(Dreissena polymorpha) Byssal Adhesion Apparatus

126 Pages·2010·8.84 MB·English

Investigation of the Protein Components of the
Zebra Mussel (Dreissena polymorpha) Byssal
Adhesion Apparatus

by
Trevor Gilbert
A thesis submitted in conformity with the requirements
for the degree of Masters of Applied Science
The Institute of Biomaterials and Biomedical Engineering
University of Toronto
© Copyright by Trevor Gilbert 2010
Investigation of the Protein Components of the Zebra Mussel
(Dreissena polymorpha) Byssal Adhesion Apparatus
Trevor Gilbert
Masters of Applied Science
The Institute of Biomaterials and Biomedical Engineering

University of Toronto
2010

Abstract
The byssal adhesion mechanism of the biofouling species Dreissena polymorpha was
investigated using a combination of studies on synthetic peptide mimics of tandem repeat
sequences from byssal component Dreissena polymorpha foot protein 1 (Dpfp-1) and
characterization of the regions of the byssus. A 20-residue fusion peptide incorporating two
Dpfp-1 repeat sequences adopts a random coil and β-turn conformation in solution, and
spontaneously forms a film at the solid-liquid interface in the presence of iron (III) cations.
Infrared characterization of the byssus Amide I region showed that β-sheets dominate its
secondary structure, although the proportion of different secondary structures varies between
regions. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of intact byssal
regions identified previously unknown differences in the composition of byssal threads, plaques,
and the adhesive interface, which are believed to correlate to the different roles of these
components in the overall structure.
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Acknowledgments
There are a number of people I would like to acknowledge for their guidance, support, and
assistance over the course of this project. First, I would like to thank my supervisor, Professor
Eli Sone, for his mentorship and guidance throughout the project, as well as the other students in
the Sone Lab (Nikrooz Farsad, Varuna Prakash, Nick Fonseca, Hamzah Qureshi, Ryan Singh,
Gurpreet Dhaliwal, and other Sone Lab colleagues) for their collaborative efforts.

I would also like to thank Professor Christopher Yip, for valuable advice on infrared
spectroscopy experiment design and access to his lab’s instruments, as well as Professor Yip’s
students (Jocelyne Verity, Gary Mo and Michelle Edwards for providing training and debugging
assistance on the Yip Lab’s equipment. I would also like to express my gratitude towards
Professor Warren Chan and his post-doctoral fellow Dr. Fayi Song, for access to and training on
their lab’s UV-Vis spectrometer.

Additionally, I wish to acknowledge the valuable technical support provided by Feryal Saraf
(Faculty of Dentistry) for cryo-microtome operation; Dr. Jian Chen and Suzanne Ackloo
(Ontario Cancer Biomarker Network) for MALDI analytical services; Dr. Alex Young (Faculty
of Chemistry) for ESI mass spectrometry of synthetic peptides; and Dr. Mostafa Hatam and his
colleagues at Aapptec for their valuable assistance with the peptide synthesizer from their
company.

Finally, I would like to thank my family for their continued encouragement and support
throughout my studies.
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Table of Contents

Acknowledgments .......................................................................................................................... iii 
Table of Contents ........................................................................................................................... iv 
List of Tables ................................................................................................................................ vii 
List of Figures .............................................................................................................................. viii 
List of Appendices (if any) ........................................................................................................... xii 
Chapter 1 Introduction and Literature Review ............................................................................... 1 
1  Introduction ................................................................................................................................ 1 
1.1  Introduction to Zebra Mussels – Biology, Mode of Life, and Invasive Properties ............ 1 
1.2  The Zebra Mussel Byssus and Foot .................................................................................... 5 
1.3  Byssal Composition in Marine Mussels ............................................................................. 9 
1.4  Byssal Composition in Zebra Mussels .............................................................................. 13 
1.5  Comparison of Dpfp-1 to Marine Mussel Adhesion Proteins .......................................... 18 
1.6  Mussel-mimetic Peptides based on Marine Foot Proteins ................................................ 20 
1.7  Long-term rationale for studying byssal adhesion ............................................................ 22 
1.8  Objectives ......................................................................................................................... 24 
1.8.1  Overall Objectives ................................................................................................ 24 
1.8.2  Specific Objectives ............................................................................................... 25 
1.9  Fmoc Solid-Phase Peptide Synthesis ................................................................................ 27 
1.10 Secondary Structure Determination from Fourier-Transform Infrared Spectroscopy ...... 28 
1.11 Matrix-Assisted Laser Desorption Time-of-Flight (MALDI-TOF) Mass Spectrometry . 31 
Chapter 2 Materials and Methods ................................................................................................. 35 
2  Materials, Techniques and Methods: ....................................................................................... 35 
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2.1  Materials, Instruments and Suppliers ................................................................................ 35 
2.2  Fmoc-protected peptide synthesis ..................................................................................... 35 
2.3  Fourier-transform infrared (FTIR) absorbance spectroscopy: .......................................... 38 
2.4  Deconvolution / spectral feature sharpening ..................................................................... 40 
2.5  MALDI-TOF Mass Spectrometry Sample Preparation .................................................... 41 
Chapter 3 Mimetic Peptides Based on Dpfp-1 Repeat Sequences ............................................... 44 
3  Mimetic Peptides Based on Dpfp-1 Repeat Sequences: .......................................................... 44 
3.1  Introduction ....................................................................................................................... 44 
3.2  Single Copies of Dpfp-1 Consensus Sequences ............................................................... 46 
3.3  Peptide interactions with Iron (III) chloride ..................................................................... 49 
3.4  Double Tridecameric-Repeat Mimetic Peptide ................................................................ 53 
3.5  Dpfp-1 Consensus Sequence Fusion Peptide .................................................................... 56 
Chapter 4 Spatial Approaches to Analyzing the Byssus ............................................................... 62 
4  Spatial Approaches to Analyzing the Byssus ........................................................................... 62 
4.1  Spatially Selective Byssal Characterization by Fourier-Transform Infrared
Spectroscopy: .................................................................................................................... 62 
4.1.1  Introduction ........................................................................................................... 62 
4.1.2  Adhesive interface layer ....................................................................................... 63 
4.1.3  Plaque Matrix and Thread Cores .......................................................................... 66 
4.2  MALDI-TOF Mass Spectrometry ..................................................................................... 69 
4.2.1  Introduction ........................................................................................................... 69 
4.2.2  Interface layer composition ................................................................................... 70 
4.3  Pair-wise comparisons of MALDI-TOF data from different regions ............................... 73 
4.3.1  Footprint vs. upturned plaque ............................................................................... 73 
4.3.2  Adhesive Interface vs. Plaque Matrix: .................................................................. 74 
4.3.3  Threads vs. plaque matrix: .................................................................................... 76 
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4.3.4  Interface vs. Threads: ............................................................................................ 77 
4.3.5  Summary of Mass Peaks and Localization ........................................................... 78 
4.4  Attempted sequencing of MS peaks ................................................................................. 79 
Chapter 5 Conclusions .................................................................................................................. 81 
5  Conclusions: ............................................................................................................................. 81 
5.1  Mimetic Peptide Investigations ......................................................................................... 81 
5.2  Byssal Characterization .................................................................................................... 82 
Chapter 6 Recommended Future Work ........................................................................................ 84 
6  Recommended Future Work: ................................................................................................... 84 
6.1  Future directions for mimetic peptide investigations ........................................................ 84 
6.2  Future directions for byssal characterization .................................................................... 85 
References ..................................................................................................................................... 87 
Appendix I: Peptide Synthesis Protocols ...................................................................................... 94 
Appendix II: Kaiser and Chloranil Test Protocols ...................................................................... 100 
Appendix III: ESI Mass Spectra and Analytical HPLC Chromatograms of Synthesized
Peptides .................................................................................................................................. 101 
Appendix IV: SEM Sample Preparation ..................................................................................... 106 
Appendix V: List of Suppliers for Chemicals and Equipment ................................................... 107 
Appendix VI: Table of MALDI peaks by region ........................................................................ 110 
Copyright Acknowledgements .................................................................................................... 111 

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List of Tables

Table 1: Locations and functions of known marine mussel byssal proteins ................................. 11 
Table 2: Zebra Mussel Foot Proteins Dpfp-1 through -3 .............................................................. 15 
Table 3: Primary sequence of foot-derived Dpfp-1; Uniprot ID Q9NB54. Repeat sequences are
italicized and underlined. C-terminal repeats alternate blue and unhighlitghted; N-terminal
repeats alternate yellow and unhighlighted. .................................................................................. 16 
Table 4: Consensus repeat sequences in Dpfp-1. Sites of post-translational modifications
(hydroxylation to DOPA for tyrosine residues and glycosylation with galactosamine for serine
and threonine residues) are indicated by *. ................................................................................... 17 
Table 5: Tandem repeat sequences from other species ................................................................. 19 
Table 6: FTIR Amide I peak assignments for D O and H O ....................................................... 28 
2 2
Table 7: Mimetic Peptide Sequences ............................................................................................ 36 
Table 8: Coupling protocols used for each amino acid ................................................................. 36 
Table 9: List of synthesis reagents and suppliers ........................................................................ 107 
Table 10: List of general-purpose reagents and suppliers .......................................................... 108 
Table 11: List of Instruments ...................................................................................................... 109 
Table 12: Summary of MALDI-MS peak locations in the regions of the byssus ....................... 110 

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List of Figures

Figure 1: Zebra mussels; showing foot and siphons. Inset: typical size of an adult zebra mussel. 2 
Figure 2: Internal organ structure of zebra mussel; reproduced with permission [1] ..................... 3 
Figure 3: Schematic and micrograph of the hierarchical ultrastructure of the byssus .................... 5 
Figure 4: Zebra mussel foot. A: Photograph of foot extruded from shell to deposit a byssal
thread on the shell of another mussel. B: Cross-section of foot (F), showing byssal gland (BG)
and byssal canal (BC); reproduced with permission [1] ................................................................. 7 
Figure 5: Schematic of enzymatic formation and reactions of protein-bound DOPA (other modes
of covalent cross-linking are also possible) .................................................................................... 8 
Figure 6: Interconversion of bis (purple) and tris (pink) iron (III) DOPA complexes in Mefp-1.
Reproduced with permission [23]. ................................................................................................ 10 
Figure 7: Spatial distribution of byssal proteins in marine mussels ............................................. 12 
Figure 8: Mature zebra mussel byssal plaque on a glass substrate, showing brown pigment
characteristic of quinone tanning .................................................................................................. 14 
Figure 9: Snell's Law .................................................................................................................... 29 
Figure 10: Attenuated Total Reflection ........................................................................................ 30 
Figure 11: A: tandem MALDI (TOF/TOF) mass spectrometer schematic. B: Schematic of triple
quadrupole TOF/TOF analyzer, used for more sophisticated ion sorting. Both reproduced with
permission [72]. ............................................................................................................................ 34 
Figure 12: Schematic of ATR study of interface adhesive layer - mussels remain attached
throughout the experiment, but are not depicted in this figure. .................................................... 39 
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Figure 13: MALDI-TOF Sample Preparation. A: Adhered byssal material may be either shaved
and sectioned to produce footprint and bulk samples (left path) or treated to dissolve the
substrate, allowing the plaque to be fixed in gelatin with the interface upturned for matrix
preparation (right path). B: Plaques prior to shaving. C: Footprints left from (B). D:
Cryomicrotome section of bulk plaque material embedded in gelatin. E: Cryomicrotome section
of thread cross-sections embedded in gelatin. .............................................................................. 43 
Figure 14: Gaussian fit Amide I absorbance of single-copy tridecameric repeat peptide mimic in
D O (15 mg/mL) ........................................................................................................................... 47 
2
Figure 15: Gaussian fit of heptameric repeat peptide mimc in D O (15 mg/mL) ........................ 48 
2
Figure 16: Comparison of Amide I band profile in single-copy peptide mimics and co-solution at
standard concentration 15 mg/mL in D O .................................................................................... 48 
2
Figure 17: Peak splitting of characteristic DOPA ring-stretch in the single-copy tridecameric
repeat peptide mimic (15 mg/mL), in the presence of iron (III) cations (at a ratio of two
equivalents iron (III) chloride to one DOPA). Peak splitting was not observed in the presence of
calcium cations at the same molar ratio. ....................................................................................... 50 
Figure 18: Quantitative fit of DOPA ring stretch peak-splitting of the single-copy tridecameric
repeat peptide mimic in the presence of iron (III) cations ............................................................ 50 
Figure 19: UV-Vis titration of tridecameric repeat into BisTris-buffered iron (III) chloride
solution. Step size of peptide: iron cation ratio is based on titration steps of 0.5 equivalents
peptide per equivalent of iron (III) chloride in the starting solution. ............................................ 52 
Figure 20: Manually constrained Gaussian fit of the Amide I band and adjacent aspartic acid
carbonyl stretch in the double tridecameric repeat peptide mimic ............................................... 53 
Figure 21: Iron (III) chloride has no major effect on the Amide I band shape of the double
tridecameric repeat peptide mimic ................................................................................................ 54 
Figure 22: Titration of double tridecameric peptide into BisTris-buffered iron (III) chloride. Step
size of peptide: iron cation ratio is based on titration steps of 0.5 equivalents peptide per
equivalent of iron (III) chloride in the starting solution. Ratios higher than 4 peptide: 1 iron were
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examined without observing an absorbance shift to the tris region, but were not shown because
they presented non-linearities at 535 nm which disrupted fitting. ................................................ 55 
Figure 23: Gaussian fit of fusion peptide in D O solution, 10 mg/mL ......................................... 57 
2
Figure 24: Iron (III) chloride addition to fusion peptide in BisTris-buffered D O does not affect
2
Amide I band shape ...................................................................................................................... 57 
Figure 25: Fusion peptide + FeCl films; 3 equivalents peptide: 2 equivalents iron.
3
A: Peptide 1 mg/mL. B: Peptide 10 mg/mL. ............................................................................... 59 
Figure 26: Scanning electron micrograph of a fold in the smooth area of the free-standing film
recovered from the air-water interface .......................................................................................... 59 
Figure 27: Scanning electron micrograph of an area of the rough morphology which occupies
approximately 10% of the fusion peptide film from the air-water interface, between folds of the
smoother morphology ................................................................................................................... 60 
Figure 28: Hypothesized schematic of fusion peptide film self-assembly, incorporating iron-
DOPA complexation and side-chain attractions between individual peptide units. Two additional
non-DOPA ligands forming coordination bonds to the iron centres are not shown; see Figure 6
and Section 1.3 for details of possible ligands. ............................................................................. 61 
Figure 29: Anomalous fit of Amide I absorbance of a footprint in transmission mode. The β-
sheet peak at 1635cm-1 appears to be the true largest contribution, but the fit settled on a different
local error minimum dominated by the 1660 cm-1 helix / turn ambiguous component (marked by
arrows). The assignment of the 1619cm-1 peak (marked with ? in the table) is tenuous, since that
peak is close to the lower end of the β-sheet range ....................................................................... 64 
Figure 30: Fitted Amide I regions of ATR-FTIR scans of the underside of attached byssal
plaques .......................................................................................................................................... 65 
Figure 31: Gaussian fit of the Amide I band of a plaque .............................................................. 67 
Figure 32: Gaussian fit of the Amide I band of thread ................................................................. 67 
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