Examining the Translocation of Amphiphiles across Lipid Bilayers using a Gramicidin Channel-Based Fluorescent Assay

Wednesday, February 15, 2017 in lipid headgroup-driven nanodomain formation in PC-PS lipid mixtures. Our study shows a dependency of monovalent cation size Mþ on lipid domain formation of anionic PS and zwitterionic PC in mixed bilayers. The formation of these domains are dependent on lipid-ion binding modes and partition free energy of ions in the water-bilayer interface. Certain binding modes lead to growth of ion-mediated PS lipid clusters. A coupled relationship between lipid curvature and asymmetry is observed in highly demixed PC/PS mixed bilayers. This is the first molecular-level simulation study on the monovalent cation sizedependent lipid domain formation in zwitterionic-anionic mixed lipid bilayers. 2566-Pos Board B173 Examining the Translocation of Amphiphiles across Lipid Bilayers using a Gramicidin Channel-Based Fluorescent Assay Thasin Peyear, Radda Rusinova, Olaf S. Andersen. Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA. Detergents are common tools in molecular biology and biochemistry, including the solubilization and purification of membrane proteins. Detergents have long been known to alter the lateral organization of lipid bilayers, more recently they have been shown to increase lipid bilayer elasticity (Lundbæk et al., PNAS 2010). Some are able to cross lipid bilayers, others not (le Maire et al, Biochemistry, 1987; Heerklotz, Biophys J, 2001), but less is known about this aspect of detergent-bilayer interactions. We explored this question using gramicidin channels to detect amphiphile-induced changes in bilayer elasticity with a sequential-mixing fluorescence quench assay, which allows for determining the time course of detergent interaction with the outer leaflet of a large unilamellar vesicle (LUV), including the time is takes to cross the bilayer to intercalate into the intravesicular leaflet. Three common detergents were investigated: CHAPS; SDS; and Triton X-100. All three produced a fast (complete within 15 ms) increase in fluorescence quench rate, indicative of a shift in the gramicidin monomer4dimer equilibrium toward the conducting dimers. In the case of Triton X-100, this initial phase was followed by a second phase, complete within 500 ms, which is indicative of Triton translocation to the intravesicular leaflet. The same general pattern was observed with SDS, though the slow phase was complete only after ~1 min, whereas CHAPS appeared to reside in the extravesicular leaflet only. We tested the implications of these results using LUVs prepared in the presence of detergent, where Triton X-100 and SDS again appeared to be membrane permeant, whereas CHAPS appeared to be impermeant. The LUV size distributions were determined dynamic light scattering; though the size distributions differed when the LUVs were prepared in the presence of detergent, these differences could not account for the changes in fluorescence quench rates (or our conclusions). 2567-Pos Board B174 Calcium Ion-Mediated PIP2-Cluster Formation from All-Atom Molecular Dynamics Simulations Kyungreem Han, Richard M. Venable, Richard W. Pastor. Laboratory of Computational Biology, NIH/NHLBI, Rockville, MD, USA. Phosphatidylinositol (4,5)–bisphosphate (PIP2), a phosphorylated derivative of phosphatidylinositol, is a critical lipid in cell membranes. Not only PIP2 itself is the precursor of the essential second messengers for the cellular signal transduction such as inositol (1,4,5)–trisphosphate (IP3) and diacylglycerol (DAG), but also the cooperative action from PIP2–clusters plays indispensable roles in the onset and progression of various human diseases, including cancer, neurodegeneration, metabolic disorder, and inflammation. In the present study, we have performed all–atom molecular dynamics simulations to discover the context– dependent nature of the PIP2–cluster formation. Special attention has been paid to the hydrogen bond network formation depending on local ion and/or lipid compositions. We examine synergistic, additive, and inhibitory effects between Kþ and Ca2þ in clustering based on a series of molecular dynamics simulations on hydrated mono- and dimethylphosphate anions, molecular fragments which model properties of the PIP2 head–group. Furthermore, hypotheses which address the roles of cholesterol in the hydrogen bond network formation are tested by simulations of pure PIP2 and PIP2–rich mixed monolayers. These results are applied to the interpretation of Langmuir trough experiments. This study provides a computational framework for better understanding the fundamentals of the cation–mediated PIP2–pair, –cluster, and –network formation. 2568-Pos Board B175 Lipid Membrane Structural and Mechanical Properties Modulated by Polyglutamine Aggregates Nawal K. Khadka1, Fengyu She2, Jianfeng Cai2, Jianjun Pan1. 1 Physics, University of South Florida, Tampa, FL, USA, 2Chemistry, University of South Florida, Tampa, FL, USA. Lipid membranes are potential targets by protein aggregates. We study aggregates formed by a polyglutamine (polyQ) peptide, and their disruptive

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effect on lipid membranes. Using solution atomic force microscopy (AFM), we observe polyQ oligomers coexisting with short fibrils. Fourier transform infrared spectroscopy reveals that the content of b-sheet enriched aggregates increases with incubation time. PolyQ aggregates-induced membrane disruption is inferred from time-dependent calcein leakage of lipid vesicles. Detailed structural and mechanical perturbations of lipid membranes are revealed by solution AFM. We find that membrane disruption by polyQ aggregates proceeds by a two-step process, involving partial and full disruption. In addition to height contrast, the resulting partially and fully disrupted bilayers have distinct rigidity and adhesion force properties compared to the intact bilayer. Specifically, the bilayer rigidity increases as the intact bilayer becomes partially and fully disrupted. Surprisingly, the adhesion force first decreases and then increases during the disruption process. By resolving individual fibrils deposited on bilayer surface, we show that both the length and the number of fibrils can increase with incubation time. Our results highlight that membrane disruption could be the molecular basis of polyQ aggregates induced cytotoxicity. 2569-Pos Board B176 Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts Felix M. Goni, Aritz Garcia-Arribas, Hasna Ahyayauch, Jesus Sot, Pablo L. Lopez-Gonzalez, Alicia Alonso. Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain. The effects of increasing amounts of palmitoylceramide (pCer) on human red blood cell lipid membranes have been studied using atomic force microscopy of supported lipid bilayers, in both imaging (bilayer thickness) and force-spectroscopy (nanomechanical resistance) modes. Membranes appeared homogeneous with pCer concentrations up to 10 mol % because of the high concentration of cholesterol (Chol) present in the membrane (about 45 mol %). However, the presence of pCer at 30 mol % gave rise to a clearly distinguishable segregated phase with a nanomechanical resistance 7-fold higher than the continuous phase. These experiments were validated using differential scanning calorimetry. Furthermore, Chol depletion of the bilayers caused lipid domain generation in the originally homogeneous samples, and Chol-depleted domain stiffness significantly increased with higher amounts of pCer. These results point to the possibility of different kinds of transient and non-compositionally constant, complex gel-like phases present in RBC lipid membranes rich in both pCer and Chol, in contrast to the widespread opinion about the displacements between pCer-enriched ‘‘gel-like’’ domains and liquid-ordered ‘‘raft-like’’ Chol-enriched phases. Changes in the biophysical properties of these complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine-tuning the properties of membranes as required. 2570-Pos Board B177 Effects of Carotenoids on Membrane Bending Rigidity Rudy M. Me´ndez Reina1, Maria I. Pe´rez Lo´pez1, Chad Leidy2, Manu Forero-Shelton1. 1 Department of Physics, Universidad de los Andes, Bogota´, Colombia, 2 Student Academic Success Center, University of California, Davis, CA, USA. Carotenoids are a group of pigments found in plants and some photosynthetic organisms that play several important physiological functions. For example, carotenoids have been shown to promote virulence by stabilizing the membrane of Staphylococcus aureus during infection. In this work we focus on how carotenoids modulate the properties of the cell membrane of S. aureus as well as model membranes. We observed a dramatic decrease in the pigmentation of S. aureus biofilms when compared to planktonic cells. We quantified the carotenoid content of membranes isolated from biofilms and planktonic cultures of S. aureus by absorption spectroscopy and confirmed this is due to decreased carotenoid content. We also characterized the lipid composition in S. aureus membranes and found that the most abundant lipid was C14. For this reason, we chose a mixture of two C14 lipids (DMPC:DMPG), in order to model both the mechanical and electrostatic properties of the membrane of S. aureus. In order to understand how carotenoids affect the membrane, we first measured the phase transition of DMPC with carotenoids by monitoring GP Laurdan as a function of temperature. We observed that carotenoids lower the cooperativity of the lipid phase transition. We also measured the bending rigidity for DMPC:DMPG vesicles in the presence of carotenoids with Vesicle Fluctuation Analysis (VFA) and saw an increase in rigidity. These changes could be important in understanding the mechanism of S. aureus0 resistance to antimicrobial peptides.