Bleecker, Joan V.

Cox, Phillip A.

Keller, Sarah L.

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Monday, February 9, 2015 241aThis influx of information provides new opportunities for understanding the
chemistry of these proteins.
Using a structural bioinformatics approach, we have determined a strong asym-
metry in the charge distribution of these proteins. For the outward-facing amino
acids of the beta barrel within regions of similar amino acid density for both
membrane leaflets, the external side of the membrane contains more than three
times the number of charged amino acids as the internal side of the membrane.
Moreover, the lipid bilayer of the outer membrane is asymmetric, and the over-
all preference for amino acid types to be in the external leaflet of the membrane
correlates roughly with the hydrophobicity of the membrane lipids. This pref-
erence is demonstrably related to the difference in lipid composition of the
external and internal leaflets of the membrane. The charge asymmetry of pro-
teins in the outer membrane has important implications for how we understand
the mechanism of outer membrane protein insertion.
1215-Pos Board B166
A Fundamental Force Governing Protein Self-Assembly in Membranes
Shachi Katira1, Kranthi K. Mandadapu2, Suriyanarayanan Vaikuntanathan3,
Berend Smit4, David Chandler2.
1Bioengineering, University of California, Berkeley, Berkeley, CA, USA,
2Chemistry, University of California, Berkeley, Berkeley, CA, USA,
3Chemistry, University of Chicago, Chicago, IL, USA, 4Chemical and
Biomolecular Engineering, University of California, Berkeley, Berkeley,
CA, USA.
Using coarse-grained molecular dynamics simulations, we demonstrate the na-
ture of a proposed phenomenon that governs self-assembly of inclusions within
a lipid bilayer, inspired by the statistical mechanics of the hydrophobic effect.
We study the nature of this effect on membrane inclusions of various chemis-
tries and sizes. We identify the range of hydrophobic thicknesses over which
this phenomenon occurs and characterize the effects of the proposed phenom-
enon on small inclusions such as cholesterol versus larger, multidomain trans-
membrane proteins. Our results show that this effect can provide a force for
assembly and reorganization in a lipid bilayer based on the in-plane size and
hydrophobic thickness of the inclusion, and the melting temperature of the sur-
rounding lipids. We propose that this effect provides a physical framework that
can explain lipid raft formation.
1216-Pos Board B167
Comparing Lo/Ld Membrane Thickness Mismatch and Miscibility Tran-
sition Temperatures using Fluorescence and Atomic Force Microscopy
Joan V. Bleecker, Phillip A. Cox, Sarah L. Keller.
Chemistry, University of Washington, Seattle, WA, USA.
Micron-scale coexisting liquid phases appear in lipid bilayers composed of, at
minimum, a ternary mixture of lipids and sterols. Lipids that are predominantly
found in the liquid ordered membrane phase (the Lo phase as opposed to the
liquid disordered Ld phase) feature long, saturated carbon chains that pack
with high order at high areal densities [Veatch et al.BJ 2004]. Membrane thick-
ness is an especially interesting parameter due to its effects on the trafficking
and function of membrane proteins [Simons and Sampaio, Cold Spring
Harb. Perspect. Biol. 2011]. Experimental results [Garcı´a-Sa´ez et al. J. Biol.
Chem. 2007] present the trend that miscibility transition temperatures increase
with increasing thickness difference between the domains and the surrounding
membrane. Here we test the robustness of this result by applying the same tech-
niques (fluorescence microscopy and room temperature atomic force micro-
scopy) to different lipid mixtures than those studied by Garcı´a-Sa´ez et al.
1217-Pos Board B168
The Effects of Walp Peptides on Phase Behavior in Quaternary Lipid
Mixtures: A Molecular Dynamics Study
David G. Ackerman, Gerald W. Feigenson.
Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Quaternary lipidmixtures containing a high-melting lipid, a nanodomain-inducing
low-melting lipid, a macrodomain-inducing low-melting lipid and cholesterol
reveal behaviors not observed in ternary mixtures. Through fixing the amounts
of high-melting lipid, total low-melting lipid and cholesterol and altering only
the relative amounts of the two low-melting lipids, domain size in quaternarymix-
tures can be finely tuned from nanoscopic to macroscopic, with an intermediate
patterned phase morphology. We have previously used extensive coarse grain
and atomistic molecular dynamics simulations to study one such quaternary lipid
mixture, containing the high-melting lipid DPPC (16:0,16:0-PC), the low-melting
lipids PUPC (16:0,18:2-PC) andDUPC (18:2,18:2-PC), and cholesterol. In partic-
ular, we quantified the effect of the two low-melting lipids on domain size, align-
ment, lipid order and lipid tilt. Using those simulations as a control, we are
currently evaluating howaddingWALPpeptides to the quaternarymixture affects
the sizes, alignment and properties of coexisting phases using coarse grained mo-
lecular dynamics.Weexaminehow these properties are affectedbyboth the lengthof the WALP peptide as well as their concentration. A main focus of our work is
analyzing the extent to which theWALPs alter the onset of large-scale phase sep-
aration and domain alignment. The addition of WALP to the quaternary systems
makes these simulations some of the most complex and biologically relevant
membranes studied to date with molecular dynamics.
1218-Pos Board B169
Appearance of Modulated Bilayer Morphology for Coexisting LD and LO
Phases is Correlated with Line Tension
Sanjula P. Wickramasinghe1, David G. Ackerman2, Gerald W. Feigenson2.
1Cornell University, Ithaca, NY, USA, 2Molecular Biology and Genetics,
Cornell University, Ithaca, NY, USA.
Giant Unilamelar Vesicles (GUVs) appear uniform for the brain-SM/POPC/
Chol bilayer mixture, whereas GUVs exhibit macroscopic domains of coexist-
ing liquid-disordered þ liquid-ordered phases for the brain-SM/DOPC/Chol
mixture. We can travel through composition space in order to study this tran-
sition from nanodomains with POPC to macrodomains with DOPC. Using r
defined as the ratio [DOPC]/[DOPC]þ[POPC], the domain morphology un-
dergoes a transition regime where ‘‘modulated phases’’ appear as a function
of r. The formation of these different morphologies on a GUV can be under-
stood as a competition between line tension, which favors large domains,
and bending energy, which favors small domains (Amazon et al). We measured
the r values where modulated phases appear for different 4-component mix-
tures, using brain-SM, or egg-SM, or palmitoyl-SM as the high-melting lipid,
and DOPC/POPC or DOPC/SOPC as the low melting lipids, for a total of six
different 4-component mixtures. We then measured the line tension of the
macroscopic domains vs r, and found the same line tension at r values at
the transition between modulated phases and macroscopic phases. This finding
implies that line tension has major control over domain morphology. We are
currently studying how the transmembrane peptide, GWALP-23, changes the
morphology by changing the values of the competing interactions.
1219-Pos Board B170
Membrane Bending Modulus for Ternary Mixture Models of the Cell
Plasma Membrane
Rebecca Simpson, David Ackerman, Gerald Feigenson.
Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol
exhibit a region of liquid-ordered þ liquid-disordered phase coexistence analo-
gous to raft þ non-raft behavior in cells. These coexisting phases manifest
domain sizes that range from a few nanometers to many microns, depending
strongly on the nature of the low-melting lipid. When POPC, which gives rise
to nanodomains, is replaced by DOPC, which yields macrodomains, an interme-
diate region is observed of patterned, or modulated phases. This domain
morphology can be explained as a competition between line tension and bending
energieswith patterns occurringwhen the two are nearly balanced.Necessary for
testing thismodel aremeasurements of line tension, and bendingmoduli for both
phases. Here we report the bending moduli of coexisting Lo and Ld phases from
mixtures that produce domains ranging in size from nanoscopic to macroscopic.
Measurements were made by shape analysis of giant unilamellar vesicles with
both fluorescence and phase contrast microscopy. Vesicles of a single phase
were made by the gentle hydration method to obtain a more narrow distribution
of vesicle tensions than is obtained by electroformation. A transmembrane heli-
cal peptide, WALP23, strongly partitions to the liquid disordered phase, and
changes the size of coexisting Loþ Ld domains. We are currently investigating
the effects of WALP23 on membrane mechanical properties and line tension.
1220-Pos Board B171
Minor Changes in Sterol Structure Impact theMiscibility Temperatures of
Model Cell Membranes Significantly
Ranee C. James, Jonathan P. Litz, Sarah L. Keller.
Chemistry, University of Washington, Seattle, WA, USA.
Evolution selected for an array of structurally similar sterols in cell membranes.
While mammalian cells rely on cholesterol, yeast and fungi utilize ergosterol
and plants incorporate a variety of phytosterols. Although the chemical struc-
ture of ergosterol and phytosterols differ only slightly from cholesterol, these
small differences have been shown to significantly impact the solubility limit
of the sterols in model cell membranes [Stevens & Keller, 2010, 5882]. In
our current study, we use fluorescence microscopy to correlate the structural
features of ergosterol, stigmasterol, and ß-sitosterol with the miscibility tem-
perature of ternary membranes containing DOPC, DPPC, and a sterol. We
map the full ternary phase diagram of each system and compare it with that
of the well-characterized DOPC/DPPC/cholesterol system. Our data provides
insight into the intermolecular interactions taking place between sterols and
PC-lipids and reveals that seemingly minor changes in sterol structure have a
large impact on membrane miscibility temperatures.


Comparing Lo/Ld Membrane Thickness Mismatch and Miscibility Transition Temperatures using Fluorescence and Atomic Force Microscopy 

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Comparing Lo/Ld Membrane Thickness Mismatch and Miscibility Transition Temperatures using Fluorescence and Atomic Force Microscopy

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