Membrane Phases Flashcards
Lipid Raft
Definition
- the fluid membrane contains regions, or domains, of a less fluid, more stable phase in which some proteins are located, termed lipid rafts
- sub-domains in lipid bilayers rich in sphingomyelin, cholesterol and saturated lipids
Lipid Raft
Function
- sites in the membrane where functional proteins tend to be located
- however some membrane proteins seem to be specifically excluded from the rafts
- biological function (and even existence) of lipid rafts in cell membranes is still a very controversial topic
Membrane Melting
Definition
-melting processes are order transitions between two planar membrane phases or states that display different physical properties
Lipid Chains at Low vs High Temperatures
- at low temperatures, lipid chains are ordered into an all-trans configuration
- at high temperatures these chains are disordered due to rotations around the C-C bonds within the lipid chains
- the membranes are in an ordered ‘gel’ phase at low temperatures while they are in a disordered ‘fluid phase’ at high temperatures
Heat Exchange During Membrane Melting
- in the chain melting process, the molecules absorb heat (enthalpy) and the entropy increases due to the increase in the number of possible chain configurations
- the melting process is cooperative, the lipids do not melt independently of each other
- absorption of heat is typically monitored by measuring the heat capacity, cp=(dH/dT)|p
- at melting temperature Tm, is displays a pronounced maximum
Lipid Membranes
Gel Phase
- high density
- highly ordered, extended chains
- low mobility
- area per molecule ~0.40nm²
- depth of bilayer ~5-5.5 nm
- stiff, solid
- long range translational and positional order
- some rotational modes allowed
Lipid Membranes
Fluid Phase
- lower density
- well ordered, especially at chain ends
- higher mobility
- area per molecule ~0.5-0.67 nm²
- depth of bilayer ~4-5nm
Lipid Phase in Real Cells
- the inside of a cell is not clear free flowing ‘bulk’ water, rather a very complex mix of proteins, ions, nucleic acids and sugars which also interact with the water as well as the bilayer
- little free water exists
- internal phase structure of the lamellar bilayer is probably affected
- the existence of stable raft domains in cells is still a controversial area
- however, single particle tracking has recently indicated that zones of high stability exist
Head-Group vs Acyl Chain Interactions
- head-group interactions, they are hydrated / ionised species: steric interactions, dipolar and electrostatic interactions, H-bonding
- acyl chains interactions, they are homogeneous regions of hydrocarbon chains: van der Waals attractive and steric repulsive forces
Single Component vs. Binary Phase Diagram
- single component: graph showing the phases of a substance as they relate to temperature and pressure
- binary: diagram showing the phases in a system of two pure components, one variable, T or P, must be fixed in order to render the diagram in 2D
Lever Rule
-phase separation to:
a/(a+b) quantity of gel phase, composition B
b/(a+b) quantity of fluid phase, composition A
-if one segment is significantly shorter than the other then you must be near the phase boundary
Direct Evidence for Phases in Model Sustems
- creating giant unilamellar veciles using electroformation from two different phospholipids, DPPC and DOPC
- using fluorescence microscopy, gel-liquid coexistence is observable
- if both co-existing phases are fluid then domains will coalesce eventually merging into only two domains, one of each phase, making up the entire vesicle
Is it possible for two liquid phases Lo and Ld to coexist?
- in a binary system, no
- in a ternary system, i.e. add a third element, with cholesterol a phase transition between high and low temperatures is easy to observe
Effect of Cholesterol on Lipid Phase Behaviour
- cholesterol induces ordering of lipid acyl tails but retains the liquid-like structure in the plane of the bilayer, a new liquid-ordered phase
- so we now have:
- -solid gel phase
- -liquid ordered phase
- -liquid disordered phase
Liquid Ordered Phase
- slightly lower density than gel phase, ~0.42-0.5nm²
- less order
- not closely packed
- 5.5nm depth
- lower elastic modulus
- high viscosity liquid
- short range translational order
- no positional order
- highly viscous
Liquid Disordered Phase
- low density, 0.5-0.75nm²
- no order
- not closely packed
- 4.5nm depth
- much lower elastic modulus
- low viscosity liquid
- liquid, no translational or positional order
- low viscosity
Adding Cholesterol to the Solid Phase
- cholesterol acts to moderate the structural order in both solid and liquid phases
- it inserts into the closely packed saturated chains of the solid phase
- breaking down perfect order and pushing chains apart
- this weakens van der Waals interactions between adjacent chains enough to break down long range translational order
- it becomes an ordered liquid phase, Lo
Adding Cholesterol to the Liquid, Lα/Ld, Phase
- cholesterol acts to moderate the structural order in both solid and liquid phases
- the planar cholesterol molecule brings a degree of order to the highly disordered hydrophobic core
- the properties of both phases converge resulting in liquid-liquid coexistence
Ternary Phase Diagram of a Synthetic Model Cell Membrane
- triangle, each component of the system at a point on the triangle
- percentage is measured from 100% in the corner to 0% on the opposite edge for each component
- fixing temperature and pressure allows the diagram to be represented as a flat 2D triangle
AFM on Supported Bilayers
- evidence for phases in model systems
- measures differences in height 0.2-1.4nm due to a difference in density between the phases
- in Ld phase, hydrophobic acyl tails are disordered so each lipid molecule occupies more area laterally and is therefore thinner in the vertical plane
- the more ordered Lo or gel domains occupy less area per lipid molecule in the membrane and hence are deeper and protrude from the Ld phase
What are the driving forces for phase separation into Lo and Ld phases, they are both liquid, why do they not mix?
- the shape factor, if the two components have different shape factors
- head group hydrogen bonding between cholesterol and sphingomyelins
Is a binary or ternary lipid system at equilibrium when in the process of separating, or even long after the structure has seemingly stabilised?
- often NO
- the derivation behing phase diagrams assumes that phase boundaries do not contribute to the free energy but actually, the interface between two boundaries always carries a free energy contribution
- in a 2D membrane if domains of finite size exist, these contributions can be quite large
- sometimes the domains can become kinetically trapped freezing them in an equilibrium state
- this could be due to high membrane protein content, hindering domains from coalescing or by proximity of the cytoskeleton
Critical Points
- near the critical point, the physical / chemical difference between the phases is very small
- tension ar boudaries is therefore reduced and phase domains become more fractal in appearance
- at the critical point, the phases are either in the phase co-existence region or a single phase somewhere between Lo and Ld
- the system fluctuates from one to the other depending on small imbalances in temperature, up to microns in scale
- as composition moves away from critical point (or T increases) the size of fluctuations reduces to 100s then 10s of nm, this is the regime that cell membranes could possibly exist within
Spinodal Decomposition
- if the temperature is reduced and the mixture moves from a single phase melt into a two-phase region and the boundary is crossed in the vicinity of the critical point (where the line tensions are low and boundary penalty is minimal) then the structure that forms will be spinodal in morphology (long, convoluted maze-like boundaries)
- over time this structure will develop as the larger domains absorb the smaller ones