1. Computation for this work was supported by the University of Southern California Center for High-Performance Computing and Communications (www.usc.edu/hpcc). Special thanks to MOSIS for providing additional resources and funding for this project.www.usc.edu/hpcc Introduction Methods All simulations were performed using the GROMACS 4.0.5 software package on the University of Southern California High Performance Computing and Communications Linux cluster. Bilayers consisted of 128 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipids parameterized by OPLS/Berger force fields and 35 SPC water molecules per lipid at an integration time-step of 2 fs. Pore life cycle times were determined using custom Perl scripts as previously reported 1. Temperature was modulated using a modified Berendsen thermostat with corrected velocities known as the ‘v_rescale’ thermostat with a time constant of 0.1 ps. An NPT ensemble was maintained using the Berendsen barostat which applied 1 bar in xy:z semi-isotropically with a compressibility of 4.5E-5 bar -1. Bond lengths were constrained using the LINCS algorithm for lipids and SETTLE for water. Short-range electrostatic and Lennard- Jones interactions were cut off at 1.0 nm. Long-range electrostatics were calculated by the PME algorithm using fast Fourier transforms and conductive boundary conditions. Reciprocal- space interactions were evaluated on a 0.12-nm grid with fourth-order B-spline interpolation. Periodic boundary conditions were employed to mitigate system size effects. Conclusions Consistent with experimental values, the MD phase transition for POPC appears at about -3 ° C. Temperature does not significantly modify the pore creation time, though the pore annihilation time is reduced at higher temperatures. At low temperatures, quasi-stable pores develop when the field is removed. Cold pores take longer to minimize than warmer pores and retain more water, though overall bilayer thickness is not affected. 1 MOSIS, Information Sciences Institute, Viterbi School of Engineering (VSoE), University of Southern California (USC), Marina del Rey, USA 2 Department of Physics and Astronomy, College of Letters, Arts, and Sciences, USC, Los Angeles, USA 3 Ming Hsieh Department of Electrical Engineering, VSoE, USC, Los Angeles, USA, Molecular-scale details of the mechanism of electric field-driven pore formation in phospholipid bilayers are not well understood, in part because experiments cannot access the nanoscopic domain at which individual pore formation occurs. Analytical and numerical models can help to fill this void. Previous studies using molecular dynamics (MD) simulations characterized the life cycle of an electropore as a function of the externally-applied electric field, from the formation of an initial water defect to the restoration of the intact bilayer 1, but this is far from a complete description of pore creation and annihilation. Here we continue our characterization of electropores, modulating the temperature at which electropermeabilization occurs, allowing us to observe how temperature-related physical properties of lipids affect the timescales of pore formation and annihilation. These results can be compared to and reconciled with existing mathematical models of electroporation which incorporate temperature, presenting a more unified and complete framework for future studies. Temperature Modulation of Phospholipid Bilayer Electropore Creation and Annihilation Zachary A. Levine 1,2 and P. Thomas Vernier 1,3 Physical Properties of POPC Bilayers at Various Temperatures Temperature (°C) Diffusion (10 -11 m 2 /s) Lipid Lateral Diffusion versus Temperature POPC SN-2 Order Parameter (S z ) POPC SN-1 Order Parameter (S z ) T(°C) Carbon Number SzSz SzSz Temperature (°C) Area per Lipid (nm 2 ) Area per Lipid versus Temperature (A) (B) (C) (D) C p (kJ/mol K) Heat Capacity versus Temperature (E) (A) POPC lateral diffusion decreases as temperature is lowered. (B) Area per lipid also decreases with temperature, with a phase transition at about -3 °C, consistent with experiment. (C) (D) Order parameters for the sn-1 and sn-2 tails confirm the phase transition around -3 °C. (E) Similarly, heat capacity shows dynamic behavior around -3 °C (longer sampling times are required for higher resolution). Temperature (°C) Pore Creation Pore Annihilation At high temperatures pores reseal quickly, but at low temperatures pores are quasi-stable out to 125 ns. Pores at every temperature shrink to a minimum size when the field is removed. Pore creation time is not significantly affected by temperature over the range -5 °C to 50 °C, even near the critical temperature. (about -3 °C). Pore expansion takes longer at lower temperatures. E = 400 MV/m E = 0 MV/m T = 0 ° CT = 10 ° CT = 20 ° C T = 30 ° C T = 40 ° C T = 50 ° C T = -5 ° C T = -4 ° C T = -3 ° C T = -2 ° C Des Electropore Structure and Pre-Pore Pedestals – Future Work T = 0 ° C T = 10 ° C T = 20 ° C T = 30 ° CT = 40 ° C T = 50 ° C T = -1 ° C Pore Waters versus Time Bilayer Thickness Time (ns) At 30 °C and below, each quasi-stable pore retained a constant number of pore waters over 125 ns. These ‘cold’ electropores also take longer to minimize than warmer ones, and contain more water. Although temperature modifies the rate of pore expansion and resealing, the time it take to initially create the pore is strongly dependent on the local electric field profile in the water/lipid interface 2. We observe in MD, consistent with theory 3, that electropores originate from a transient pre-pore structure which acts as a meta- stable pedestal over multiple picoseconds. We are parameterizing this structure and characterizing its role in pore formation. 1.Levine, Z. A. and P. T. Vernier. 2010. Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation. Journal of Membrane Biology 236:27-36. 2. 2.Ziegler, MJ and P.T. Vernier. 2008. Interface Water Dynamics and Porating Electric Fields for Phospholipid Bilayers. Journal of Physical Chemistry B. 112 (43), 13588-13596 3.Okuno Y, Minagawab M, Matsumotob H, et al. Simulation study on the influence of an electric field on water evaporation. Journal of Molecular Structure: THEOCHEM. 904(1-3), 83-90. Water Orientation Expansion Construction Expansion Initiation Stabilization Settling Deconstructio n Dissolution Settling Stabilization