Influence of a Central Tryptophan and of Cholesterol on the Properties of Defined Transmembrane Helical Peptides

Wednesday, February 19, 2014 Protein-Lipid Interactions IV 3604-Pos Board B332 To be or not to be in Membrane Domains: Transbilayer Asymmetry and Sphingomyelin-Dependent Preferential Partitioning of the Acetylcholine Receptor Vanesa L. Perillo1,2, D. Alejandro Peñalva1,2, Marta I. Aveldaño1,2, Francisco J. Barrantes3, Silvia S. Antollini1,2. 1 Instituto de Investigaciones Bioquimicas de Bahia Blanca, Bahia Blanca, Argentina, 2Universidad Nacional del Sur, Bahia Blanca, Argentina, 3 Biomedical Research Institute, UCA-CONICET, Buenos Aires, Argentina. The preferential partitioning of the nicotinic acetylcholine receptor (AChR) in liquid-ordered (Lo) domains, heterogeneous membrane domains commonly known as rafts, is thought to be a part of its clustering mechanism. Previous studies from our group have shown that AChR lacks preference for Lo domains when reconstituted in sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model systems (Bermúdez et al., 2010). Here we study the effect on the possible Lo-preferential partitioning of purified AChR reconstituted in two different model systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1) under: a) induced transbilayer asymmetry, resulting from addition of brain sphingomyelin (bSM) to the external hemilayer; and b) the presence of different pure SM species in the model membrane (bSM, 16:0-SM, 18:0-SM or 24:1SM). AChR distribution was evaluated by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and Laurdan or dehydroergosterol fluorescence, and also by determining the presence of AChR in detergent-resistant and detergent-soluble domains (1% Triton X-100, 4 C). Both studies show that the induction of transbilayer asymmetry or the presence of 16:0-SM or 18:0-SM, as opposed to bSM or 24:1-SM, leads to a preferential partitioning of AChR in Lo domains. Thus, the localization of AChR in Lo domains strongly depends on the characteristics of the host lipid membrane. 3605-Pos Board B333 Formation of Giant Unilamellar Vesicles Containing Active Proteins Isabelle Motta, Vladimir Adrien, Andrea Gohlke, Pincet Frederic. Ecole Normale Superieure, Paris, France. Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model for cell membranes. However the study of proteomembrane interactions in this system is limited because the incorporation of integral and lipid-anchored proteins into the GUVs remains challenging. All preexisting protocols either produce membranes with a very low protein density or are tailored to specifically support the inclusion of a particular protein. We recently developed a simple generic method to incorporate protein in GUVs. It does not require specific lipids or reagents, works in physiological conditions with high concentrations of protein, and the resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is not limited to a narrow range of protein substrates; indeed we have already successfully incorporated two trans-membrane proteins and one lipid-anchored peripheral protein. These first proof-of-principle proteins present different types of challenges and thus demonstrate the broad utility of our method. TolC is an integral membrane protein and part of a heterotrimer, that together comprise a major multidrug efflux pump in E coli. The neuronal t-SNARE is a protein complex with a single transmembrane domain that mediates membrane fusion. Because of its propensity to aggregate t-SNARE is usually not functional after insertion in GUVs. To study lipidated proteins, we incorporated a modified form of the autophagy protein GABARAP L1, which we anchored it to the membrane via a cysteine-maleimide covalent bond. In each case, we verified that the proteins remain active after incorporation. We also verified their mobility by performing diffusion measurements via fluorescence recovery after photo bleaching (FRAP) experiments on micromanipulated GUVs. The diffusion coefficients are in agreement with previous data. 3606-Pos Board B334 Influence of a Central Tryptophan and of Cholesterol on the Properties of Defined Transmembrane Helical Peptides Vasupradha Suresh Kumar, Bethany P. Doss, Denise V. Greathouse, Roger E. Koeppe II. University of Arkansas, Fayetteville, AR, USA. GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) provides a favorable host framework for investigations of the influence of guest amino acids, for example a third, centrally located, Trp residue, within the hydrophobic core of a well characterized transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to singleresidue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We introduced a Trp res- 711a idue into position 12 or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solidstate 2H NMR spectra of GWALP23-W12 reveal that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation with only a low extent of dynamic averaging, comparable to GWALP23 itself. Indeed, the tilt and dynamics of GWALP23-W12 are quite similar to the values observed for GWALP23 in DMPC bilayer membranes. We have analyzed the dynamics of the peptide helices using a modified Gaussian treatment as well as a semi-static treatment. The results indicate that a central Trp residue at position 12 does not appreciably perturb the properties of bilayer-spanning GWALP23. (By contrast, Arg-12 or Lys-12, when charged, induces multistate behavior for GWALP23 in bilayer membranes [PNAS 110, 1692].) Additionally, we are investigating the influence of cholesterol upon the properties of membrane-spanning GWALP23, GWALP23-W12 and GWALP23-K12. 3607-Pos Board B335 Regulation of K-RAS Membrane Association: Calmodulin Versus PDEd Katrin Weise1, Benjamin Sperlich1, Shobhna Kapoor1, Gemma Triola1,2, Herbert Waldmann1,2, Roland Winter1. 1 TU Dortmund University, Dortmund, Germany, 2Max Planck Institute of Molecular Physiology, Dortmund, Germany. K-Ras is a small GTPase that plays a critical role in human cancer cell biology. Selective membrane localization and clustering of K-Ras4B into microdomains are mediated by its polybasic farnesylated C-terminus. The importance of the subcellular distribution for the signaling activity of K-Ras4B became apparent from recent in vivo studies [1]. PDEd and the Ca2þ-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins, leading to a modulation of the spatiotemporal organization of K-Ras4B. The latest study of our group showed that PDEd is not able to extract K-Ras4B from model raft membranes; instead, an effective delivery of PDEdsolubilized K-Ras4B to the plasma membrane was proposed [2]. Compared to PDEd, CaM exhibits additional interaction sites to the G-domain of K-Ras4B and was shown not to be required for the transport of K-Ras4B to the plasma membrane. Thus, it was suggested that calmodulin dissociates K-Ras4B from membranes [3]. However, the exact role of CaM in the intracellular localization and dynamics of K-Ras4B still remains elusive. In the present approach, the influence of CaM on the interaction of GDP- and GTP-loaded K-Ras4B with anionic model raft membranes has been investigated by a combination of different spectroscopic and imaging techniques. The results suggest that binding of the acidic CaM to the polybasic stretch of K-Ras4B reverses its charge, leading to repulsion of the complex from anionic membranes. Since one farnesyl anchor alone is not sufficient to stably anchor Ras proteins to membranes, CaM would be able to dissociate K-Ras4B from plasma membranes, contrary to PDEd. References: [1] Ismail SA et al. (2011) Nat. Chem. Biol. 7: 942-949. [2] Weise K et al. (2012) J. Am. Chem. Soc. 134: 11503-11510. [3] Bhagatji P et al. (2010) Biophys. J. 99: 3327-3335. 3608-Pos Board B336 Influence of Glutamic Acid Residues on the Properties of Model Membrane-Spanning Helices Venkatesan Rajagopalan, Denise V. Greathouse, Roger E. Koeppe II. University of Arkansas, Fayetteville, AR, USA. GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- amide) is a constructive model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J.Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged glutamic acid side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments showed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 9.0, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The E16-containing peptide seems to exhibit multi-state behavior at pH 10.9, in bilayers formed by ether-linked lipids, suggesting a pKa that is above pH 9 for the E16 side chain. The rather modest shift in the 2 H quadrupolar splittings suggests that the orientation of the transmembrane peptide helix changes rather little at high pH. It is conceivable that the close proximity of E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the pKa of E16. The molecular cousin having E14 instead of E16 shows multi-state behavior from pH 4.0 to pH 10.9 rendering pKa