Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and ...

314 Pages·2013·6.37 MB·English

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Student Research Projects, Dissertations, and
Chemistry, Department of
Theses - Chemistry Department
Winter 11-29-2010
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Matthew D. Shortridge PhD
department of chemistry, [email protected]
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Shortridge, Matthew D. PhD, "Nuclear Magnetic Resonance Affinity Screening Methods for Functional
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- Chemistry Department. 14.
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NUCLEAR MAGNETIC RESONANCE AFFINITY SCREENING METHODS FOR
FUNCTIONAL ANNOTATION OF PROTEINS AND DRUG DISCOVERY

By:

Matthew D. Shortridge

A DISSERTATION

Presented to the Faculty of

The Graduate College at the University of Nebraska

In Partial Fulfillment of Requirements

For the Degree of Doctor of Philosophy

Major: Chemistry.

Under the Supervision of Professor Robert Powers

Lincoln, Nebraska

December, 2010
NUCLEAR MAGNETIC RESONANCE AFFINITY SCREENING METHODS
FOR FUNCTIONAL ANNOTATION OF PROTEINS AND DRUG DISCOVERY

Matthew D. Shortridge, Ph.D.

University of Nebraska, 2010

Advisor: Robert Powers

With nearly 1,350 complete genome sequences available our understanding of
biology at the molecular level has never been more complete. A consequence of these
sequencing projects was the discovery of large functionally unannotated segments of
each genome. The genes (and proteins they encode) found in these unannotated regions
are considered “hypothetical proteins”. Current estimates suggest between 12%-50% of
the known gene sequences are functionally unannotated. Incomplete functional
annotation of the various genomes significantly limits our understanding of biology.
Pragmatically, identifying the functions of these proteins could lead to new therapeutics;
making functional annotation of paramount importance.
This dissertation describes the development of new methods for protein functional
annotation independent of homology transfer. The hypothesis is proteins with similar
function have significantly similar active sites. Nuclear magnetic resonance ligand
affinity screening was employed to identify and define protein active sites. The methods
developed were tested on a series of functionally diverse, annotated proteins including,
serum albumins (H. sapiens, B. taurus), and amylases (B. licheniformis, A. oryzae, B.
amyloliquefaciens H. vulgare, I. batatas), primase C-terminal domain (S. aureus),
nuclease (S. aureus) and the type three secretion system protein PrgI (S. typhirium).
Functional annotation using protein active sites require a high-resolution three-
dimensional structure of the protein. In addition to method development, this dissertation
describes the NMR solution structure of Staphylococcus aureus primase carboxy-
terminal domain (CTD). The primase CTD is essential for bacterial DNA replication and
distinctly different from eukaryotes. With the rapid rise in antibiotic resistance, the
primase CTD of S. aureus is an attractive antibiotic target. The methods used for
functional annotation were used to screen S. aureus primase CTD to identify the
compound acycloguanosine as a binding ligand to primase CTD.
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Copyright 2010
iv

Dedicated to:
Orville E. Miller

Acknowledgements:
The successful completion of this long academic journey required focus,
enthusiasm and above all else an extreme amount of support from colleagues, family and
friends. Without their helpful encouragement I would not be writing this dissertation. I
would first like to extend my greatest gratitude to my research advisor Dr. Robert
Powers. Your belief in me and constant encouragement lead to my success at Nebraska.
With your guidance and patience I was able to take an interest in NMR and turn it into an
understanding of NMR. Additionally, you have provided me with skills beyond the
technical that will continually assist me on the even larger academic journey I now begin.
My interest in science stretches beyond the halls of Hamilton and started at an
early age. This is because of the excellent science education I was lucky to receive early
in life. Thank you to all my teachers and professors at all levels of my education that
pushed me to excel. I would specifically like to thank my committee members, Dr. Mark
Griep, Dr. Liangcheng Du, Dr. James Takacs, and Dr. Greg Somerville for giving me the
freedom to explore and the focus to finish.
While working as a teaching assistant in the instrumentation facility I was
fortunate to work with two very talented spectroscopists. A warm and special thank you
goes to Dr. Joe Dumais for teaching me much more than how to shim a magnet. Your
guidance, mentorship and friendship were pivotal to my success both academically and
personally at Nebraska. An equally warm thank you goes to Sara Basagia. Sara, our
time spent filling magnets with helium and occasionally our bellies with beer (after the
v

fills of course) will be some of the happiest memories I leave with. I will truly miss you
both.
I was lucky to work with a number of talented colleagues and collaborators here
at Nebraska and throughout the scientific community. In the Powers lab I would like to
thank Andy, Bo, Jamie, Jenni, Kate, Kelly, Lisa, Mark, Mike, Paxton, Steve, and Visu,
each of you helped make our days analyzing spectra, going to conferences, and studying
for classes much more fun and rewarding. I was able to work with two very talented
undergraduate students, Andy Kichner and Michael Bokemper; both contributed greatly
to my research and this dissertation. Mike good luck in medical school and when you get
done with that I have a gel for you to run. Thank you to Dave Nelson and Chris Frey for
helping me with all my protein expression and purification questions, your experience
and insight was greatly beneficial and appreciated. I would also like to extend a warm
thank you to the talented collaborators I was lucky to work with, Dr. Peter Revesz and
Thomas Triplet both were pivotal in the development of the PROFESS database and the
structure comparison work described in chapter 6.
To all my family and friends who have supported me during this long and
rigorous journey I am in much debt to you all. Mom and Dad, thank you and I love you
both. You have always believed in me and have helped keep me on tract so that I could
reach my goals. Every day I strive to model my life after your high level of honesty,
integrity, unconditional love and determination. To my sisters Megan and Mindy, I love
you both and wish you luck and fortune in the upcoming years. I know you both will go
far.
vi

To my grandparents, you have been the constant and solid foundation in my life
and I love you all. The positive environment the four of you provided me while I was
young has contributed greatly to my current success. Specifically, my love of nature and
science is a direct consequence of my time spent outdoors with my grandparents, Orville,
and JoAnn. Together they instilled in me a respect of the natural world that cannot be
learned in any textbook. Additionally, I must attribute most of my mechanical abilities to
my grandparents Harley and Rose. The summers spent working in their shop provided me
with the troubleshooting skills and hard work ethic I now use every day.
To my new family Ron, Roxanne, and Veronica, you have taken me in and treated
me as your own from day one. I love you and look forward to building new memories
with you. I will even start rooting for the USC Trojans on football Saturdays, at least
when they are not playing the Huskers!
Lastly, to my wife Ray, I love you with all my heart. Meeting you has been the
luckiest and best thing to ever happen to me. Being able to share this journey with you
and our two boys, Watson and Beaker, is truly a gift.
vii

TABLE OF CONTENTS:

CHAPTER 1
GENERAL INTRODUCTION…………………………………...….........……….…....1
1.1 General introduction to functional genomics…………………..…....….…......1
1.2 Introduction to protein functional annotation…………………………..……..3
1.3 Annotation of function using ligand binding……………………….......……..9
1.4 General principles of high-throughput
nuclear magnetic resonance screening.............................................................11
1.5 Summary of work………………………………………………......………..14
REFERENCES………..........................................................................................………16

CHAPTER 2
ESTIMATING PROTEIN-LIGAND BINDING AFFINITY USING HIGH-
THROUGHPUT SCREENING BY NMR..……………………………… ……..……41
2.1 INTRODUCTION………………………………………………………..…....…….41
2.2 THEORY……………………………………………………..……....................…...43
2.2.1 Single point K measurements………………………………….................43
D
2.3 EXPERIMENTAL…................................................................................……..........46
2.3.1 Materials…………………………………...............................................................46
2.3.2 Apparatus………………………………………………...……....………...46
2.3.3 Sample Preparation……………………………………...………....………47
2.3.4 1D 1H NMR binding curves…………………………….………...…..…...47
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2.3.5 Measuring a free ligand NMR linewidth ……………………......…...…48
F
2.3.6 Simulated high-throughput screening by NMR………….…………...........48
2.4 RESULTS AND DISCUSSION……………………………..……………................49
2.4.1 Measuring K from 1D 1H NMR line-broadening experiments…...……....49
D
2.4.2 Co-variance of K and the NMR linewidth ratio …………….....…..…......53
D
2.4.3 Sensitivity of K and NMR linewidth Ratio c…….....……………........….57
D
2.4.4 Comparison of estimated K values with literature values……..…………57
D
2.4.5 Estimating K based on single-point
D
1D 1H NMR line-broadening Measurements……….…...........................…60
2.5 REFERENCES………................................................................................................64
APPENDIX…………...................................................................................................….72

CHAPTER 3
STRUCTURAL AND FUNCTIONAL SIMILARITY BETWEEN THE
BACTERIAL TYPE III SECRETION SYSTEM NEEDLE PROTEIN
PRGI AND THE EUKARYOTIC APOPTOSIS BCL-2
PROTEINS.......................................................................................................................79
3.1 INTRODUCTION..………… ……………………..………...........................…...…79
3.2 EXPERIMENTAL……………………………...……………................................…81
3.2.1 FAST-NMR screen of PrgI……………...…………................................…81
3.2.2 Structure similarity searching……………………..…………….................85
3.2.3 Sequence similarity searching using BLAST and T-Coffee……….............85
3.2.4 Secondary binding site similarity between Bcl-xL and PrgI……................86