Solubilized Nicotinic Acetylcholine Receptor: Lipid Composition and Requirements for Activity

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Tuesday, March 1, 2016

proteins (perilipin 1, 2, 3, and 5), apoE and apoLp-lll all have these bundles, and all are found on neutral lipid particles. In this work we investigate binding of the model amphipathic a-helix bundle protein, apoLp-III, onto different phospholipid monolayers at the aqueous solution-oil interface to shed light on lipid binding of this important protein domain. The effects of charge and acyl chain length were studied separately, using a series of carefully-chosen lipids and a home-built droplet tensiometer. 2080-Pos Board B224 Computational Prediction of Lipid Binding Profiles for Sec14-Like Domains Lauren C. Heller1, Mwangala Akamandisa2, Donald E. Elmore3. 1 Department of Mathematics, Wellesley College, Wellesley, MA, USA, 2 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 3 Department of Chemistry and Biochemistry Program, Wellesley College, Wellesley, MA, USA. Sec14 is a phosphatidylinositol (PI) transfer protein that functions in transport from the Golgi apparatus and regulation of lipid metabolism in Saccharomyces cerevisiae. This protein is the namesake of the Sec14 family of proteins that includes relatives in a wide variety of eukaryotic organisms. Structural models of the Sec14 lipid binding domain predicted it would have increased selectivity for particular phosphorylated PI derivatives, such as PI5P. Lipid binding assays for Sec14 confirmed this previously unreported preference, which has also been observed for the homologous Sec14 protein Patellin from Arabidopsis thaliana. We also have extended our computational modeling to Sec14 proteins from the moss Physcomitrella patens in order to predict their lipid binding profiles. Ongoing work is aimed at experimentally confirming these computational predictions. A robust computational model for Sec14 lipid binding would simplify functional predictions, providing insight into the physiological function for these proteins. 2081-Pos Board B225 Modulation of Gel Phase Model Membranes by Vitamin D-Related Proteins Sidra Rashid1, Robert V. Law2, Abdel F. Isakovic1, Vivian Stojanoff3, Deborah L. Gater1. 1 Khalifa University, Abu Dhabi, United Arab Emirates, 2Imperial College, London, United Kingdom, 3Brookhaven National Laboratory, Upton, NY, USA. Severe vitamin D deficiency has long been linked to dysfunctions of the musculoskeletal system, and more recent studies have also associated vitamin D deficiency with other conditions, ranging from certain cancers to cardiovascular disease and diabetes. Vitamin D is a steroidal hormone that can be biosynthesized from 7-dehydrocholesterol by the action of UV light on skin (vitamin D3 only), or obtained from dietary sources (vitamins D3 and D2). Vitamins D3 and D2 are inactive precursor molecules that must be successively hydroxylated in the kidney and liver in order to become active. The vitamin D binding protein (DBP, GC-globulin) is responsible for transporting the lipophilic vitamin D throughout the body, and the vitamin D receptor (VDR) is the transcription factor that exerts the most well-established activities of vitamin D in the cell nucleus. Here, we present data based on synchrotron X-ray diffraction, 31P NMR and 2H NMR describing the effects of DBP and VDR in modulating the phase behavior of model membranes composed of DPPC, brain-sphingomyelin and cholesterol. Initial results suggest that DBP particularly interacts with the bilayers to reduce the gel-fluid lamellar phase transition and increase lipid mixing in ternary membranes, with the effect of VDR being somewhat less significant. These data have consequences for our understanding of how the action of soluble proteins that bind lipophilic, membrane-based molecules may alter bilayer structure and dynamics as they interact with the membrane surface to identify and extract their target. 2082-Pos Board B226 Effect of the Novel Amyloid Inhibitor ‘‘anle145c’’ on Aggregation of Islet Amyloid Polypeptide and how it is Modulated by Membranes Saravanan Manikam Sadasivam1, Sergey Ryazanov2, Andrei Leonov2, Steven Roeters3, Sander Wouterson3, Armin Giese4, Christian Griesinger2, J. Antoinette Killian1. 1 MBB, Utrecht University, Utrecht, Netherlands, 2NMR Based Structural Biology, MPI for Biophysical Chemistry, Gottingen, Germany, 3Laboratory of Molecular Photonics, Van’t Hoff Institute for Molecular Sciences, UVa, Amsterdam, Netherlands, 4Zentrum fu¨r Neuropathologie und Prionforschung, Universita¨t Mu¨nchen, Munchen, Germany. Aggregation of islet amyloid polypeptide (IAPP) in the human pancreas is linked to the pathogenesis of type-II diabetes. Interactions of IAPP with mem-

branes may play an important role in the disease, because membranes may catalyze IAPP aggregation and because amyloid formation of IAPP results in membrane permeabilization, and hence cytotoxicity [1]. In order to control this cytotoxicity, there is an intense focus on the development of amyloid inhibitors. Here, we studied the interaction of a novel amyloid inhibitor ‘‘anle145c’’ [2,3,4] on IAPP aggregation in the absence and presence of model membranes using a range of biophysical techniques. Our results show that in both cases the compound acts as an efficient inhibitor, but that it has a distinctly different mode of action in the presence of membranes. Importantly, similar results were obtained with the structurally related natural inhibitor EGCG but with less efficiency. Our complementary experimental approaches reveal that the inhibitors target a different species in the absence and presence of membranes. We propose a model to explain our findings and we discuss its implications for in-vivo studies on these inhibitors. [1] Engel M et al., PNAS. (2008) - 105:6033. [2] Wagner J et al., Acta Neuropathologica (2013) - 125:795. [3] Levin J et al., Acta Neuropathologica (2014) - 127:779. [4] Wagner J et al., Acta Neuropathologica in press (2015) ‘‘Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies’’. 2083-Pos Board B227 Structure and Lipid Binding Preferences of the Alternatively Translated Region of PTEN-Long Anne-Marie Bryant. Worcester Polytechnic Institute, Fall River, MA, USA. PTEN-Long is a 576-amino acid translational variant of phosphatase and tension homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits an alternative translation region (ATR) with additional 173 N-terminal amino acids to the normal PTEN open reading frame. Several studies have demonstrated that PTEN-Long is secreted from cells, can exist outside the cell and can enter other cells via a yet to be determined mechanism. PTEN-L may have therapeutic uses by restoring a functional tumor suppressor protein to tumor cells. A computational analysis reported that the N-terminal ATR part of PTEN-L is largely unstructured and has the potential to interact with a variety of proteins. The ATR region of PTEN-L is evolutionary conserved and contains a polyarginine stretch with homology to cell permeable peptides. Using CD and FTIR spectroscopy, we confirmed that the ATR region of PTEN-L is intrinsically disordered. It is not very well understood if PTEN-L ATR shares the same protein-protein, protein-lipid activity and mechanism of regulation as normal PTEN. Our FTIR studies with lipid mixtures of PC, PS, and PI(4,5)P2 did not reveal an observable structural change to PTEN-L ATR upon lipid binding. We are currently delineating the lipid binding preferences of the ATR part of PTEN-L as well as the fulllength protein. 2084-Pos Board B228 Solubilized Nicotinic Acetylcholine Receptor: Lipid Composition and Requirements for Activity Juan C. Mercado1, Jose´ A. Lasalde Dominicci1, Orestes Quesada2, Jose´ O. Colo´n1. 1 Biology, University of Puerto Rico San Juan, San Juan, PR, USA, 2 Chemistry, University of Puerto Rico San Juan, San Juan, PR, USA. This study is the first detailed report about the bulk molecular species of lipid species from the Torpedo californica (Tc) electric tissue and six nAChR detergent complexes (nAChR-DCs) that examines the correlation of lipid composition of the nAChR solubilized state with functional channel activity. We first evaluated the efficiency of Folch and the Bligh & Dyer (B&D) methods in the extraction of Tc tissue lipids. Comparison of both methods reveals that the B&D provides the most consistent and efficient method for the extraction of lipid species including cholesterol from the Tc electric organ. Lipidomics analysis of extracted lipids using Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS) shown Seventy six (77) phospholipid species were found in the Tc tissue with different degree of fatty acids saturation but preference for palmitic acid. Phospholipid profile of nAChR-DCs prepared with phospholipid analog detergents alkylphosphocholine (FC) and Lysofoscholine (LFC) of 12-16 carbon chains display a range of 12-6 phospholipid species per complex. Among all six of the nAChR-DCs, two of them display full functional activity similar to the crude Tc membranes (LFC14-nAChR and LFC16-nAChR) and one (FC12nAChR) display significant amount of macroscopic current but with a substantial alteration in macroscopic channel kinetics (activation and desensitization).

Tuesday, March 1, 2016 A qualitative cross correlation analysis of the lipid species present in each of these six nAChR cannot dissect which lipid species are essential for nAChR function in the soluble state, however, the partially functional nAChR-FC12 detergent complex lack phospholipids species with polyunsaturated fatty acids that are present in the fully functional nAChR-DCs. 2085-Pos Board B229 Detergent-Free Isolation, Characterization and Functional Reconstitution of a KD Channel: The Power of Native Nanodiscs Jonas M. Do¨rr, Marre Scha¨fer, Martijn C. Koorengevel, J. Antoinette Killian. Chemistry, Utrecht University, Utrecht, Netherlands. A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid (SMA) copolymer. This polymer self-inserts into membranes and is capable of directly extracting membrane patches in the form of nanosize discoidal proteolipid particles or ‘‘native nanodiscs.’’ Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Finally, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. To further elucidate the preferential interactions of lipids with KcsA, these studies were extended to an artificial system with reconstituted protein in liposomes of well-defined composition. This allows for better quantification of the interactions and more detailed insights into both headgroup and chainlength preferences of the channel. Together, our findings highlight the potential of the use of native nanodiscs as a tool in the study lipid-protein interactions. Reference: Do¨rr JM, et al., 2014, PNAS 111(52):18607-18612. 2086-Pos Board B230 Electrostatic Forces Govern Assembly and Disintegration of the Influenza Virus Protein Scaffold to Provide Tension for Membrane Fusion Oleg V. Batishchev1,2, Liudmila A. Shilova1,2, Michael V. Kachala1, Vsevolod Yu Tashkin1, Valerij S. Sokolov1, Natalia V. Fedorova3, Liudmila A. Baratova3, Denis G. Knyazev4, Joshua Zimmerberg5, Yury A. Chizmadzhev1. 1 A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of RAS (IPCE RAS), Moscow, Russian Federation, 2Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation, 3A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation, 4Johannes Kepler University Linz, Institute of Biophysics, Linz, Austria, 5Section on Cellular and Membrane Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. Influenza A virus is an enveloped negative strand RNA virus. Its outer envelope consists of the lipid membrane with incorporated glycoproteins and proton channel M2. The inner envelope of the virion is a membrane-associated scaffold of matrix protein M1, which contacts both the viral RNP and the lipid envelope. Formation and disintegration of the protein scaffold are essential processes for influenza replication and infection. Both involve interaction of M1 with the lipid membrane; both are controlled by pH. We investigate the physico-chemical mechanism of these processes using the combination of electrochemical and fluorescent measurements with AFM. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. Acidification drives conformational changes in M1 molecules and increase of their charge leading to partial desorption due to increased repulsion between M1 monomers still stuck to the membrane. This repulsive force could generate tension for membrane rupture, as it was demonstrated for lipid vesicles coated with M1. Thus, electrostatic forces could explain M1 protein scaffold disintegration at low pH and most likely stretch the lipid membrane, promoting fusion pore widening for RNP release.

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2087-Pos Board B231 Stochastic Fluctuation Sensing in a Bistable Phosphatidylinositol-Based Reaction Diffusion System Scott D. Hansen, William Y. Huang, Young Kwang Lee, Jay T. Groves. Chemistry & QB3, UC Berkeley, Berkeley, CA, USA. Bistability is an underlying principle of many cellular processes including: signaling transduction, gene expression, and cell polarity. One important class of membrane constituents that act as master regulators of signal transduction is the large family of phosphatidylinositol lipids, which selectively recruit cytosolic proteins to intracellular membranes and locally control their activity. Although many regulators and downstream effectors of phosphatidylinositol synthesis pathways have been identified, it remains unclear how bistability emerges from the competition between opposing lipid kinases and phosphatases. To better understand this fundamental cellular process, we biochemically characterized the emergent properties of a simple two-component reaction diffusion system based on the synthesis of PI(4)P and PI(4,5)P2 lipids. Using supported lipid bilayers as a mimetic to cellular membranes, we demonstrate that competition between lipid kinases and phosphatases with either intrinsic or engineered positive feedback loops is sufficient to generate bistability in phosphatidylinositol lipid synthesis. Using single particle tracking of fluorescently labeled enzymes we demonstrate that bistability emerges from ultrasensitivity to phosphatidylinositol lipid densities and stochastic fluctuation sensing. We purpose a model for fluctuation sensing using stochastic chemical kinetics. 2088-Pos Board B232 Cardiolipin Membranes as Photoreduction Inhibitors in Ferricytochrome C: A Resonance Raman Study with Soret Band Excitation Dmitry Malyshka, Reinhard Schweitzer-Stenner. Drexel University, Philadelphia, PA, USA. The binding and behavior of ferricytochrome c to cardiolipin (CL) containing membranes is important to characterize to fully understand the protein’s involvement in apoptosis. Upon binding, it has been shown that the protein loses its Met80 ligand and ultimately gains peroxidase activity, a vital step in its dissociation from the inner mitochondrial membrane (IMM). Multiple binding studies have been done on the protein binding to CL containing liposomes, which serve as IMM mimics, using spectroscopic techniques including UVCD, UV-VIS absorption, and fluorescence, among others. These techniques, however, provide rather limited information about the heme group which serves as the active site for the protein’s peroxidase activity. Resonance Raman (RR) spectroscopy on ferricytochrome c allows us to selectively probe the heme vibrational modes, which are very sensitive to changes in the heme’s conformation and changes in the ligation, spin, and oxidation states. However, it has been found that Soret bands excitation leads to photoreduction of the protein proving proper analysis very difficult. We investigated whether the binding of ferricytochrome c to CL containing liposomes results in the inhibition of photoreduction due to a sharp decrease in redox potential arising from the dissociation of the Met80 ligand. It was found that the extent of photoreduction is lowered by increasing cardiolipin concentrations, with most spectra obtained at low cardiolipin concentrations showing a clear mixture of bands assignable to both the photoreduced and oxidized protein. There was little to no photoreduction at high lipid concentrations, along with a clear disappearance of the low frequency RR modes, a sign of the heme becoming more planar. 2089-Pos Board B233 Elucidation of Electrostatic Determinants in Cytochrome C-Cardiolipin Binding Margaret M. Elmer-Dixon. University of Montana, Missoula, MT, USA. Recently, there has been great interest in understanding the role of cytochrome c (Cytc) as it pertains to the intrinsic apoptotic pathway. Positively charged domains on Cytc interact with the anionic phospholipid cardiolipin. Although the exact mechanism of this interaction is still under investigation, Cytc undergoes a conformational change upon association with cardiolipin. In the presence of reactive oxygen species, these conformational changes lead to increased peroxidase activity of Cytc. Cytc is capable of oxidizing cardiolipin acyl chains leading to Cytc dissociation from the membrane, and downstream initiation of apoptosis through apoptosome formation. Using circular dichroism, fluorescence and UV-visible spectroscopy methods, we investigate the cytochrome c/cardiolipin interaction. To date, three potential cardiolipin interaction sites have been proposed on cytochrome c. Through environmental manipulation, we are able to isolate and study the cytochrome c/cardiolipin interaction at anionic binding site A. Using mutagenesis we are able to investigate the role of specific positively charged amino acids in site A during electrostatic