Effects of Cholesterol on Membranes: Physical Properties Removes gel to liquid crystal phase transition New “intermediate” phase called liquid ordered - ordering of the membrane lipids due to condensation (closer packing, less trans-gauche isomerizations) Cholesterol “thickens” bilayers with 16 C or less Cholesterol “thins” bilayers with 18 C or more Modulation of physical properties and minimizing permeability to small molecules

Suppression of phase transition to LC state NMR, EPR, fluorescence, DSC

FRAP : Flourescence Recovery After Photobleaching Used to determine viscosity: relevant to effects of cholesterol, lateral phase separation and RAFTS

Permeability: Hypothesis: gauche-Trans isomerizations lead to small gaps or holes of “free volume” space. The idea is that the constant movement of the bilayer will make a series of connected holes to allow small molecules (smaller than glucose) to pass. Cholesterol reduces the number of gauche-trans isomerizations by ordering the hydrocarbons, thus limiting the permeability.

Other interesting facts: cholesterol rotates along it’s long axis with a correlation time ~ 100ps phospholipids rotate with longer correlation times ~ 10ns. Therefor, there are no long lived complexes between phospholipids and sterol Increased elastic stiffness -allows membranes to get bigger and withstand stress, ie erythrocyte membrane (45-50% cholesterol) increased viscosity -changes the diffusion rate of membrane proteins, can also effect function of membrane proteins: Na+K+ATPase and growth, rhodospsin etc Cholesterol Movement Between Membranes: collision mediated, partitioning although cholesterol has low solubility in water (sub nM), it is believed that cholesterol will partition in and out of the membrane (like a detergent or fatty acid) and can transfer between membranes via this mechanism

Cholesterol Location In Cells: Plasma membrane : mol% ER : 10-12% inner mitochondrial < 10% Cholesterol is also heterogeneously distributed within given membranes/organelles. Question becomes: what is controlling separation and trafficing of cholesterol? Physical properties of the membrane? Cholesterol prefers membranes of PC over PE, hydrophobic effect as discussed early for PE bilayers PC PE

Common Sterol Structure Question for exam: why doesn’t lanosterol or epicholesterol have the same effect on the structural properties of phospholipid bilayers? 18 steps biologically

Cholesterol Modulates Membrane Structure, dynamics and or function through three major molecular mechanisms: Membrane protein function via sterol-protein interactions internal properties of the bilayer and cell membrane (hence can also affect protein function) alters lateral distribution of components in cell membranes (raft formation) Mammalian cells require cholesterol for growth mycoplasma mycoides also require cholesterol S. cerevisiae (yeast) require ergosterol Cannot substitute cholesterol for ergosterol in various cells, therefor there must be some specific protein-sterol interaction that can disctriminate

Studies on Na + K + ATPase -responsible for maintaining the Na+ and K+ gradients, single greatest user of ATP in many cells Found in plasma membrane, hightened activity with higher concentrations of cholesterol. Extrapolation to zero cholesterol from experiments indicates enzyme would be inactive if the plasma membrane had no cholesterol

Other proteins affected by cholesterol: Na+Ca2+ exchange ATP-ADP exchange glutamate transport GABA transport mediated lactate transport acetylcholine receptor galanine binding to galanine receptor Specific binding to protein binding to protein in the bilayer (annulus of sterol) modification of bilayer properties Ergosterol