Insights into lipid structure and dynamics: some neutron scattering case studies

1. Insights into lipid structure and dynamics: some neutron scattering case studies Paul Butler CHEG 867 SCATTERING METHODS

2. What can you study with neutrons? Shear thickening fluids Superconductivity Marbles Geological formations Polymer composites Self assembled systems Explosives Protein complexes Magnetic nanoparticles Hydrogen storage materials Pressure vessel steels Monoclonal Antibodies Colloid Physics Etc. Center for Neutron Sciences

3. When is it useful? Not the best Survey instrument – not very good for unknowns. But … can be very powerful tools, particularly when using deuteration, for quantitatively answering questions about structure (size, shape, orientation) or dynamics: • Pose the question clearly (propose hypothesis) • Carefully design experiment • Careful analysis Center for Neutron Sciences

4. Neutrons don’t lie (tales from the crypt) Wrong solvent Wrong cell materials Unstable sample Impurities Quality of sample (monodisperse, spheres, etc.) Center for Neutron Sciences

5. Insights into lipid structure and dynamics Self assembled system of double tail amphiphiles. Head groups can be ionic or not and tails can be saturated or not. Saturated lipids have gel to liquid transitions Mixed lipid systems and the formation (or not) of pores -structure Cholesterol exchange - kinetics Membrane fluctuations - dynamics Center for Neutron Sciences

6. Insights into lipid structure and dynamics Mixed lipids (bicelle) and pores? bilayers DMPC 14 C chain Bilayer+MicelleBicelle DHPC 6 C chain micelles Center for Neutron Sciences

7. Insights into lipid structure and dynamics Mixed lipids (bicelle) and pores? Mixing/Segregation confirmation Center for Neutron Sciences

8. Insights into lipid structure and dynamics Lamellar MLV T(°C) Worm like micelles?? ~ Tm Bicelle q Mixed lipids (bicelle) and pores? 50C Center for Neutron Sciences

9. Insights into lipid structure and dynamics Mixed lipids (bicelle) and pores? T = 50ºC T = 15ºC h/d DMPC T = 50ºC DHPC Matched solvent h/d DMPC Center for Neutron Sciences Matched solvent DHPC

10. Insights into lipid structure and dynamics Mixed lipids (bicelle) and pores? Match point • For this experiment: • Match point using mixed h/d lipid to match tail and head • Match mixture to D2O/H2O … at 50C!! (76% D2O) Center for Neutron Sciences

11. Insights into lipid structure and dynamics Mixed lipids (bicelle) and pores? Using equation for effective ration of short to long and measured SLD, 94% ± 6% of DHPC in vesicle is evenly distributed in sphere. Center for Neutron Sciences

12. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Inside a Cell To ER From food Mitocondria Endoplasmic reticulum (ER) To PM Late Endosome To liver Plasma Membrane (PM) Early Endosome Golgi 0.05 - 1.0% Cholesterol content 60-70% Center for Neutron Sciences

13. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport In model membrane Rodrigueza et. al, Biochemistry (34),1995,6208-17 Lipids Lipids+chol Days Hours Flip Exchange Minutes Isolation Seconds Leventis et. al BJ (81), 2001, 2257–67 Center for Neutron Sciences

14. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport H2O D2O Measuring Cholesterol Exchange Measuring Lipid Exchange Cholesterol + D-lipids D-lipids H-lipids D-lipids 56% D2O Invisible Vesicle Just after mixing Invisible Vesicle After a finite time Center for Neutron Sciences

15. Insights into lipid structure and dynamics 10 6 t=0 ) 4 t=600 -1 2 1 Intensity (cm 6 4 2 4 5 6 7 8 9 2 3 0.01 q (A-1) Understanding Intra-cellular Cholesterol Transport =number of particles =Volume of particle =Form-factor of solute Center for Neutron Sciences

16. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Half life for exchange: ~100min Half life for flipping: ~250min (slow!) Arrhenius equation Center for Neutron Sciences

17. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Donor Acceptor Donor Acceptor S=SLD=f(x) x=Fraction of chol. Exch. 0.5 Kex Kf 0.25 Center for Neutron Sciences

18. Insights into lipid structure and dynamics Cholesterol-Sulfate 1.00 Cholesterol I(t)/I(t=0) 1.0 0.9 0.625 0.8 I(t)/I(t=0) 0.7 0 10000 Time (sec) 0.6 0.5 3 0 20 40 60 80x10 Time (sec) Understanding Intra-cellular Cholesterol Transport Origin of Second Process x=0.25 I=0.625 Center for Neutron Sciences

19. Insights into lipid structure and dynamics 1.0 1.0 Chol 1.0 DHE 0.9 0.9 Chol with Chol 50nm Cyclodextrine 0.9 0.8 0.8 I(t)/I(t=0) 100nm 0.7 0.7 I(t)/ I(t=0) 200nm I(t)/I(t=0) 0.8 Fit 0.6 0.6 0.7 0.5 0.5 0.6 0 10 20 30 3 0 10 20 30 3 40x10 40x10 Time (sec) Time (sec) 0.5 0 200 400 600 800 1000 t (min) Understanding Intra-cellular Cholesterol Transport Effect of Typical Additives on Transport Kinetics Center for Neutron Sciences

20. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Effect of Varying the Sterol Center for Neutron Sciences

21. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Changing the Lipid – POPS Highly Enriched in the Inner Leaflet of the Cell Membrane Center for Neutron Sciences

22. Insights into lipid structure and dynamics Understanding Intra-cellular Cholesterol Transport Is there a Cholesterol Solubility Limits? POPC POPS Center for Neutron Sciences

23. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Lipid membranes are self-assembled highly flexible structures that have the ability to undergo an array of conformational and dynamic transitions which are essential for many biological functions. Spectroscopic length scale: Microscopic length scale: Cell signal transduction is affected by molecular lateral diffusion within the lipid membrane.(1) Membrane stiffness and fluidity have been shown to have a large impact on cellular uptake and release.(2) Intermediate length scale : Membrane thickness fluctuations are thought to be involved in membrane protein insertion and have been proposed as a mechanism for pore formation.(3) Center for Neutron Sciences

24. Insights into lipid structure and dynamics THEORY Membrane Thickness Fluctuations Breathing model of a lipid bilayer by Miller Miller, Top. Bioelectrochem. Bioenerg. 4, 161 (1981).; Bach and Miller, Biophys. J. 29, 183 (1980).; Miller, Biophys. J. 45, 643 (1984). Amplitude of the fluctuations reaches ≈ 15 Å or more from the geometrical constraints (volume conservation) Thickness fluctuations by Hladky and Gruen Hladky and Gruen, Biophys. J. 38, 251 (1982). Thickness fluctuations occur, but the amplitude is small. Long wavelength fluctuation amplitude is negligible Short wavelength fluctuations (< 30 Å) are severely limited Intermediate wavelength fluctuation amplitude < 10 Å Deformation free energy of bilayer membranes by Huang Huang, Biophys. J. 50, 1061 (1986). Center for Neutron Sciences

25. Insights into lipid structure and dynamics SIMULATION Membrane Thickness Fluctuations Lindahl and Edholm, Biophys. J. 79, 426 (2000). Simulation time scale: 10 or 60 ns Monolayer RMS amplitude for peristaltic (thickness fluctuation): 2.5 Å Suppressed thickness fluctuations in the gel phase West and Schmid, Soft Matter6, 1275 (2010). Thickness fluctuation amplitude is much smaller than that in the fluid phase Center for Neutron Sciences

26. Insights into lipid structure and dynamics SURFACTANT Membrane Thickness Fluctuations thickness Thickness fluctuations are observed as an enhancement of a motion on top of the bending bending Deff αq2 κ αq3 Nagao, Phys. Rev. E80, 031606 (2009).; Nagao et al., Soft Matter7, 6598 (2011).; Nagao, J. Chem. Phys. 135, 074704 (2011). Center for Neutron Sciences

27. Insights into lipid structure and dynamics SURFACTANT Membrane Thickness Fluctuations experiment Courtesy of Takumi Hawa MD simulation Nagao et al., Soft Matter7, 6598 (2011). Center for Neutron Sciences

28. Insights into lipid structure and dynamics Membrane Thickness Fluctuations DMPC (C=14) DPPC (C=16) DSPC (C=18) 10 wt% lipids form large unilamellar vesicles (LUVs) by extruding through 100 nm polycarbonate filter in D2O To contrast match the tail region of the lipids to D2O, tail deuterated and hydrogenated lipids were mixed at appropriate mixing ratio Density measurement to confirm the melting transition temperature, Tm #from Avanti Polar Lipid web site Woodka et al., Phys. Rev. Lett. 109, 058102 (2012). Center for Neutron Sciences

29. Insights into lipid structure and dynamics Membrane Thickness Fluctuations r(r) dt dh 0 rc r NSE q range qmin Center for Neutron Sciences

30. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Bending motion for a planar membrane is described by the Zilman Granek/Watson model Zilman and Granek, Phys. Rev. Lett. 77, 4788 (1996).; Zilman and Granek, Chem. Phys. 184, 195 (2002). G: decay rate, b=2/3 ~ k: effective bending modulus, h: solvent viscosity, a ≈ 1 Center for Neutron Sciences

31. Insights into lipid structure and dynamics Membrane Thickness Fluctuations DMPC T = 35 ˚C Above Tm Excess enhancement = thickness fluctuations Bending no excess enhancement = suppressed thickness fluctuations DMPC T = 16 ˚C Below Tm Bending Center for Neutron Sciences

32. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Approximately an order slower (≈ 100 ns) than surfactant membranes (a few ns) Center for Neutron Sciences

33. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Width of Lorentz peak relates to the fluctuation amplitude Mean amplitude = 3.7 Å ± 0.7 Å experiment Huang’s mean amplitude ≈ 4.5 Å Huang, Biophys. J. 50, 1061 (1986). theory Lindahl & Edholm’s amplitude ≈ 5 Å Lindahl and Edholm, Biophys. J. 79, 426 (2000). simulation ≈ 8 % of the membrane thickness; close to the value seen in surfactant membranes (≈ 12 %) Is amplitude defined by geometrical constraints, like volume conservation? Center for Neutron Sciences

34. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Can we increase the amplitude? Can we tell if fluctuations are frozen or only slowed down below Tm? DMPC (C=14) DSPC (C=18) Equimolar mixture between DMPC and DSPC, so that we can compare with pure DMPC or DSPC thickness fluctuations Center for Neutron Sciences

35. Estimation of thickness fluctuation parameters Nagao et al., in preparation. Fit to the empirical relation Nagao, Phys. Rev. E80, 031606 (2009).; Nagao et al., Soft Matter7, 6598 (2011).; Nagao, J. Chem. Phys. 135, 074704 (2011).; Woodka et al., Phys. Rev. Lett. 109, 058102 (2012).; Lee et al., Phys. Rev. Lett.105, 038101 (2010). Center for Neutron Sciences

36. Insights into lipid structure and dynamics Membrane Thickness Fluctuations fluid gel gel-fluid Gradual decrease of k during the phase segregation Center for Neutron Sciences

37. Insights into lipid structure and dynamics Membrane Thickness Fluctuations slow down Fluid phase the time scale is similar to the pure cases Gel-Fluid coexistence significantly slower than in the fluid phase Center for Neutron Sciences

38. Insights into lipid structure and dynamics Nagao et al., in preparation. Membrane Thickness Fluctuations Around Tu and below: the fluctuation amplitude is equivalent to the pure ones --> domain formation makes the thickness fluctuations the same as pure larger Well above Tu: DSPC is confined --> possible larger fluctuation amplitude Center for Neutron Sciences

39. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Effects of segregation on bilayer dynamics Veatch and Keller, Phys. Rev. Lett.94, 148101 (2005). Center for Neutron Sciences

40. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Effects of protein insertion on the bilayer dynamics Center for Neutron Sciences

41. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Pore forming peptides in DMPC-ULV Gramicidin 0 to 1.25 mol% + 0 to 2.5 mol% + DMPC Alamethicin Alamethicin 4 to 6 molecular bundle Gramicidin dimer http://people.ucalgary.ca/~tieleman/gallery.html Lundbæk, Collingwood, Ingolfsson, Kapoor1 and Andersen, J. R. Soc. Interface 7, 373 (2010). Center for Neutron Sciences

42. Insights into lipid structure and dynamics Woodka et al., in preparation. Gramicidin Alamethicin Center for Neutron Sciences

43. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Increase in the bending rigidity with protein concentration Center for Neutron Sciences

44. Insights into lipid structure and dynamics Woodka et al., in preparation. Membrane Thickness Fluctuations Almost constant with Alamethicin Becomes faster with Gramicidin, then slows down Center for Neutron Sciences

45. Insights into lipid structure and dynamics Membrane Thickness Fluctuations Enlarged with Gramicidin, then becomes smaller Almost constant with Alamethicin Are the Different pore forming mechanisms between Alamethicin and Gramicidin responsible for this difference? Enhanced thickness fluctuations to form dimer for Gramicidin? Helfrich and Jakobsson, Biophys. J.57, 1075 (1990). Center for Neutron Sciences

46. Insights into lipid structure and dynamics Concluding Remarks • Neutrons can be a very powerful tool if used correctly • NS can help answer questions of structure and dynamics over a very wide range of length and time scales • The more work you put into designing the experiment the higher the reward • We have lots of fun!!! • ….. Neutrons Don’t Lie Center for Neutron Sciences