2797-Pos Board B227 Binding of Daptomycin to Lipid Bilayers is not Significantly Altered by the Inclusion of Lysyl-Phosphatidylglycerol Tala Khatib 1 , Hannah Lineberry 1 , Heather Stevenson 1 , Bayer S. Arnold 2 , Michael R. Yeaman 2 , Antje Pokorny 1 . 1 Chemistry and Biochemistry, Univ. North Carolina Wilmington, Wilmington, NC, USA, 2 Division of Adult Infectious Diseases, University of California at Los Angeles Medical Center, Los Angeles, CA, USA. In many bacterial cytoplasmic membranes, including that of Staphylococcus aureus, approximately 20 mol% of the diacylphosphatidylglycerol fraction in the cytoplasmic membrane is esterified to the amino acid lysine. This secondary modification leads to the formation of a net-cationic membrane lipid, which re- duces the negative surface charge of the bacterial membrane. Elevated levels of lysyl-phosphatidylglycerol (LPG) are associated with increased bacterial resis- tance to a number of cationic antibacterial peptides, including the clinically important calcium-dependent lipopeptide antibiotic daptomycin. Inclusion of lysylated phospholipids in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphogly- cerol (POPG) has been shown to effectively protect lipid vesicles against leakage of contents induced by the alpha-helical antimicrobial peptides RP-1, mastoparan X, and cecropin A. However, peptide binding to the lipid bilayer was only significantly reduced at LPG fractions exceeding 30 mol%. Here, we investigated whether binding of daptomycin to lipid vesicles containing mixtures of POPG, POPC and LPG is affected in ways similar to that of alpha-helical peptides. We find that daptomycin binds calcium ions in aqueous solution with an apparent dissociation constant on the order of 10 mM. In gen- eral, binding of daptomycin to lipid bilayers depends strongly on calcium and the fraction of POPG in the mixture. However, when 20 mol% of POPG are replaced by LPG, daptomycin binding is only minimally affected. We also show that daptomycin does not cross the lipid bilayer into the vesicle lumen in giant lipid vesicles. These data suggest that clinical daptomycin resistance in S. aureus strains is caused by mechanisms beyond an LPG-mediated reduc- tion in daptomycin binding. Proteins, Lipids, and Small Molecules 2798-Pos Board B228 Phosphatidylserine, a Lipid Present at the Outer Membrane Leaflet of Cancer Cells, Hinders the Insertion of pHLIP, a Potential Cancer Cell Marker Haden Scott. BCMB, University of Tennessee, Knoxville, TN, USA. The pH-Low Insertion Peptide (pHLIP) has shown the ability to insert into membranes when acidic conditions are present. pHLIP can be found in three different states: a random coil in pH 8 solution devoid of lipids (state I), a sur- face bound form when lipids are present (state II), and a transmembrane helix in acidic conditions (state III). From this ability of insertion based on pH, pHLIP has been shown to effectively target diseases in which the extracellular pH of the tissue has become acidic. Cancer is an example of such a disease, and it has been shown that pHLIP can target cancerous cells. One concern with this tar- geting is that some cancerous cells are known to display phosphatidylserine (PS) in the outer leaflet of the their plasma membranes, as opposed to normal cells. However, little is known about how pHLIP’s tumor targeting is influ- enced by this increased PS content. Here, we study how pHLIP properties are influenced by the presence of PS by using phosphatidylcholine (PC) large unilamellar vesicles (LUV’s) that contain PS. Circular dichroism data shows that the characteristic three states are maintained in the presence of PS. Our re- sults show that pHLIP has higher affinity to PC than to PS. We also found that when LUV’s contain PS, the insertion pKa decreases compared to LUV’s only containing PC with a midpoint at 0.950.4 percent. This decrease in insertion pKa might limit pHLIP’s targeting of mildly acidic tumors. We added sodium chloride to screen any electrostatic repulsion; however, no change in the pKa was observed. This suggests that the effect of PS on the binding and insertion of pHLIP is not merely due to electrostatic repulsion. 2799-Pos Board B229 Understanding Molecular Complexity in Protein and Peptide-Lipid Systems Hannah M. Britt, Vian S. Ismail, Aruna S. Prakash, Jackie A. Mosely, John M. Sanderson. Dept. of Chemistry, Durham University, Durham, United Kingdom. Peptide addition to lipid membranes has been shown in some cases to lead to significant molecular complexity, principally as a consequence of the chemical processes that occur subsequent to the addition (http://dx.doi.org/10.1016/ j.jmb.2013.07.013). Foremost amongst these is acyl transfer from lipid constit- uents of the membrane to acceptor sites on the peptide, generating lipidated peptides and lyso-lipids. As an example, addition of the peptide melittin to a two-component lipid membrane leads to the potential formation of up to 20 different peptide species and 8 different lyso-lipids, many of which have been detected, some in significant quantities. The lipidation by-products, lyso-lipids, may also serve as acyl group donors for further lipidation. This pro- cess is not restricted to peptides; some proteins and low molecular weight com- pounds exhibit the hallmarks of this lipidation activity. In a proof-of-principle study, we have examined the lipidation profiles of candidate proteins and shown that they closely resemble the fatty acyl composition of the membrane in which they are generated, supporting the hypothesis that their lipidation arises from membrane lipid precursors. At the other extreme, low molecular weight compounds have been found to undergo similar lipidation processes. Current work to understand the factors that dictate this reactivity, and the con- sequences of lipidation, will be discussed. 2800-Pos Board B230 pHLIPÒ Targeting and Delivery of PNA to Silence MicroRNA in Tumor Cellsx Donald M. Engelman 1 , Christopher J. Cheng 2 , Raman Bahal 3 , Imran A. Babar 4 , Zachary Pincus 5 , Francisco N. Barrera 6 , Connie Liu 7 , Alexander Svoronos 1 , Demetrios T. Braddock 8 , Peter M. Glazer 3 , W. Mark Saltzman 9 , Frank J. Slack 7 . 1 Dept Molec Biophys/Biochem, Yale University, New Haven, CT, USA, 2 Departments of Molec, Cell and Devel Biol, Biomed Eng, Molec Biophys/Biochem., Yale University, New Haven, CT, USA, 3 Department of Therapeutic Radiology, Yale University, New Haven, CT, USA, 4 601 Lexington Avenue, 54th Floor, OrbiMed Advisors LLC, New York, NY, USA, 5 Departments of Developmental Biology and Genetics, Washington University, Saint Louis, MO, USA, 6 Dept. of Biochemistry and Cellular and Molecular Biology, University of Tennessee–Knoxville, Knoxville, TN, USA, 7 Dept Molec/Cell/Devel Biol, Yale University, New Haven, CT, USA, 8 Department of Pathology, Yale University, New Haven, CT, USA, 9 Dept biomed eng, Yale University, New Haven, CT, USA. MicroRNAs (miRNAs) are short non-coding RNAs that, when expressed in different tissue and cell types, suppress the expression of complementary genes. Certain miRNAs, called oncomiRs, play a causal role in the onset and mainte- nance of cancer when overexpressed. Tumors that depend on these miRNAs are said to display oncomiR addiction. Inhibition of oncomiRs using antisense oligomers (i.e. antimiRs) is an evolving therapeutic strategy. The efficacy of current antimiR technologies in vivo is, however, hindered by barriers to target- ing and delivery into tumor cells. pHLIPs are peptides that insert across cell membranes in acidic environments, delivering cargoes into the cytoplasm, and releasing them by disulfide cleav- age. We have used a pHLIP as an antimiR delivery platform that targets the acidic tumor microenvironment, evades systemic clearance by the liver, and fa- cilitates cell entry via a nonendocytic pathway. We found that the disulfide attachment of peptide nucleic acid (PNA) antimiRs to the inserting end of a pHLIP peptide produced a construct that could transport these antimiRs across plasma membranes under acidic conditions such as those found in solid tumors (pH ~6). This conjugate effectively inhibited the miR-155 oncomiR in vitro and in vivo by targeting lymphoid tumors in mouse models. In a large B-cell lym- phoma mouse model, silencing miR-155 reduced tumor burden, prevented lymphocyte metastases, and derepressed targets of miR-155 identified using RNA-seq analysis, with no apparent toxicity to the mice. This study introduces a new paradigm in the targeted delivery of polar molecules, such as antimiR PNAs, as anticancer drugs, which can have broad applicability in the field of targeted drug delivery. x Nature, 2014, accepted. Supported by NIH grants CA133890, CA131301, GM073857, ES005775, CA148996, HL007974, and the Yale Comprehensive Cancer Center. 2801-Pos Board B231 Membrane Cholesterol Association and Structure of Two Leukotoxin Peptides Cayla M. Miller, Angela C. Brown, Jeetain Mittal. Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA. The study of membrane effects on protein structure and functioning is relevant to understanding many native cell processes as well as the cytotoxic effects of some proteins. The cholesterol recognition/interaction amino acid consensus (CRAC) motif is a primary structure pattern that is common among many pro- teins which interact with cholesterol, a key membrane component. However, the CRAC pattern alone is not sufficient to determine the cholesterol-binding 552a Wednesday, February 11, 2015