Outline

1. Influence of Cholesterol on the Bilayer Properties ofMonounsaturated Phosphatidylcholine Unilamellar VesiclesNorbert KučerkaNSERC Visiting Fellow in CNBC – NRC

2. Outline • Significance of the Cholesterol • Small-Angle Neutron-Scattering • DOPC Bilayers • Short vs. Long Chain Bilayers • Conclusions

3. Motivationand Expectation • Function of membrane proteins depends on the membrane’s state and lipid composition • Addition of the cholesterol modulates protein activity • Addition of the cholesterol increases acyl chain order • Addition of the cholesterol affects membrane (hydrophobic) thickness • Cholesterol’s influence depends on the chain length, saturation level, and thermodynamic phase of lipid bilayer • Cholesterol increases order and thickness of saturated lipids, while its effect on unsaturated lipids is not clear

4. SANS Bilayer structure was determined using a model with the details that are appropriate for a low-resolution SANS. It provided the bilayer’s thickness, per lipid area and hydration level. Experimental SANS data obtained from ULVs dispersed in D2O. DOPC (diC18:1PC) bilayers containing 0, 17, 29, 38, and 45 mol % of cholesterol. N. Kučerka, J. F. Nagle, S. E. Feller and P. Balgavý: Models to analyze SANS from ULVs. Phys Rev E 69 (2004)

5. SANSDOPC Bilayers One dimensional profiles of the total scattering length density (SLD). Bilayer thicknessincreases with an increase of cholesterol concentration.

6. DOPC Bilayers • Area per lipid • Total thickness • Hydrocarbon region thicknessand • Hydration level • exhibit a quadratic dependence as a functionof cholesterol concentration. N. Kučerka, J. Pencer, M.-P. Nieh, and J. Katsaras: Influence of Cholesterol on the Bilayer Properties of Monounsaturated PC ULVs. accepted by EPJ E (2007)

7. Cholesterol as a spacer Addition of the cholesterol causes: • thickening of the hydrocarbon region (by increasing the order of acyl chains) • increased hydration of lipid headgroups (by increasing the separation between lipid headgroups) water  Cholesterol acts as a spacer molecule

8. diC22:1PC diC14:1PC DOPC cholesterol Short vs. Long Chain Bilayer Molecular structures adapted from Avanti Polar Lipids, inc (www.avantilipids.com)

9. Short vs. Long Chain Bilayer • Area per lipid • Total thickness • Hydrocarbon region thicknessand • Hydration level • increases as a functionof cholesterol concentration for short- chain bilayers. both and long- The striking similarities are suggestive of the sameinfluence of cholesterol. N. Kučerka, J. Pencer, M.-P. Nieh, and J. Katsaras: Influence of Cholesterol on the Bilayer Properties of Monounsaturated PC ULVs. accepted by EPJ E (2007)

10. Conclusions • Addition of the cholesterol resulted in a monotonic changes in DOPC bilayer structure. Cholesterol acted as a spacer molecule. • Addition of the cholesterol induced similar changes in bilayers made of short- and long- chain monounsaturated lipids. • This was an unexpected result for the long-chain lipid bilayers. • The ordering effect of cholesteroldominates over the bilayer’s ability to reduce the hydrophobic mismatch in the thick bilayers.

11. Acknowledgement • Jeremy Pencer • John Katsaras • Mu-Ping Nieh • CNBC – NRC, Chalk River, ON • Jonathan Sachs • University of Minnesota, Minneapolis, MN