydrophobic anchors protruding into the lipid bilayer. They are integral proteins (very stable) although not membrane-spanning. Prostaglandin synthase Biochemistry, J. Berg et al., 5 th ed.,

30 Membrane proteins topologies Some proteins covalently attached membrane lipids by means of attached hydrophobic groups. Biochemistry, J. Berg et al., 5 th ed.,

31 Membranes are fluids Membranes are highly flexible and dynamic objects Lipid dynamics include: 1. Chain conformational transitions and defect motions 2. Rotational diffusion around long axes 3. In-plane lateral diffusion (local and jumps) 4. Heads motions 5. Out-of-plane vibrations 6. Collective undulations 31

32 Membranes are fluids Lipid and proteins lateral diffusion Lateral diffusion obeys Einstein rule: Diffusion coefficient of various (fluid) membranes ~ 1µm 2 s -1 (1/100 of water). <s> ~ 2 µm in 1s a lipid can go from one end to the other of a bacterium in 1 s! Proteins lateral mobilities can vary from very low to lipid-like values: µm 2 s -1 for fibronectin, a glycoprotein anchoring cells to extracellular matrix (ECM) through linkages with integrin, in turn linked to cytoskeleton through interaction with actin fibers µm 2 s -1 for bacteriorhodopsin (fast movement essential for efficient signaling). s = 4Dt 32

33 Membranes are fluids Lipid and proteins transverse diffusion (flip-flop) At opposite to lateral diffusion, flip-flop of lipids occur very rarely (once in several hours), and that of proteins has never been observed. Very fast Very rare 33

34 Membranes are fluids Lipid and proteins transverse diffusion (flip-flop) Transverse diffusion important mechanism involved in building-up new membranes from existing patches (in eukaryotes plasma membranes are build from ER ones). Flippase enzymes present in ER membrane of eukaryotes and in plasma membrane of prokaryotes transfer some of newly formed phospholipids to opposite monolayer. Flippases do not bind all phospholipids equally well, thus two membrane leaflets end up having different distributions of phospholipid (asymmetric!) Membrane building in eukaryots Flippase action 34

35 Membranes are fluids Fluidity depends on composition and presence of non-fatty-acids lipids Membranes can exist in different phases: two extreme ones are gel (solid-like) and fluid liquid. Gel to liquid transition can be caused by conformational changes in packing of phospholipids acyl chains from trans to gauche conformations. Membrane-mediated processed depends crucially on fluidity! Gel phase Liquid phase 35

36 Membranes are fluids Fluidity depends on composition and presence of non-fatty-acids lipids Transition from gel-like to fluid state occurs abruptly at melting temperature T m Biochemistry, J. Berg et al., 5 th ed., 2001 Additional phases also found (ripple and different fluid-like ones) Transitions among different phases depend on many factors including temperature, ph, ionic strength, pressure and membrane composition (e.g. saturated/ unsaturated lipids ratio and content of molecules such as cholesterol) 36

37 Membranes are fluids Fluidity depends on composition and presence of non-fatty-acids lipids Some phospholipids (e.g. PC) undergo two steps transition. First transition at few degrees below T m, due to changes in vicinity of polar heads (e.g. increased interaction with solvent). Two step transition consequence of simultaneous changes in lipid order and membrane curvature lipids that exhibit a pre-transition temperature display additional lamellar phase (ripple). Ripple phase partially disordered, displays periodic one dimensional undulations on surface of lipid bilayer (arising from periodic arrangements of linear ordered and disordered lipid domains). 37

38 Membranes are fluids Fluidity depends on composition and presence of non-fatty-acids lipids T m depends on length of fatty acyl chains and on their degree of unsaturation: cis double bond induces bending, interfering with ordered packing T m is lowered. Long hydrocarbons interact more strongly than do short ones. Each additional CH 2 group adds ~0.5 kcal/mol to free energy of interaction of two adjacent chains. Biochemistry, J. Berg et al., 5 th ed.,

39 Membranes are fluids Membrane fluidity regulation in bacteria Bacteria regulate fluidity of their membranes by varying number of double bonds and length of fatty acyl chains. Ratio of unsaturated to saturated fatty acyl chains in E. coli membrane increases from 0.35 to 1.6 as growth T is lowered from 42 C to 27 C. Decrease in the proportion of saturated residues prevents membrane from becoming too rigid. 39

40 Membranes are fluids Fluidity regulation in animal eukaryotic cells Cholesterol is key regulator of fluidity, allowing bilayers to adopt extra lamellar phase (liquid-ordered), intermediate between gel and fluid. Opposite effects depending on T being below or above T m : - Inserting cholesterol in gel phase disrupts packing, reducing ordering of lipid chains. - In liquid phase rigid hydrophobic moiety of cholesterol intercalates between lipid chains, favoring trans conformation. In liquid-ordered phase: - Lateral and rotational diffusion similar to liquid-disordered phase. - Conformational order similar to solid-ordered. 40

41 Membranes are fluids Ordering effect of cholesterol in DPPC bilayer 41

42 Membrane flexibility: a model Flexibility and hetereogenity of membrane particles (different kinds of lipids and proteins) reflect on macroscopic shape and rigidity of bilayer Membrane geometry deformations include stretching, bending, shrinking or expansion, shearing. The free energy cost associated to membrane deformation can be roughly estimated by means of elastic continuum theory. 42

43 Membrane flexibility: a model Flexibility and hetereogenity of membrane particles (different kinds of lipids and proteins) reflect on macroscopic shape and rigidity of bilayer Stretching can be described by a simple area function Δa(x 1,x 2 ), function of the position on the plane (reflects eventual inhomogeneity in the membrane). Compression and expansion can be described by simple thickness function w(x 1,x 2 ). Shearing can be describe through an angle θ formed by two sides of surface elements. 43

44 Membrane flexibility: a model Flexibility and hetereogenity of membrane particles (different kinds of lipids and proteins) reflect on macroscopic shape and rigidity of bilayer Bending can be described by a simple height function h(x 1,x 2 ) calculated from a reference plane. For practical purposes, best plane is the tangent to point (x 1 0,x2 0 ) of interest quadratic expansion of h using Taylor series in local reference frame: h(x 1, x 2 ) = 2 i, j=1 κ ij x i x j 44

45 Membrane flexibility: a model Flexibility and hetereogenity of membrane particles (different kinds of lipids and proteins) reflect on macroscopic shape and rigidity of bilayer Bending can be described by a simple height function h(x 1,x 2 ) calculated from a reference plane. For practical purposes, best plane is the tangent to point (x 1 0,x2 0 ) of interest quadratic expansion of h using Taylor series in local reference frame: h(x 1, x 2 ) = Can be done for every point of even complex surfaces, but sub-dividing surface and introducing many local reference frames. 2 i, j=1 κ ij x i x j 45

46 Membrane flexibility: a model Flexibility and hetereogenity of membrane particles (different kinds of lipids and proteins) reflect on macroscopic shape and rigidity of bilayer Curvature (bending) of membrane can be calculated from the knowledge of h, namely from the 2 nd order derivatives which account for surface curvature matrix κ:! κ = # " κ 11 κ 12 κ 21 κ 22 $ & % 2 h κ ij = x i x j Diagonalization of κ corresponds to identify reference frame giving two principal axes of curvature. 46

47 Membrane flexibility: a model ΔG of membrane deformation as functional of shape functions Δa, h, w, θ Area stretching modeled by means of spring network. Deformation from reference area associated to increase in free energy: G stretch = K a 2 Δa( x 1, x 2 ) a 0 If relative areal strain Δa/a constant: " $ # K a stretch modulus: k B T/nm 2 ~ mn/m % ' & 2 da G stretch = K a 2 Δa 2 a 0 47

48 Membrane flexibility: a model ΔG of membrane deformation as functional of shape functions Δa, h, w, θ Bending (deformation from planar surface) modeled by means of spring network. Free energy as a functional of principal curvatures: G bend = K b 2 ( ) +κ 2 ( x 1, x 2 ) da!" κ 1 x 1, x # 2 $ 2 K b bending rigidity: k B T 48

49 Membrane flexibility: a model ΔG of membrane deformation as functional of shape functions Δa, h, w, θ Change in thickness modeled by means of elastic network. Deformation from reference thickness associated to increase in free energy: G thickness = K t 2 ( ) w 0 a0 " da w x, x 1 2 $ # % ' & 2 K t stiffness modulus: 60 k B T/nm 2. Shear deformation also quadratic in θ. 49

50 Vesicles Vesicles are closed spherical shells of lipids that can be used as tools to investigate elastic properties of membranes Two main experiments Micropipette aspiration Membrane pulling 50

51 Vesicles Micropipette aspiration Small pipette (~ 10 µm diameter d) used as a solution probe to grab vesicles. Pressure difference Δp between micropipette and solution allows suction that deforms the vesicle membrane. Knowledge of Δp and geometric parameters R v, R 1 (~d/2) and l allows determination of surface tension τ and of moduli K a and K b. Δp Δp in Laplace-Young relation gives the pressure needed to maintain sphere of given radius: Δp out = 2τ R V Δp in = 2τ R 1 Δp out Since: We have: Δp = Δp in Δp out τ = Δp 2 R 1 ( R V ) 1 R 1 51

52 τ = Δp 2 R 1 ( R V ) 1 R 1 Vesicles Micropipette aspiration For each Δp measure geometric parameters and calculate τ From τ calculate moduli K a and K b. E.g. K a calculated recalling that τ corresponds to ΔG per surface area: Δa determined from expt. images, or approximated (assuming in particular that R v does not vary upon suction) by: τ = K a Δa a 0 Δa = 2π R 1 l + 2π R 1 2 a 0 = 4π R V 2 τ = K a Δa a 0 = K a R 1 ( ) 2R V l R 1 52

53 τ = K a Δa a 0 = K a R 1 ( ) 2R V l R 1 Slope of τ vs. Δa/a 0 gives the stretch modulus K a Vesicles Micropipette aspiration Typical values for pure phospholipid bilayers ~250 mn/m 53

54 Vesicles Micropipette aspiration Two main limitations: Formula linking tension to stretch modulus ignore thermal fluctuations in the membrane (entropic effect). This is valid at high enough tensions ironing out spontaneous undulations. Stress-stretch relationship in entropic regime allows determination of bending constant K b. Values obtained for ideal single-lipid membranes, not accounting lipid heterogeneity, membrane proteins and intra- and extra-cellular connections. 54

55 Vesicles Membrane pulling Closely mimic real world situation, where formation of tubules (e.g. in ER and trans- Golgi network) occurs driven by forces different than those arising from Δp. 55

56 Vesicles Membrane pulling Closely mimic real world situation, where formation of tubules (e.g. in ER and trans- Golgi network) occurs driven by forces different than those arising from Δp. Experiments with optical tweezers show that applying a force to a single point of the membrane results in tether formation. The forces are in the range 5-20 pn for a variety of different membranes. 56

57 Vesicles Membrane pulling Free energy of vesicle+tether system can be written as sum of four terms: G bend = 8πK + b Vesicle G stretch = K a 2 πk L b r Tether body ( a a 0 ) 2 a 0 + 4πK b Tether end # G pv = Δp 4 3 π & % R3 + r 2 π L( $ ' G load = fl 57

58 Vesicles Membrane pulling Minimizing the sum of the free energy terms with respect to geometric variables r, L and R gives a relation between the applied force and the tension and constant K b f = 2π 2K b τ 58