wide angle x-ray scattering probes chain order and identifies liquid-liquid phase coexistence in ...

294 Pages·2007·12.26 MB·English

by  Thalia Mills

WIDE ANGLE X-RAY SCATTERING PROBES CHAIN ORDER
AND IDENTIFIES LIQUID-LIQUID PHASE COEXISTENCE IN
ORIENTED LIPID MEMBRANES

A Dissertation
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

by
Thalia Tilden Mills
August 2007
© 2007 Thalia Tilden Mills
WIDE ANGLE X-RAY SCATTERING PROBES CHAIN ORDER
AND IDENTIFIES LIQUID-LIQUID PHASE COEXISTENCE IN
ORIENTED LIPID MEMBRANES
Thalia Tilden Mills, Ph.D.
Cornell University 2007

We have used grazing incidence wide-angle x-ray scattering (GIWAXS) on oriented
lipid multilayers to measure chain order and to examine liquid-liquid coexistence in
the system DOPC/DPPC/cholesterol, a model for the outer leaflet of the cell plasma
membrane. Coexistence of liquid-disordered (Ld) and liquid-ordered (Lo) domains is
thought to be related to "rafts" in the cell membrane, cholesterol-rich lipid
heterogeneities which provide platforms for protein sorting. Many of the methods
used for measuring liquid-liquid coexistence in model membranes require a potentially
perturbing probe, while x-ray scattering is probe-free. In unoriented (powder) x-ray
data, scattering from the Ld and Lo phases looks very similar, whereas in GIWAXS
patterns from oriented samples, these phases are easily distinguishable because of the
differences in their chain orientational order. By using a simple analytical model to
relate the GIWAXS data to the chain orientational distribution, we fit our data to
obtain the average chain orientational order parameter, S . While this type of
mol
analysis has been well-used for liquid crystals, it is not commonly applied to model
membrane systems. For DOPC/cholesterol and DPPC/cholesterol mixtures,
composition and temperature dependent trends in S determined by GIWAXS are
mol
consistent with earlier NMR data. Addition of 40% cholesterol to liquid-phase DPPC
or DOPC more than doubles S . In addition to measuring chain orientational order
mol
parameters for binary mixtures of DOPC/cholesterol and DPPC/cholesterol, we have
measured GIWAXS for ternary mixtures where fluorescence microscopy and NMR
indicate the coexistence of Ld and Lo phases below the miscibility transition
temperature, T . In order to fit to the GIWAXS data for these mixtures at low
mix
temperature, we required two values of S , which we interpret as evidence of
mol
coexisting Ld and Lo phases. Our T values based on x-ray work agree reasonably
mix
(to within the 5-10˚C temperature steps used) with the T values based on the NMR
mix
and microscopy work of Veatch et al. (Veatch and Keller, 2003b; Veatch et al., 2004;
Veatch et al., 2007b). This approach provides a new method for examining phase
coexistence in model membranes without the need to add a potentially perturbing
probe.
BIOGRAPHICAL SKETCH
Thalia Tilden Mills attended Swarthmore College, where she received a B.A.
in 2000 with majors in physics and chemistry. She started Cornell University in the
physics Ph.D. program in the Fall of 2000. Her most significant accomplishment
during seven years of graduate school was making (and eating) many, many pans of
brownies from Rosie's cookbooks (introduced to her by her friend Otavia). Thalia
highly recommends purchasing both cookbooks by Judy Rosenberg: Rosie's Bakery
All-Butter, Fresh Cream, Sugar-Packed, No-Holds-Barred Baking Book and Rosie's
Bakery Chocolate-Packed Jam-Filled Butter-Rich No-Holds-Barred Cookie Book.
Her favorite recipe is probably the peanut butter brownies, but the most popular seem
to be the cream cheese brownies ("boom booms").
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To Mom ("D." or "S.") and Dad

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ACKNOWLEDGMENTS
Many thanks to my thesis advisor, Dr. Gerald Feigenson, and Drs. Stephanie
Tristram-Nagle and John Nagle, my current employers and collaborators on the thesis
project who were very helpful with both experiments and supervision of the thesis
writing process. I learned much from the collaboration between the Feigenson and
Nagle labs. This work greatly benefited by being able to combine expertise in lipid
phase diagrams (Feigenson lab) with expertise in lipid x-ray scattering (Nagle lab).
I owe a great debt to Gil Toombes. I am very happy that Gil shared his idea
for the GIWAXS project with me. In addition to giving me the idea for the project,
Gil also helped with the analysis. Without Gil's help, I may have ended up with a very
big pile of pretty x-ray data and no way to analyze it.
This work would not have been possible without the support of my labmates in
the Feigenson lab: Nelson Morales, Fred Heberle, Jiang Zhao, and Jing Wu. They
spent many hours at CHESS helping to collect data and preventing me from melting
down. Dr. Norbert Kučerka (formerly Nagle lab) also helped collect data.
I thank Drs. Detlef Smilgies and Arthur Woll for their help with the CHESS
experiments. I appreciate that the CHESS staff are so willing to take risks on people
and projects. Two years ago Detlef got the ball rolling on this project by offering me a
week of beamtime after a brief meeting.
I thank Drs. Mark Tate and Gil Toombes for their help with the rotating anode
work. I particularly appreciated their patience.
I thank Dr. Sarah Veatch, Dr. Adam Hammond, Dr. Jonathan Sachs, and
Elaine Farkas for their encouragement and interest in this project. Sarah was kind
enough to supply me with her NMR data before publication.
Jianjun Pan, my office-mate for the bulk of the thesis writing process, has
definitely lived up to the gold standard set by my former labmates.
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I thank my committee members, Drs. Sol Gruner, Carl Franck, and Jim Sethna
for critical reading of the thesis.
I thank the following sources of support for this thesis work: NIH Molecular
Biophysics Training Grant, NSF MCB-0315330 to G. W. Feigenson, NIH Grant GM
44976 to the Nagle group, NSF DMR-0225180 to CHESS, and DOE DE-FG02-
97ER62443 for support of the Gruner anode.
In addition to the work presented in this thesis, I was fortunate to work with a
number of people on a variety of projects at Cornell. While in the Feigenson lab, I
learned about neutron scattering from lipids from Drs. Jeremy Pencer, David
Worcester, Susan Krueger, and John Katsaras. While in the Pollack group, I learned
about nanofabrication and x-ray scattering from nucleic acids from my advisor, Dr.
Lois Pollack, from my labmates Lisa Kwok (Dr. Lisa now) and Greg Maskel, and
from CHESS scientists Drs. Ken Finkelstein and Ernie Fontes. I thank Lisa and Greg
for putting up with me and for providing me with some of my best memories from
graduate school. Drs. Sol Gruner, Don Bilderback, and David Barbano mentored me
during a summer of x-ray scattering from cheese, which makes a great conversation
starter.
Thanks to my undergraduate thesis advisors: Profs. Robert Pasternack and
Peter Collings. They and my other Swarthmore professors were always encouraging.
My family has been supportive throughout this process. Mom, Dad, Debby,
Stephanie, Nate, Alicia, and Tilden: I thank you for continuing to send me postcards
and phone me despite serious neglect on my part.
Thanks to Elizabeth and Dana for being great housemates for 6.5 years;
because of you, Ithaca became home. And thanks to Gil for looking after me, for
writing my family postcards and for sending them cookies.
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And thanks to everyone who helped make great memories for me during
graduate school including: "The Roadtrip" to start out graduate school, Swarthmore
reunions in Ithaca, the Fit Bird Society, hiking and camping adventures, Cortland
trips, IM basketball, berry picking, Spike's, Mrs. Hysters, the Mermaid Inn, the
Gilmore Girls, and trips to the Dairy Bar. Special thanks to Henri, Anthony Trollope,
Rosie's cookbooks, and Lisa's Rum Cookery for providing me with endless sources of
entertainment.
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TABLE OF CONTENTS

Biographical Sketch................................................................................................... iii
Dedication.................................................................................................................. iv
Acknowledgments......................................................................................................v
Table of Contents....................................................................................................... viii
List of Figures............................................................................................................ xii
List of Tables............................................................................................................. xv
List of Abbreviations................................................................................................. xvi
List of Symbols..........................................................................................................xviii

1 Introduction................................................................................. 1
1.1 Overview............................................................................................ 1
1.2 Cell membranes and the raft hypothesis............................................ 1
1.3 Lamellar lipid phases......................................................................... 5
1.4 Chain order.........................................................................................9
1.4.1 Definitions of orientational order, conformational order,
and lateral positional order................................................................. 9
1.4.2 Chain order parameters.......................................................... 11
1.5 Role of cholesterol in phospholipid phase behavior.......................... 14
1.6 Phase coexistence in model membrane systems................................ 17
1.6.1 The DOPC/DPPC/cholesterol phase diagram........................ 17
1.6.2 Comparison of methods for detecting phase coexistence...... 21
1.7 X-ray scattering from model membranes.......................................... 24
1.7.1 X-ray scattering basics........................................................... 24
1.7.2 Structure in model membranes: lamellar and chain-chain
ordering.............................................................................................. 26
1.7.3 Types of samples: oriented vs. powder.................................. 26
1.7.4 The different lipid lamellar phases: what do x-rays see?....... 29
1.7.5 Potential of GIWAXS on oriented lipid multilayers in the
context of previous x-ray work on model membranes.......................32
1.8 Thesis summary................................................................................. 34

2 Experimental............................................................................... 36
2.1 Introduction........................................................................................ 36
2.2 X-ray sources..................................................................................... 38
2.3 GIWAXS on oriented lipid multilayers............................................. 39
2.3.1 Preparation of oriented lipid multilayers............................... 39
2.3.2 Sample chamber..................................................................... 43
2.3.3 Beamline description............................................................. 45
2.3.3.1 Overall schematic....................................................... 45
2.3.3.2 X-ray optical setup..................................................... 48
2.3.3.3 Beamstop....................................................................49
2.3.3.4 Detectors.................................................................... 52
2.3.3.5 Calibration of sample-to-detector distance................ 53
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