Dissertation by Aaron T. Dossey - Things Bugs

227 Pages·2006·7.67 MB·English

CHEMICAL BIODIVERSITY AND SIGNALING:
DETAILED ANALYSIS OF FMRFamide-LIKE NEUROPEPTIDES AND OTHER
NATURAL PRODUCTS BY NMR AND BIOINFORMATICS

By

AARON TODD DOSSEY

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2006
Copyright 2006

by

Aaron Todd Dossey
To my family; to members of the laboratory of Dr. Arthur S. Edison; and to God
Almighty and the magnificent natural world he created which has given me much joy and
a basis for my career in science.
ACKNOWLEDGMENTS
As with any endeavor one may pursue in life, I cannot take sole credit for anything
I have done and, thus, thanks are certainly in order to those who have helped make my
PhD possible. First and foremost, I would like to thank my family. In particular, I thank
my grandparents, Jerry and Emma Dossey, and mother, Teresa (Dossey) Scott, for
instilling in me three key components of my successes in life thus far: determination, a
strong work ethic, and faith in myself. I would also like to thank God the creator for my
life and the bountiful life forms of this earth that I enjoy studying every day.
I would also like to thank Oklahoma State University and others who helped foster
the early stages of my career in biochemistry. I thank Dr. Eldon C. Nelson, my
undergraduate advisor, for showing great care about my career and keeping me focused
and motivated on the career related aspects of my tenure there. For many interesting and
encouraging conversations about entomology from which I learned a lot, I would like to
thank Don C. Arnold, the curator of the Oklahoma State University Entomological
Museum. I also thank the Southwestern Bell Telephone Corporation and Sylvia Coles
Denebeim for supporting me through generous scholarships which helped tremendously
with school related costs and allowed me to focus on my education.
At the University of Florida (UF), I would like to thank Dr. Arthur Edison, my
supervisory committee chair, for providing a research atmosphere that has allowed me to
develop as a scientist. I also thank Dr. Edison for his patience while I was training in
protein NMR and learning how to write scientific articles. I thank James R. Rocca in the
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UF AMRIS (Advanced Magnetic Resonance Imaging and Spectroscopy) facility for
countless hours of help and discussion. His tireless efforts in NMR training were an
invaluable complement to the training I received from Dr. Edison. Pat Jones, the
Biochemistry department secretary, was also invaluable to me by keeping me in line with
deadlines and course registration and I thank her for that as well. I also thank my
supervisory committee members (Drs. Arthur S. Edison, Ben M. Dunn, Brian D. Cain,
Joanna R. Long, and Stephen A. Hagen) for always being available to help guide me
through my PhD studies. I also thank the University of Florida for awarding me the
Grinter Fellowship for the first three years of my tenure there.
For help with specific projects, others certainly deserve my thanks. I thank Dr.
Cherian Zachariah for help, training, and experiments in protein expression and
purification and NMR. For experiments performed and exciting discussion on phasmid
insect pheromone chemistry, I thank Dr. Spencer Walse and James Rocca. For data
resulting in my first publication (Chapter 3), I thank Drs. Mario de Bono, Peter Evans,
and Vincenzina (Reale) Evans, and Heather Chatwin for bioassay experiments on FLP-18
related neuropeptides. For their support and friendship, I would like to thank Omjoy
Ganesh, Iman Al-Naggar, Ramazan Ajredini, Fatma Kaplan, Dr. James Smith, and Dr.
Terry B. Green (all fellow members of Dr. Edison’s lab).

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TABLE OF CONTENTS

page
ACKNOWLEDGMENTS.................................................................................................iv
LIST OF TABLES...............................................................................................................x
LIST OF FIGURES...........................................................................................................xi
ABSTRACT.....................................................................................................................xiii
CHAPTER
1 INTRODUCTION........................................................................................................1
Importance of Nematodes.............................................................................................1
FMRFamide-Like Neuropeptides (FLPs).....................................................................5
FLP Precursor Proteins..........................................................................................6
Receptors and Functions........................................................................................6
FLPs as Natural Products......................................................................................9
Natural Products.........................................................................................................10
Dissertation Outline....................................................................................................13
2 BIOCHEMICAL PROPERTIES OF FLPS AND THEIR PRECURSOR PROTEINS
FROM THE NEMATODE Caenorhabditis elegans..................................................17
Introduction.................................................................................................................17
Experimental Methods................................................................................................18
Data Mining for Nematode flp Precursor Protein Sequences.............................18
Alignment and Phylogenetic Analysis of flp Precursor Proteins........................20
Analysis of Biochemical Properties of flp Precursor Proteins and Figure
Generation........................................................................................................21
Analysis of FLP Mature Peptide Biochemical Properties...................................22
Results.........................................................................................................................23
Analysis of Biochemical Properties of flp Precursor Proteins............................23
Sequence Repetition Patterns.......................................................................34
Charge Distribution......................................................................................35
Unstructured Propensity...............................................................................36
Other Features..............................................................................................40

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Biochemical Properties of Mature Processed FLPs............................................41
Peptide Charge.............................................................................................44
Peptide Length and Amino Acid Conservation............................................44
Discussion...................................................................................................................47
Grouping of FLP Subfamilies by Precursor and Peptide Properties...................47
The flp-1 Group............................................................................................48
The flp-6 Group............................................................................................49
The flp-7 Group............................................................................................49
The flp-20 Group..........................................................................................50
The flp-21 Group..........................................................................................51
Charge Compensation and Possible flp Precursor Structure...............................53
3 NMR ANALYSIS OF C. elegans FLP-18 NEUROPEPTIDES: IMPLICATIONS
FOR NPR-1 ACTIVATION.......................................................................................55
Introduction.................................................................................................................55
Experimental Procedures............................................................................................56
Peptide Synthesis.................................................................................................56
Peptide Sample Preparation.................................................................................56
Biological Activity Assays:.................................................................................57
NMR Spectroscopy.............................................................................................58
Results.........................................................................................................................59
Peptide Design Rationale and Physiological Responses:....................................59
NMR Chemical Shifts Reveal Regions of flp-18 Peptides with Significant
Structure:..........................................................................................................63
pH Dependence of Amide Proton Chemical Shifts Reveal Regions of flp-18
Peptides with Significant Structure:.................................................................67
pH Dependence of Arginine Side-Chains Reveal Long-Range Interactions:.....70
Quantitative Determination of pKa Reveals Multiple Interactions:....................70
Temperature Dependence of Amide Chemical Shifts Corroborates Regions with
H-Bonding:......................................................................................................74
Overall Peptide Charge is Correlated With Activity on NPR-1:.........................75
Discussion...................................................................................................................75
The backbone structure of the conserved PGVLRF-NH is predominantly
2
unstructured......................................................................................................78
DFDG forms a structural loop stabilized by H-bonding.....................................78
The DFDG loop may interact with the second loop to form a dynamic bicyclic
structure which reduces binding to NPR-1......................................................79
Charge is also important in determining the activity of flp-18 peptides on
NPR-1..............................................................................................................79
4 ANISOMORPHAL: NEW INSIGHTS WITH SINGLE INSECT NMR.................83
Introduction.................................................................................................................83
Experimental Procedures............................................................................................88
Animal Collection and Rearing...........................................................................88
Sample Collection and Handling.........................................................................88
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NMR Spectroscopy.............................................................................................90
High Pressure Liquid Chromatography – Mass Spectrometry (LC-MS)............92
Gas Chromatography (GC)..................................................................................93
Gas Chromatography – Mass Spectrometry (GC-MS).......................................94
Results.........................................................................................................................94
NMR of Single Milkings Shows a New Component and Isomeric Heterogeneity94
Glucose Verified by Chromatography and Colorimetric Assay........................104
Stereoisomeric Heterogeneity Verified by Gas Chromatography and Mass
Spectrometry..................................................................................................105
Discussion.................................................................................................................107
Glucose Discovered in Stick Insect Defensive Spray – Potential Functions....107
Isomeric Heterogeneity in Phasmid Defensive Compounds – Chemical
Biodiversity....................................................................................................108
Peruphasmal – A Novel Phasmid Defensive Compound Isomer......................109
5 CONCLUSIONS AND FUTURE DIRECTIONS...................................................111
Conclusions...............................................................................................................111
Future Directions......................................................................................................115
Evolutionary History of FLPs and Other Neuropeptides..................................116
Neuropeptide Structure/Function Analyses.......................................................118
Anisomorphal and Other Insect Natural Products.............................................119
APPENDIX
A ACCESSION NUMBERS (WITH CORRESPONDING SEQUENCE NAMES)
FOR ALL FLP PRECURSOR PROTEIN SEQUENCES FROM ALL NEMATODE
SPECIES USED IN WORK RELATED TO CHAPTER 2.....................................121
B ALIGNMENTS OF FLP PRECURSOR PROTEINS FROM Caenorabditis elegans
AND OTHER NEMATODE SPECIES...................................................................128
C 1H NMR ASSIGNMENTS FOR ALL PEPTIDES EXAMINED IN CHAPTER 3.163
D PKA VALUES CALCULATED FOR RESONANCES WITH PH DEPENDANT
CHEMICAL SHIFTS...............................................................................................175
E 1H AND 13C NMR CHEMICAL SHIFT ASSIGNMENTS FOR DOLICHODIAL-
LIKE ISOMERS FROM THE WALKING STICK INSECT SPECIES Anisomorpha
buprestoides AND Peruphasma schultei..................................................................178
F HLPC-MASS SPEC IDENTIFICATION OF GLUCOSE FROM DEFENSIVE
SECRETIONS OF Anisomorpha buprestoides........................................................179
G 2D NMR SPECTRA OF DEFENSIVE SECRETIONS OF Anisomorpha
buprestoides AND Peruphasma schultei..................................................................181
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H GAS CHROMATOGRAPHY TRACES AND MASS SPECTRA OF DEFENSIVE
SECRETIONS OF Anisomorpha buprestoides AND Peruphasma schultei AND
EXTRACTS OF Teucrium marum...........................................................................192
LIST OF REFERENCES.................................................................................................195
BIOGRAPHICAL SKETCH...........................................................................................212

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LIST OF TABLES
Table page

1-1 Global numbers of the major human nematode infections........................................4
1-2 Sample quantities required for analysis using the three most powerful analytical
techniques for chemical structure determination......................................................14
1-3 Mean values for a selection of molecular properties among natural, drug, and
synthetic compounds................................................................................................15
2-1 Sequence patterns and properties common to various groups of flp precursor
proteins in C. elegans...............................................................................................33
3-1: Peptides examined by NMR and their activities on NPR-1.......................................61

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