Single-Molecule Studies of Ion Channels Expressing Unnatural Amino Acids Thesis by

361 Pages·2011·9.45 MB·English

by  Brandi Shanata

Single-Molecule Studies of Ion Channels

Expressing Unnatural Amino Acids

Thesis by:

Jai Anand Pattur Shanata

In Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

California Institute of Technology

Pasadena, CA 91125

2011

(Defended June 1, 2011)
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© 2011

Jai Anand Pattur Shanata

All Rights Reserved
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Dedicated to Brandi Shanata
The best partner I could hope for
Whose kindness, intelligence, and support
Surpass my wildest dreams everyday
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Acknowledgments
This section is important; I started it ~ 5 years ago—it is the first section of
my thesis that I began writing. In many ways it is still a work in progress, but I
hope that it now at least conveys a sense of my appreciation for the incredible
support I have received both specifically here at Caltech and broadly in getting to
this stage in my life. I will acknowledge people from several categories: research
mentors, members of the Dougherty and Lester Labs, other academic resources,
the GSC, Student Affairs, and Caltech Administration, my pre-Caltech education,
my family and friends, and a variety of other support.

Research Mentors
Professor Dennis Dougherty is a truly special person. A gifted scientist,
he is also incredibly funny and empathetic. Dennis is an awesome adviser who
was a great fit for me. I cannot possibly think of a way to express my gratitude or
convey the incredible support he provided me in just a few words. So, while
Dennis is generally a man of few words, I will take the liberty to use a few extra
words, as is my nature, to try to recognize some of the many ways in which he
supported me.
Throughout my time in the Dougherty group, I had a weekly meeting with
Dennis. On numerous occasions, I left Dennis‘ office with a significantly different
outlook than when I entered it. A specific subset of ~ 10 roughly 30 minute
conversations are probably the most insightful, revolutionary, and critical to the
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development of my scientific thinking: certainly they constitute the most
productive 5 hours in my graduate career. Dennis made those happen and knew
when to make them happen (I don‘t know how!).
Whether Dennis fully realized it or not, > 95% of the time he gave exactly
what I needed: interesting discussions of science, encouragement, and even the
occasional gentle but firm push to counteract my perfectionist nature and propel
me to the finish line. In an extremely important way, Dennis taught me not to
care so much about details, when they aren‘t important—his comments on my
inveterate editing were very didactic! Dennis‘ capacity to motivate, reassure, and
ultimately inspire the best in his graduate students is, as far as I can tell,
unparalleled. In many ways, this seems to arise from the fact that Dennis highly
tailors his mentoring to each of his graduate students as individuals. At the core,
his expectations of us are what we need to reach the goals that we each
articulate for ourselves. Yet, they are consistently high expectations. As Dennis
put it once—a good sign that we‘re engaged in our research project is that we‘re
thinking about our science in our morning showers. And yet, Dennis also
strongly demonstrates the value and importance of time with family and friends,
as well as taking vacation.
Perhaps one of Dennis‘ qualities that has consistently impressed me most
is his understanding of the mundane (i.e., banal notation) and also scientific
concepts at a general level. This makes communication with him facile (one
doesn‘t have to sweat the details), but also highlights his approach to science.
So-called experts in a field may thrive based on their ‗deep‘ understanding of
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their subject, but Dennis adds to this an important desire to understand broader
impacts of our research, and the role of science and teaching in the world.
Dennis regularly impressed me with his ability to see from different perspectives
—he doesn‘t need standardized forms of presentation, he just gets it.
Early in my time at Caltech, Dennis opened my eyes to an unfortunate
reality—we have far too little time both in graduate school and life. This forced
me to think more carefully about the commitments that I took on both in lab and
more broadly in life. I still find this limiting reality to be deeply saddening, but
Dennis‘ clear early guidance on this issue helped me to overcome a frantic effort
to take on every worthwhile experiment and task and focus on a few of the most
important. I particularly want to recognize Dennis‘ resolute support of my
interests outside of lab—TAing, GSC academics committee, GSC Chair, and
taking a sabbatical for an external teaching experience. These greatly enhanced
my career. Dennis has been a stalwart advocate of his students, even when it
was not necessarily in the immediate best interest of that student‘s experiments
in lab. Perhaps this is because he recognizes the importance of the whole
person to the development of good scientists.

Professor Henry Lester is great on a log. By this, I mean that my one-on-
one conversations with Henry have always been fantastic, personalized learning
experiences. I particularly enjoyed the few occasions that Henry jumped into lab
with me. Henry also made it a point to specifically inquire about how I felt about
my projects, progress, and life. His questions sparked organic conversations,
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which gave me an opportunity to reflect on my time in the lab and how I felt both
intellectually and emotionally—something that I always wanted and planned to do
more of but which otherwise rarely happens.
While Henry is well known for his infectious passion for science, I had the
opportunity of seeing Henry in other capacities beyond the lab. From his time as
Chair of the Faculty Board, Henry made a strong impression on me when he
commented ‗if a few students come to me with a concern, then I have to do
something about it‘. Later, as Chair of the Caltech Mental Health Task Force
(MHTF), Henry and some of his committee members attended dinners that the
Graduate Student Council hosted to discuss graduate student mental health and
invited us to present our findings to the full task force.
From sharing his excitement about science, to caring about me as a
person, to working together on quality of life issues for graduate students, Henry
has been an instrumental part of my time here at Caltech.

Bruce Cohen taught me both about performing single-channel recording
and data analysis in QuB. He also shared his deep understanding and
knowledge of background literature with me. For the first year-and-a-half at
Caltech, Bruce was, in many ways, the direct mentor to whom I was closest and,
as a result, shaped the scientist I am today.
Bruce helped me to think beyond my immediate experiments to what the
implications are, and how my results fit (or don‘t) into the bigger picture of
science. At the same time, he helped me to realize the importance of every
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single piece of data, especially for single-channel recording. I also greatly
appreciate that, throughout my time at Caltech, Bruce has clearly been interested
in my success and willing to give strong advice about how to achieve my career
goals.

My science and professional life have greatly benefitted from
conversations with the three other members of my Thesis Committee: Professor
Peter Dervan, Professor Doug Rees, and Professor Bil Clemons. As my
Committee Chair, Peter has given me useful advice and taken a clear interest in
my career. In addition to being on my Thesis Committee, I had the opportunity to
TA Chem 1B with Doug for two years. I appreciate Doug‘s thoughtful approach
both to the specific course material and to the way we taught it.

Dougherty and Lester Labs
In addition to these fabulous mentors, I would like to acknowledge the
support, both scientifically and personally, that I received from other graduate
students, SURFs (Summer Undergraduate Research Fellows), and high school
student interns, in the Dougherty group as well as postdocs and graduate
students in the Lester lab. Although for advice and discussion of single-channel
experiments and results, my three mentors were my first stop, for everything
else, I turned primarily to those in my bay. My time at Caltech was greatly
enhanced by hundreds of conversations with Mike Torrice, Amy Eastwood,
Kiowa Bower, Katie McMenimen, Ariele Hanek, Sean Kedrowski, Angela Blum,
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Nyssa Puskar, Darren Nakamura, Maggie Thompson, and Jinti Wang. My
science benefited significantly from collaborations with several lab-mates,
including separate projects with: Kristin Gleitsman and Shawna Frazier, Joanne
Xiu and Nyssa Puskar, Ximena Da Silva, Angela Blum, and Nyssa Puskar, and
Maggie Thompson.
I would like to thank Lori Lee for taking the time to be my first mentor in
the Dougherty group, before I had even officially joined, and introducing me to
some of the lab‘s molecular biology as well as the people. Mike Torrice was a
cohesive, nucleating force in lab. I had the fortune of sitting next to him for my
first 3 years and it wasn‘t unusual for our conversation topics to range from free
will, to race, to improving the editorial standards of Chemical and Engineering
News photographs, to race, to creating graphs of the distribution of the political
affiliations of the lab. And we would start again the next day. Overall, Mike
taught me that people are more resilient than they might seem and my life is
richer for talking and laughing with him. Mike is also a talented scientist, and I
appreciate his feedback on my candidacy proposals and numerous discussions
about our science.
Ariele Hanek taught me to use the OpusXpress early in my time in lab.
Then, for the next few years, she continued to be a considerate lab mate with
great advice on science and life. Ariele also provided useful feedback on my
candidacy research report. Kiowa Bower was a good friend and supportive lab
mate throughout my time in the Dougherty group. He brought a very positive
attitude to lab every day and is an incredibly caring person. He, too, provided
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thoughtful feedback on my candidacy research report. Kristin Gleitsman is as
inspired a scientist as she is a mother. I genuinely enjoyed working with her on
what turned into the ‗ELFCAR‘ project (Chapter 3) and the hard work she puts
into her science makes her a great collaborator. She also has a breadth of
interest and talents in science—from lab to computational work. Kristin‘s
dedication to her students as a TA encouraged me to work harder as a TA for
Chem 1B and helped to keep up my interest and enthusiasm in teaching during
graduate school. In addition to the graduate students who I worked directly with
on single-channel recording (Shawna Frazier, Maggie Thompson, and Ximena
Da Silva), I strongly appreciate the effort that Kristin, Ariele, and Kiowa made to
learn the strengths and weaknesses of single-channel recording and help to
integrate this new type of data into our lab.
While our research projects never significantly overlapped, Sean
Kedrowski and I did bond over our enthusiasm for talking about stocks, science,
and politics. I enjoyed working with Kay Limapichat (as well as Katy Muzikar and
Brinton Seashore-Ludlow) to TA Chem 3A in 2005–2006. Angela Blum shaped
the lab with her rigorous work ethic and strong critical thinking skills. She is
tenacious and often argues persuasively for her views. Angela and I made time
on several occasions, though regrettably with diminishing frequency over the
years, to discuss our shared interest of teaching at small liberal arts colleges. I
treasure those conversations that we did have, and I look forward to continuing
these mutually beneficial conversations about teaching and research in the
liberal arts environment in the coming years.