THEORETICAL AND EXPERIMENTAL APPROACHES By ADAM SCOTT GOLER A dissertation ...

213 Pages·2015·14.14 MB·English

UNDERSTANDING BIMOLECULAR MACHINES: THEORETICAL AND
EXPERIMENTAL APPROACHES
By
ADAM SCOTT GOLER
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
Department of Physics and Astronomy
MAY 2015
(cid:13)c Copyright by ADAM SCOTT GOLER, 2015
All Rights Reserved
(cid:13)c Copyright by ADAM SCOTT GOLER, 2015
All Rights Reserved
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To the Faculty of Washington State University:
The members of the Committee appointed to exam-
ine the dissertation of ADAM SCOTT GOLER find it
satisfactory and recommend that it be accepted.
James A. Brozik, Ph.D., Chair
Susan Dexheimer, Ph.D.
Philip Marston, Ph.D.
Matthew McCluskey, Ph.D.
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DEDICATION
To Ollie and Baxter,
who taught me to stop and smell the roses.
They are forever loved,
and deeply missed.
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ACKNOWLEDGEMENTS
This dissertation represents the culmination of my career as a student, which began
long before I matriculated to Washington State University. In fact, it began with my parents
Lori and Andy Goler, who deserve the deepest and most profound acknowledgments and
thanks. For without their unwavering support, love and guidance I would have never been
abletosucceed. Ialsooweagreatdebtofgratitudetomyextendedfamilyandgrandparents.
In particular I would like to thank my grandmother and grandfather Harriet and George
Goler, whoalwaysencouragedandfacilitatedmyloveforreadingduringchildhood. Grandpa
George would certainly have loved to read this dissertation!
My grandfather George Braunstein also deserves incredible thanks for his constant
reminders not to forget to enjoy the finer things in life, and for teaching me to play cards.
Cousins and fellow scientists Jonathan Goler and Alexander Braunstein also deserve great
thanksfortheirsupportandadviceaboutnavigatingthepitfallsofgraduateschoolingeneral
and for being living proof that there is much adventure still to be had afterwards.
Next, no academic acknowledgments section could possibly be complete without ac-
knowledging the academic personnel who made this work possible. My expertise in physics
gained over the past several years is largely due to the diligent instruction by many talented
individuals. I would like to thank professors Fred Gittes and David Keller for many very
helpful discussions on topics related to biophysics. I would also especially like to thank my
committee members Philip Marston, Matthew McCluskey, and Susan Dexheimer for their
advice and input on shaping this dissertation and throughout graduate school in general. In
addition to professors, a deep and profound thank you is due to the administrative personnel
of the WSU Physics and Astronomy department, who assisted me in all stages of my grad-
uate studies with seemingly iron resolve and abundant expertise. Thank you very much to
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Sabreen Dodson, Laura Krueger, Kris Boreen, Mary Guenther, Leslee Kimbleton, and Tom
Cowger. To Sabreen, enjoy your retirement!
My undergraduate professors Nathan Harshman, Philip Johnson, William Parsons,
Michael Black, Teresa Larkin, Stephen Casey, Ali Enayat, Joshua Lansky, Dan Kalman,
Jeffrey Adler, and Angela Wu deserve extreme gratitude for their patience, effort, and total
dedication to their students–a set I was most fortunate to be an element thereof. Their
combined effort greatly reduced the friction during the transition from undergraduate to
graduate education.
Despite the long list of academic professionals mentioned so far, so many thanks are
due to great friends who have always supported my efforts and made time spent outside of
lablotsoffun, whetherbysharingabeer, playingboardgames, orpartakinginmiscellaneous
adventures. Thanks to the "food science crew" Ford and Mariah Childs, Jesse and Abbey
(and Sparty!) Zuehlke, Lauren Schopp, Blake Herron, and Anne Zwink. Thanks also to
my great friend Josef P. Felver (esquire) and his dear wife Kate, with whom I shared the
brightest and darkest days of graduate school. Josef remains the Spock to my Kirk, the
Chewbacca to my Han. To Josef, I have been and always shall be your friend. Thanks are
also due to my dear friend Starbuck, who was always ready to accommodate a break from
my research with hikes, games of fetch and general revelry.
Further profound thanks are due to my stalwart spiritual advisor, hotshot strength
coach, Captain America historian and Japanese cultural guru Jordan C. Kessler, and no less
to Beth Kessler, and of course to my K9 nephew Big Lou. The Kesslers have always been
there for me through thick and thin with gracious love and support in times of darkness
and shadow. They’ve also enjoyed a lot of ridiculous puns that I’ve made without remorse
over the years, which is certainly no small feat. Master Evan Douglas Wong is a friend
who similarly enjoyed my puns (sarcasm, too) and besides always being ready with highly
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systematic, logical and friendly advice, was also sometimes my go-to for odd combinatorics
and math related queries. I would also like to thank John Lang, who was among one of the
first students I met at WSU, and who provided significant guidance in the early stages of
my graduate career.
In addition to being friends, my fellow group-members have been excellent colleagues
willing to lend their expertise to me on many occasions, which was of extreme value in
such interdisciplinary research. I am deeply grateful to Adam Barden (and his excellent
beard), Samaneh Tabatabaei, Sara Humphreys, Elsa Silva-Lopez, and Angela Rudolph for
their support in this regard.
I would also like to thank my girlfriend, Courtney, for her constant support and love
throughout the latter parts of my graduate school career. I feel profoundly lucky to have met
her during my tenure at WSU, and even more lucky to have been inspired by her motivation
and her endless capacity for humor. Her love and companionship have truly been a blessing
without which the world would be that much darker a place. Frankly, I am sick and tired of
not being married to this woman.
Finally, I would like to thank my advisor, professor James Brozik. Dr. Brozik has been
a great friend and mentor to me over the past few years, and has given me countless oppor-
tunities as a developing scientist. For his dedication, expertise, mentorship and friendship, I
am grateful to the utmost.
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UNDERSTANDING BIMOLECULAR MACHINES: THEORETICAL AND
EXPERIMENTAL APPROACHES
Abstract
by Adam Scott Goler, Ph.D.
Washington State University
May 2015
Chair: James A. Brozik
This dissertation concerns the study of two classes of molecular machines from a phys-
ical perspective: enzymes and membrane proteins. Though the functions of these classes of
proteinsaredifferent, theyeachrepresentimportanttest-bedsfromwhichnewunderstanding
can be developed by the application of different techniques.
HIV1 Reverse Transcriptase is an enzyme that performs multiple functions, including
reverse transcription of RNA into an RNA/DNA duplex, RNA degradation by the RNaseH
domain, andsynthesisofdsDNA.Thesefunctionsallowfortheincorporationoftheretroviral
genes into the host genome. Its catalytic cycle requires repeated large-scale conformational
changes fundamental to its mechanism. Motivated by experimental work, these motions
were studied theoretically by the application of normal mode analysis. It was observed that
the lowest order modes correlate with largest amplitude (low-frequency) motion, which are
most likely to be catalytically relevant.
Comparisons between normal modes obtained via an elastic network model to those
calculated from the essential dynamics of a series of all-atom molecular dynamics simu-
lations show the self-consistency between these calculations. That similar conformational
motions are seen between independent theoretical methods reinforces the importance of
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large-scale subdomain motion for the biochemical action of DNA polymerases in general.
Moreover, it was observed that the major subunits of HIV1 Reverse Transcriptase interact
quasi-harmonically.
The 5HT3A Serotonin receptor and P2X1 receptor, by contrast, are trans-membrane
proteins that function as ligand gated ion channels. Such proteins feature a central pore,
which allows for the transit of ions necessary for cellular function across a membrane. The
pore is opened by the ligation of binding sites on the extracellular portion of different protein
subunits. In an attempt to resolve the individual subunits of these membrane proteins
beyond the diffraction limit, a super-localization microscope capable of reconstructing super-
resolution images was constructed. This novel setup allows for the study of discrete state
kinetic mechanisms with spatial resolution good enough to distinguish individual binding
sites of these membrane proteins. Further use of this technique may allow for the study of
allostery and subunit specific stoichiometry in the presence of agonist or antagonist ligands
relevant to pharmacology.
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Contents
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Introduction 1
1.0.1 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.0.2 Ligand Gated Ion Channels (LGICs) . . . . . . . . . . . . . . . . . . 8
1.1 Theoretical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.1.1 Normal Mode Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.1.2 Radial Distribution Functions and the Potential of Mean Force . . . . 24
1.2 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
1.2.1 Fluorescence Recovery After Photobleaching (FRAP) . . . . . . . . . 29
1.2.2 Total Internal Reflection Fluorescence Microscopy (TIRFM) . . . . . 30
1.2.3 Super-resolution and Super-localization Microscopy . . . . . . . . . . 34
1.3 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2 Functional properties of HIV1 Reverse Transcriptase from the Anisotropic
Network Model and Essential Dynamics 49
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.2.1 Selection of Crystal Structures . . . . . . . . . . . . . . . . . . . . . . 53
2.2.2 Molecular Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . 55