Physical Properties of Model Membranes Containing Pope and Phytosterol


Sunday, February 16, 2014 91a
483-Pos Board B238
Hiv-1 Tat Membrane Translocation Probed by Low- and Wide-Angle X-
Ray Scattering, Neutron Scattering, CD Spectroscopy and MD Simula-
tions
Kiyotaka Akabori1, Bradley W. Treece1, Michael S. Jablin1, John F. Nagle1,
Brian Maranville2, Kun Huang3, Angel E. Garcia3, Stephanie Tristram-
Nagle1.
1
Biological Physics Group, Physics Dept., Carnegie Mellon University,
Pittsburgh, PA, USA, 2NIST Center for Neutron Research, Gaithersburg,
MD, USA, 3Department of Physics and Astronomy, Rensselaer Polytechnic
Institute, Troy, NY, USA.
In an effort to understand membrane translocation of a cell-penetrating peptide,
interactions of HIV-1 Tat peptide (GRKKRRQRRRPPQ) with DOPC, DOPC/
DOPE, DOPC/DOPS, and nuclear membrane mimics were investigated using
low- and wide-angle x-ray scattering (LAXS and WAXS), neutron scattering,
and circular dichroism (CD) spectroscopy. The diffuse scattering analysis
applied to LAXS collected at CHESS revealed that Tat-membrane interactions
reduce the membrane thickness by ~1 Å. In DOPC and DOPC/DOPE mem-
branes, the position of Tat was found to transition from the vicinity of the
glycerol-carbonyl headgroup to the phosphate headgroup as Tat mole fraction
was increased from 0.009 to 0.06. The area per lipid for DOPC and DOPC/
DOPE membranes increased by ~2 Å2 at the highest Tat mole fraction. The
membrane bending modulus was found to decrease by roughly a factor of 2
at the highest Tat mole fraction except for the nuclear mimic. The chain-
orientational order parameter, Sxray, calculated from WAXS and corrected
for mosaic spread, showed Tat slightly disordered chains. Neutron scattering
collected at NIST from fully hydrated samples consisting of DOPC:DOPE
(3:1) membranes and Tat at 0.06 mole fraction showed a prominent, broad
peak corresponding to a Tat-membrane correlation of ~100 Å. The secondary
structure of Tat calculated from CD spectra using DichroWEB was found to be
the same in pure water as in lipid thin films and primarily consisted of b-sheet
and random coil with small helical content. Our findings are consistent with the
results from MD simulations by Herce and Garcia, which suggested that Tat in-
teracts with phosphate headgroups across the bilayer, facilitating the formation
of pores. The ensemble of configurations obtained from a new MD simulation
allows visualization of Tat/membrane interactions. Funded by GM44976,
GM86801, DMR-0936384(CHESS), and DOE(NIST).

484-Pos Board B239
A Systematic Study of Phase Changes Induced by Trans-Membrane Pep-
tide Gramicidin-A in Multi-Component Lipid Membranes
Ebrahim Hassan-Zadeh, Juyang Huang.
Physics, Texas Tech University, Lubbock, TX, USA.
What are the effects of proteins on lipid membrane domains? In order to answer
this question, we systematically investigated the phase changes induced by
trans-membrane peptide gramicidin-A in 16:0-18:2PC(PLPC)/
di18:0PC(DSPC)/cholesterol and 16:0-18:2PC/di16:0PC(DPPC)/cholesterol
lipid bilayers. Quaternary giant unilamellar vesicles (GUV) were prepared us-
ing our recently developed Damp-Film method. The phase boundaries of
liquid-ordered and liquid-disordered (LoþLd) coexisting region as well as
the critical points were determined using video fluorescence microscopy.
Within the phase coexisting regions, thermodynamic tie-lines were determined
using a fluorescence assay. Our results show that adding 1 mol% of gramicidin
produces significant and complex phase changes to the lipid bilayers: at some
lipid compositions, gramicidin can induce lipid domains; at others, gramicidin
completely abolish the phase separation; even if the phase separation is pre-
served, gramicidin significantly alters the lipid compositions of membrane do-
mains and tie-lines. In the biological relevant critical region, these changes
could be quite dramatic. We also measured gramicidin-A partition coefficients
between coexisting LoþLd lipid phases. Away from the critical point, the co-
efficient is close to 2, indicating that gramicidin slightly prefers the disordered
Ld lipid domains with smaller bilayer thickness. However, the partition coeffi-
cient continuously changes with lipid composition. Near the critical point, the
partition coefficient approaches to the theoretical value of 1.

485-Pos Board B240
Physical Properties of Model Membranes Containing Pope and Phytos-
terol
Ya-Wei Hsueh, Yen-Chun Chen.
Dept. of Physics, National Central University, Jung-li, Taiwan.
We have studied the effect of phytosterol on the physical properties of 1-palmi-
toyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multilamellar vesi-
cles using deuterium nuclear magnetic resonance (

2
H NMR). The sn-1 chain

of POPE is deuterium labeled. The NMR spectra were taken as a function of
temperature and phytosterol concentration. The order of POPE-d31 mem-
branes, measured through the spectral first moment, is almost not affected by
the addition of phytosterol in the gel phase, while it increases with phytosterol
concentration in the liquid-crystalline phase. A significant difference in the
ability of phytosterol to disorder the gel-phase and to order the liquid-phase
POPE membranes is observed. This finding differs from those observed in
POPE/chol and other lipid/sterol systems. Furthermore, the temperature-
composition phase diagram will be discussed.

486-Pos Board B241
Measuring the Dimerization Propensities of Mucin1 Transmembrane and
Juxtamembrane Domains
Edwin Li, Christopher Moll, Bernadette Eichman, Jessica King.
Biology, Saint Joseph’s University, Philadelphia, PA, USA.
Overexpression of the membrane protein mucin 1 (MUC1) has been linked to
75% of all human solid tumor cancers, including 90% of breast carcinomas. In
cancer cells, MUC1-MUC1 homodimerization has been associated with cell
migration and adhesion. Furthermore, this interaction is necessary for forming
complexes with growth factor receptors and targeting to the nucleus, where
MUC1 can interact with effector proteins regulating gene expression. Thus, un-
derstanding how MUC1 forms homodimers is essential for developing novel
therapeutic strategies to block its oncogenic effects. A recent study has shown
that the membrane proximal CQC motif promotes dimerization under oxidizing
conditions, suggesting that the motif may act as a redox switch in response to
changes of cytosolic oxidant levels. Aside from these few studies focusing on
the CQC motif, very little is known regarding the mechanism of MUC1 homo-
dimerization. Currently, we are using the ToxR and AraTM assays to investi-
gate if the transmembrane domain, without the cytosolic CQC motif, is able to
dimerize by itself. We are also measuring if the dimerization propensity of the
TMD changes with the membrane proximal CQC motif. The two assays allow
us to compare the dimerization propensity when the CQC motif is in reducing
and oxidizing environments.

487-Pos Board B242
Characterizing the Curve: A Mechanistic Study of CPLA2-Mediated
Membrane Bending
Katherine E. Ward1, James P. Ropa1, Robert V. Stahelin1,2.
1Chemistry and Biochemistry, University of Notre Dame, South Bend, IN,
USA, 2Biochemistry and Molecular Biology, Indiana University School of
Medicine-South Bend, South Bend, IN, USA.
Lipid membranes play a critical role in cellular signaling through selective
protein-lipid interactions. The membrane composition of organelles often
drives specific proteins to localize in cells. Lipid binding proteins, including
those harboring BAR and ENTH domains, have been shown to shape biological
membranes into vesicles necessary to transport cargo across the membrane.
Recently, we observed that the calcium-dependent enzyme cytosolic phospho-
lipase A2 (cPLA2), bends model membranes through its N-terminal C2 domain,
which is dependent upon its membrane penetration (Ward et al. JLR, 2012).
Thus, in addition to its role in generating arachidonic acid from membrane
phospholipids, this enzyme may have a role in regulating membrane curvature
changes. This hypothesis is supported by roles for cPLA2 described in the liter-
ature including intra-Golgi trafficking, Golgi tubulation, Golgi vesiculation and
Fc-receptor-mediated phagocytosis. We found that membrane bending by
cPLA2 translated into A549 and HeLa cells, supporting the physiological rele-
vance of our earlier findings. Thus, we sought to characterize the molecular
forces driving cPLA2-dependent membrane bending in vitro and in cells. Using
a variety of mCherry and mEGFP protein chimeras, we investigated the hy-
pothesis that cPLA2 oligomerizes on membranes with a series of correlation
spectroscopy experiments. These results show that cPLA2 forms large protein
oligomers on cytoplasmic vesicles using number and brightness analysis and
with an in vitro crosslinking assay. Taken together, using a variety of biophys-
ical methods, we have consistently found cPLA2 to oligomerize through its C2
domain in vitro and in cells.

488-Pos Board B243
Cubic - Inverted Hexagonal Phase Transition Kinetics in Monoolein-
Sucrose Mixtures
Zachariah I. Strango, Caleb W. Reese, Christopher J. Ver Hoef,
Paul E. Harper.
Department of Physics and Astronomy, Calvin College, Grand Rapids, MI,
USA.
Sugars play key roles in the biology, yet much remains unknown about their
interactions with lipids. In particular, we examine the effect of different con-
centrations of sucrose-water solutions on the cubic - inverted hexagonal transi-
tion in monoolein. Using DSC (differential scanning calorimetry), we ramp the
temperature up and down through the transition and measure the ramp-rate


	Hiv-1 Tat Membrane Translocation Probed by Low- and Wide-Angle X-Ray Scattering, Neutron Scattering, CD Spectroscopy and MD ...
	A Systematic Study of Phase Changes Induced by Trans-Membrane Peptide Gramicidin-A in Multi-Component Lipid Membranes
	Physical Properties of Model Membranes Containing Pope and Phytosterol
	Measuring the Dimerization Propensities of Mucin1 Transmembrane and Juxtamembrane Domains
	Characterizing the Curve: A Mechanistic Study of CPLA2-Mediated Membrane Bending
	Cubic - Inverted Hexagonal Phase Transition Kinetics in Monoolein-Sucrose Mixtures