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Amelogenin domains in a self-assembly process

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Amelogenin domains in a self-assembly process

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AMELOGENIN DOMAINS IN A SELF-ASSEMBLY PROCESS
by
Yaping Lei
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August, 2000
Copyright 2000 Yaping Lei
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UMI Number: 1405244
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U N IV E R S IT Y O F S O U T H E R N C A U F O R N IA
T H E G R A D U A T E SCHOOL.
U N IV ER SITY RANK
LOS A N GELES. CALIFORNIA 1 0 0 0 7
This thesis, w ritten by
y a p in g l e x
under the direction o f A £ x Thesis Com m ittee,
and approved by a ll its members, has been p re
sented to and accepted by the D ean o f T h e
Graduate School, in p a rtia l fulfillment o f the
requirements fo r the degree of
Master of Science
T i n t * Augus t 8, 2000
T H E COMMITTEE
/Ux-A
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DEDICATION
To M y Family for Their Love, Understanding and
Encouragement through Those Years
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ACKNOWLEDGEMENTS
I would like to thank my mentor, Dr. Malcolm Snead who encourages
me to the master program, for his guidance and expertise.
Also thanks to my committee member, Dr. Zoltan Tokes, Dr. Robert
Stellwagen, and Dr. Norri Kasaharra for their time and effort in helping me to
prepare this thesis.
Everyone in Dr. Snead’s lab (Dr. Wen Luo, Dr. Michael Paine, Benton
Yoshida, Dr. Caroline Paine, Danhong Zhu, Suwanna J, Larry Zhou, Dr.
Chendong Huan, and Dr. Jenny Yan), thanks for all your help. Without you
guys I could not hawe done this work.
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TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract ix
Chapter 1 Introduction 1
1.1 Biomineralization 1
1.2 Dental Enamel 1
1.3 Amelogenesis 2
1.4 Amelogenin and Enamel 4
1.5 Molecular Biology of Amelogenin 6
1.6 Functional M otif of Amelogenin in Self-assembly Process 7
1.7 Hypothesis 8
1.8 Experimental Approach 8
1.9 Importance 10
Chapter 2 Material and Methods 11
2.1 Subcloning 11
2.2 Mutagenesis 14
2.3 In vitro expression, Purification of Recombinant Protein 19
2.4 Construction of Expressive Plasmids for the Yeast Two-Hybrid 25
Assay
2.5 Immunoblotting 26
2.6 Polymerase Chain Reaction 27
2.7 DNA Nucleotide Determination 28
Chapter 3 Amelogenin Interaction Properties Detected by the Yeast 29
Two-Hybrid Assay
3.1 Introduction 29
3.2 Material and Methods 32
3.3 Results 37
3.4 Conclusion 40
iv
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Chapter 4 Changed Interaction Behavior of Amelogenin Detected by 41
Plasmon Resonance Spectroscopy
4.1 Introduction 41
4.2 Material and Methods 44
4.3 Results 46
4.4 Conclusion 56
Chapter 5 Discussion 58
5.1 Removal of the A-domain Abolish Protein-protein 60
Interaction, Resulting in Failure of Amelogenin
Self-assembly
5.2 Mutation of the A-domain Resulted in Aberrant Assembly 60
5.3 The B-domain Deletion Increases the Affinity of Amelogenin 62
Interaction
5.4 Summary and Conclusion 64
References 65
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LIST OF TABLES
Table Title Page
1.1 Recombinant Constructs Used in this Study 9
2.1 Oligonucleotide Primers Used to Subclone Full Length 12
Amelogenin and Truncated Amelogenin to Bacteria Expression
Vector pQE30
2.2 Oligonucleotide Primers Used to Introduce Mutated Codons 14
to Wildtype Amelogenin
2.3 Oligonucleotide Primers Used to Subclone the Wildtype and 26
Mutant Amelogenin into the Yeast Two-Hybrid System Vector
3.1 Groups of Constructs Used in the Yeast Two-Hybrid Assay 33
4.1 Summary of Rate Constants Detected by Plasmon Resonance 56
Spectroscopy for the Interaction of Wildtype, Truncated, or
Mutated Amelogenin with Wildtype Amelogenin M179
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LIST OF FIGURES
Figure
1.1
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
3.1
3.2
3.3
4.1
4.2
Title Page
Enamel Organ Structure 3
Reading Frame for M180-pQE30, M180AA-pQE30, 13
M180AB-pQE30 Constructs Verified by DNA Sequence
Determination
DNA Sequence Verification for the T2 1 -Ile Mutation 16
in the Amelogenin T-mutant Construct
DNA Sequence Verification for P4 1 -Thr Mutation in 17
Amelogenin P-mutant Construct.
DNA Sequence Verification for T2l-Ee, P^-Thr 18
Mutation in Amelogenin D-mutant Construct
SDS-PAGE gel Analysis of Purified His-tagged Recombinant 21
Amelogenin Proteins
Western Blot Analysis of Amelogenin His-tagged M l80 22
Western Blot Analysis of Amelogenin His-tagged M180AA 23
Western Blot Analysis of Amelogenin His-tagged M 180AB 24
Yeast Two Hybrid System Principle 3 1
Filter Assay Results for p-Galactosidase Activity in Yeast 38
Two Hybrid System
P-Galactosidase Activity Detected by Liquid Assay in Yeast 39
Two Hybrid System
The Principle of Plasmon Resonance Spectroscopy 43
Sensorgram Overlaid for the Interaction of M 180-M179, 47
M180AA-M179, M180AB-M179, T-mutant-M179,
P-mutant-M179, and Double Mutant-M179 at 0.4uM
Concentration
V ll
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4.3 A Representative Overlaid Sensorgram for M l80 Binding 49
Kinetics with Wildtype Amelogenin M l79 as well Fitting
Results for 1 to 1 Binding Model
4.4 A Representative Overlaid Sensorgram for Ml80AA Binding 50
Kinetics with Wildtype Amelogenin M l79 as well Fitting
Results for 1 to 1 Binding Model
4.5 A Representative Overlaid Sensorgram for Ml80AB Binding 51
Kinetics with Wildtype Amelogenin M l79 as well Fitting
Results for 1 to 1 Binding Model
4.6 A Representative Overlaid Sensorgram for Amelogenin 52
T-mutant Binding Kinetics with Wildtype Amelogenin
M179 as well Fitting Results for 1 to 1 Binding Model
4.7 A Representative Overlaid Sensorgram for Amelogenin 53
P-Mutant Binding Kinetics with Wildtype Amelogenin
M179 as well Fitting Results for 1 to 1 Binding Model
4.8 A Representative Overlaid Sensorgram for D-mutant 54
Binding Kinetics with Amelogenin Wildtype M179 as
well Fitting Results for 1 to 1 Binding Model
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ABSTRACT
How the extracellular matrix proteins in a calcified tissue regulate and organize
calcium and phosphate into a highly organized carbonated apatite is a major concern in
the field of biomineralization. Dental enamel serves as an advantageous model for
investigation into the molecular basis underlying biomineralization. During tooth
development, extracellular matrix proteins secreted by ameloblasts regulate inorganic
crystallite initiation, propagation, maturation and termination. Amelogenin is one of
matrix proteins, which has been demonstrated to regulate enamel formation. Previously
Paine et al verified that there were two functional domains in the primary structure of
mouse amelogenin that participated in self-assembly process. One is defined by the N-
terminal amino acid 1-42 residues, and is called the A-domain. The other is defined by
the C-terminal amino acid residues 157-173, and is named the B-domain. Which domain
is more important for self-assembly, however, still remains vague. One form of the
naturally occurring genetic disorder amelogenesis imperfecta, results from point
mutations in the N-terminal A-domain and provides a hint that A-domain is critical. My
hypothesis is that The N-terminal A-domain is required for amelogenin self-assembly. In
order to test this hypothesis, a series of recombinant proteins, including wildtype
amelogenin (M l80), truncated amelogenin lacking the N-terminal A-domain (M180AA),
or lacking the C-terminal B-domain (M180AB), or mutated amelogenin containing single
amino acid changes such as T2I-He (T-mutant), P^-Thr (P-mutant), or both amino acid
mutated (e.g. T2 1 -Ile + P4I-Thr) (D-mutant), were engineered. Their behavior in the
assembly process were separately analyzed by the yeast two hybrid assay and by the
ix
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plasmon resonance spectiroscopy system. The data were interpreted to mean that the N-
terminal A-domain deletion abolished self-assembly process and that any of the three
mutations within the N-terminal A-domain reduced amelogenin-amelogenin interaction
affinity dramatically. Reonoval of C-terminal B-domain increased the interaction affinity.
These findings help in umderstanding how amelogenin may behave in vivo and serve to
control enamel hydroxyapatite mineral habit.
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CHAPTER 1: INTRODUCTION
1.1 Biomineralization
Biomineralization is a process in which discrete and organized inorganic
crystalline structures are formed within macromolecular extracellular matrices. This is a
widespread biological phenomenon happening in physiologic situation, such as shell,
bone, teeth, and in pathologic situations, for example, kidney stones and arterioscleorosis.
In all biomineralized structures analyzed to date, the process of inorganic mineral
deposition is matrix-regulated. The matrix plays two roles in this process: a nucleator for
mineral deposition, and a scaffold for regulation of the mineral crystal growth.
Biopolymers of matrix-bound, cell-secreted organic surfaces, can interact with ions to
initiate the nucleation and growth of an inorganic phase. The mineral crystals grow
within the organic phase, and the crystal habit is regulated by the matrix proteins. Thus
the organic matrix is a crucial structural component defining spatially and temporally the
space to be replaced by mineral. In bone, collagen fibrils limit the potential primary
growth of mineral crystal, and force the crystal to be discrete and discontinuous. In
dental enamel, however, the extracellular matrix forms the boundaries of a large
compartment within which the mineral phase is continuous, thereby forming individual-,
elongated-crystal aggregates.
1.2 Dental Enamel
Dental enamel, a unique and highly mineralized ectoderm-derived tissue covering
vertebrate teeth, serves as an advantageous model for investigating cellular and
1
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extracellular events leading to biomineralization (Nanci et al., 1992). Dental enamel
tissue is synthesized and secreted by specialized cells of the enamel organ (Figure 1.1),
called ameloblasts, which are lost as the tooth erupts into the oral cavity. During
development, ameloblasts differentiated from inner dental epithelium, secrete proteins
into the extracellular matrix, and these proteins regulate the initiation, growth, and the
architectural arrangement of the crystals. Proteins comprising the organic matrix of
enamel are processed and degraded extracellularly. The resulted fragments together with
water are withdrawn and replaced by an equivalent volume of mineral. “The final
product is a completely acellular and avascular extracellular matrix that contains almost
no organic material but highest mineral content” (Smith, 1998).
1.3 Amelogenesis
Amelogenesis, i.e. enamel formation, has been historically classified into three
well-defined stages: presecretory, secretory and maturation. At the presecretory stage,
ameloblasts acquire their phenotype. The differentiated ameloblasts are elongated,
polarized cells with their nuclei, Golgi apparatus, and lysosomes sitting at the proximal
end, and Tomes’ processes, a specialized apical membrane that secretes the enamel
matrix localized to the distal end. At the secretory stage the ameloblasts are rich in rough
endoplasmic reticulum, and numerous secretory vesicles of enamel proteins are present in
Tomes’ processes. These ultrastructural changes reflect that at this stage the ameloblasts
are devoted to synthesis and secretion of extracellular matrix protein, along with some
mineral ions, thus creating a partially mineralized matrix. At the maturation stage of
2
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Figure 1.1 Enamel organ structure from Smith and Nanci, 1995
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amelogenesis, ameloblasts change their ultrastructure. At this stage, the Tomes’
processes are lost and cell length is reduced, which reflects the functional alteration of the
ameloblasts. The cells now are rich in mitochondria and lysosomal granules. The major
functions of ameloblasts at this stage are degradation, removal of extracellular matrix
components previously secreted, and introduction of mineral ions, such as calcium and
phosphate, into the mineralizing extracellular matrix to replace the proteins and water
(Scott and Symons, 1998).
The secretory stage is an interesting stage in amelogenesis since organic matrix
secreted by ameloblasts provides an ideal environment for nucleation and growth of
elongated thin crystallites of hydroxyapatite (HAP). The enamel matrix proteins initially
restrict the growth of enamel crystallites, while in later stages the major proteins are
degraded permitting crystallites to extend their growth in width and thickness (Simmer et
al., 1995).
1.4 Amelogenin and Enamel
Ameloblasts secrete two major classes of enamel protein: amelogenin protein
(AMEL and various isoforms) and non-amelogenin proteins such as enamelin, tuftelin,
ameloblastin, and other uncharacterized proteins. Amelogenin comprises approximately
90% of the enamel matrix protein mass, while the other enamel proteins contribute the
remaining 10% of the mass at the secretory stage. Amelogenin is believed to provide a
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scaffolding matrix in which the initial carbonated hydroxyapatite crystals can grow
(Deutsch et al., 1995).
The essential role played by amelogenin during enamel biomineralization had
been demonstrated by the following studies:
1) Amelogenesis imperfecta (Al) is a naturally occurring genetic disorder
affecting human with prevalence 1 in 14,000 in the United State. The enamel of these
patients is hypomineralized, hypoplastic or hypomatured (Langerstrom-Fermer and
Landegren, 1995; Langerstrom-Fermer et al., 1995). The amelogenin gene locus in Al
patients exhibits defects that reduce or eliminate amelogenin expression, indicating a
direct link between amelogenin and enamel formation. More over an enamel defect
similar to amelogenesis imperfecta has been created in mice by injecting hammerhead
ribozyme designed to target amelogenin message RNA, and thus to reduce amelogenin
protein translation in developing mouse molar (Lyngstadaas et al., 1995).
2) A role for amelogenin in enamel formation comes from the experiments
performed by Doi and colleagues. In that experiment, purified amelogenin protein was
used to direct mineral deposition exclusively to the end of hydroxyapatite seed crystal
(Doi et al., 1984). Similarly, Moradian-Oldak et al. (1998) compared the influence of
amelogenin, polyproline, and phosvitin on the growth kinetics of seeded apatite crystals,
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and found that compared with the two other macromolecules, the purified full-length
amelogenin protein resulted in aggregation of the growing apatite crystals.
3) Additional evidence for a role of amelogenin in enamel formation comes from
imaging data of an in vitro experiment employing dynamic light scattering, atomic force
microscopy, and transmission electron microscopy (Fincham and Moradian-Oldak,
1995). In these experiment the authors showed that highly purified amelogenin protein
spontaneously forms supramolecular quasi-spherical aggregates (also called nanospheres)
with 15-20 nm diameters. Self-assembled amelogenin, when viewed with conventional
electron microscopy contrast reagents such as phosphotungstate or uranyl acetate,
appeared as electron-lucent structures. These in vitro structures are similar to those
observed in developing enamel marix from mouse, bovine, and hamster. Transmission
electron microscopic examination of the developing enamel matrix showed numerous
circular electron-lucent regions. These regions were interpreted as nanospheres arranged
parallel to the developing crystallites. These in vivo nanospheres are 20 nm in diameter,
and similar to those formed by purified amelogenin protein in vitro.
1.5 Molecular Biology of Amelogenin
In mice amelogenin is actually a family of proteins arising from a single gene by
differential splicing and proteolysis (Lau et al., 1992; Simmer et al., 1994; Simmer et al.,
1995). The amelogenin gene is located on the X-chromosome in mice, and is expressed
in a restricted spatial and temporal pattern during tooth development. Alternative
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splicing of the gene results in at least 7 transcripts in mice teeth. Among those transcripts
identified, the 180 amino acid (M l80) is the principal component of enamel extracellular
matrix. M l 80 is a moderately hydrophobic protein, rich in pro line, similar to collagen in
bone.
1.6 Functional Motifs of Amelogenin in the Self-assembly Process
What is the molecular basis for amelogenin proteins to undergo self-assembly to
form nanospheres? The primary structure of amelogenin contains a hydrophobic N-
terminus, and a hydrophilic C-terminus. At pH 5.3 the N-terminus of amelogenin
monomer forms a (3-sheet, whereas the C-terminus and middle portion form random coils
(Goto et al., 1993). In eleven mammalian species examined to date, the N-terminus and
C-terminus are highly conserved, suggesting that either is a functional motif contributing
to amelogenin properties.
Using the yeast two-hybrid system, Paine and Snead identified domains
facilitating amelogenin protein-protein interaction that included the N-terminal first 42
amino acids, named the A-domain, and C-terminal 17 amino acid (157-173), named the
B-domain. They proposed that the N-terminal A-domain interacted either directly or
indirectly to the C-terminal B-domain in the amelogenin self-assembly process (Paine
and Snead, 1997).
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Which domain is more important in amelogenin self-assembly process among the
two domains? Two human pedigrees with an X-linked amelogenesis imperfecta
phenotype have point mutations in the N-terminal A-domain that result in the Thr2 1 -Be
(Lench and Winter, 1995) and Pro4 1 -Thr (Collier et al., 1997) mutation in the amelogenin
protein, indicating the N-terminal A-domain might be an important domain in the
amelogenin self-assembly process. However, to date no mutation in the C-terminal B-
domain has been identified.
1.7 Hypothesis
With these background in mind my hypothesis is that the N-terminal A-domain,
e.g. the first 42 amino acids of mouse amelogenin protein, interacts with each other to
assemble, and these assemblies lead to the formation of amelogenin nanospheres.
1.8 Experimental Approach
To test my hypothesis, a series of recombinant amelogenin constructs were
engineered with molecular cloning techniques. These constructs include: 1) the wildtype
amelogenin (M l80); 2) an amelogenin with the N-terminal 42 amino acid deleted, and
substituted with Flag-tag (M180 AA); 3) an amelogenin with the C-terminal 157-173
amino acids deleted, and replaced with three Hemoglutinin A-tag (M180AB); 4) an
amelogenin with a point mutation consisting of Thr2 1 - Be (“T-mutant”); 5) an amelogenin
with a point mutation consisting of Pro4 1 -Thr (“P-mutant”); 6) an amelogenin with both
point mutations of Thr2 1 - Be and Pro4I-Thr (“D- mutant”) (Table 1.1). The yeast two
8
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hybrid assay and plasmon resonance spectroscopy were applied to analyze the interaction
among the wildtype amelogenin, the mutated amelogenin (T-mutant, P-mutant or D-
mutant), or the amelogenin deleted domain A (M180AA) or domain B (M180AB) in
order to better understand the process of amelogenin self-assembly.
Table 1.1 Amino acid sequence data for the engineered amelogenin constructs used in
this study
Amelogenin wildtype (M180)
MPLPPHPGS PG YINLS YEVLTPLKW Y QS MIRQP YPS Y G YEPMGGWLHHQI
IPVLSQQHPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH
HQPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSPLHPMQPLAPQPPLPPLF
S MQPLS PILPELPLE AWP ATDKTKREEVD *
Truncated amelogenin with B-domain deleted, and substituted with 3HA tag
(M180AB)
MPLPPHPGS PG YINLS YE VLTPLKW YQS MIRQP YPS YG YEPMGGWLHHQI
IP VLS QQHPPSHTLQPHHHLP V VP AQQP V APQQPMMP VPGHHS MTPTQH
HQPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSPLHPMQPLAPQPPLPPLF
SMOPLR (YPYDVPDYALSTKREEVD*
Truncated amelogenin with A-domain deleted, and substituted with Flag tag
(M180AA)
MPGPGADYKDDDDKGTGSDLGPMGGWLHHOIIPVLSOOHPPLOPHHHLP
WPAQQPVAPQMPVPGHHSMTPTQHHQPNIAQQPFQQPFQPQAIPPQSHQ
PQSPLHPMQPLAPQPPLPSMQPLSPELPELPLEAWP ATDKTKREE VD*
Mutated amelogenin with T2 1 -Ile (T-mutant)
MPLPPHPGS PG YINLS YE VLIPLKWYQSMIRQP YPS YGYEPMGGWLHHQII
PVLSQQHPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQHH
QPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSPLHPMQPLAPQPPLPPLFS
MQPLSPILPELPLE A WP ATDKTKREE VD *
Mutated amelogenin with P4 1 -Thr (P-mutant)
MPLPPHPGS PG YINLS YE VLTPLKWYQS MIRQP YPS YG YETMGGWLHHQI
IP VLSQQHPPSHTLQPHHHLPVVPAQQPV APQQPMMP VPGHHSMTPTQH
HQPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSPLHPMQPLAPQPPLPPLF
SMQPLSPILPELPLEAWP ATDKTKREE VD
9
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Mutated amelogenin with T2 1 -Ile and P4 1 -Thr (D-Mutant)
MPLPPHPGSPGYINLSYEVUPLKWYQSMIRQPYPSYGYETMGGWLHHQI
IP VLSQQHPPSHTLQPHHHLPWPAQQPV APQQPMMP VPGHHSMTPTQH
HQPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSPLHPMQPLAPQPPLPPLF
SMQPLSPILPELPLEAWPATDKTKREEVD*
1.9 Importance
This study will give insight as to the molecular basis for amelogenin self-
assembly to form nanosphere, and will help in our understanding how amelogenin
regulates enamel formation.
10
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CHAPTER 2. MATERIAL AND METHODS
2.1 Subcloning
Amelogenin full-length cDNA was amplified with SN176 (5’-ATC GGATCC
CCCCTACCACCTCATCCT GG-3’) and SN177 f5’-AT GAATTC TTAATCCA
CTTCTTCCCGC-3’) primers from the murine amelogenin cDNA (Genbank #D31768)
using p/I/ DNA polymerase. SN176 is the forward primer (amino acid residue 2 to 7)
containing a BamHl site, while SN177 is the reverse primer (amino acid residue 173-
180) containing an EcoRl site. The PCR product was cleaned with Bam H l, cloned into
pQE30 vector (Qiagen) at BamHl and Sm all sites, and named M180-pQE.
The M180 with the A-domain deleted (M180AA) was amplified with SN175 (5’-
ATCGGATCCCCCC TACCA CCTCATCCTGG-3’), and SN177 primers from a
previously described pFA construct (e.g., amelogenin with N-terminal amino acid 1-42
deleted and substituted with Flag-tag: Glv-Pro-Glv-Ala-Asp-Tvr-Lys-Asp-Asp-Asp-Asp-
Lys-Glv-Thr-Glv-Ser-Glu-Leu-Glv) (Paine and Snead, 1997). The M l80 with B-domain
deleted (M180AB) was amplified with SN176 and SN177 primers form the previously
created pEB construct (e.g. amelogenin with the C-terminal amino acids 157-173
replaced with three Hemoglutinin A-tag (HA-tag): Arg-(Tvr-Pro-Tvr-Asp-Val-Pro-Asp-
T vr-A laySer) (Paine and Snead, 1997). The pFA and pEB constructs were generously
provided by Dr. Michael Paine. The PCR products were cleaned with restriction
endonuclease digestion: Bgl II for M180AA, and BamHl for M180AB. The cleaned PCR
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products were cloned into pQE30 vector at the Bam H l and Small sites, and named
M180AA-pQE, and M180AB-pQE.
Table 2.1 Oligonucleotide primers used to subclone full-length amelogenin M l80
and truncated amelogenin M180AA, M180AB into the pQE vector. All
primers are written from 5’ to 3’.
Primer Name Sequence
SN 175 TT AAGATCTCC AGGGCCCGGCGCCG AC
(M180AA forward primer with Bgl II site)
SN 176 ATCGGATCCCCCCTACCACCTCATCCTGG
(Amelogenin forward primer with BamHI site)
SN 177 ATGAATTCTT AATCC ACTTCCCGC
(Amelogenin reverse primer with ECORI site
The reading frames of all three constructs were verified by manual sequencing
using dideoxy termination methods (Sequenase Version 2.0 DNA Sequencing Kit, USB)
(Figure 2.1). The primer (Type W IV primer, Qiagen) used for the sequencing is located
on the vector, upstream of the insert.
12
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ccc -
ATG-:
Figure 2.1
M180-pQE M180AA-pQE M180AB-pQE
DNA nucleotide sequence determination establishes the reading
frames for M180-pQE, M180AA-pQB, M180AB-pQE
constructs.
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2.2 Mutagenesis
Point mutations were introduced into M180-pQE construct using the Gene Edit ™
in vitro Site-Directed Mutagenesis System (Promega). Oligonucleotide primers in this
system mutate the ampicillin gene, thereby creating an enhanced resistance to antibiotics
provided by the company. A bacterial host strain, AMH71-18muts, which lacks the
ability to repair DNA mismatches, permits the mutation to be introduced, and maintained
by the bacterial host.
Two mutagenic primers, SN248 and SN247, were synthesized, and phospho-
rylated with T4 polynucleotide kinase (Roche Molecularbiochemic). Primer SN248 (5’-
TGAGG TGCTT ATCCCTTT GAAGTGG-3’) contains mutated the 61st base from C to
T which results in the 21st amino acid changed from Thr to He. Primer SN 247 (5’-
TCCACCCATGGTTTCGTAACCATAGG-3’) is a reverse primer containing thel21st
base mutated form C to A leading to the 41st amino acid changed from Pro to Thr.
Table 2.2 Oligonucleotide primers that were used to introduce mutated bases to
wildtype amelogenin
Primer Name Sequence
SN 247 5’ TCCACCCATGGTTTCGTAACCA(instead of C) TAGG 3’
(M180-Pro4l-Thr reverse primer)
SN 248 5’ TGAGGTGCTTAT (instead of C) CCCTTTGAAGTGG 3’
(MlSO-Tfrr’-Ile forward primer)
SN 254 5’ CCTAT(instead of G) GGTTACGAAACCATGGGTGGATGG 3’
(Ml80-Pro4 1 -Thr forward primer)
14
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M 180-pQE construct was introduced to and prepared from JM109 host strain.
SN248 and top-strand selective oligonucleotide primer, SN 247 and bottom-strand
selective oligonucleotide primer were annealed to the alkaline denatured M180-pQE
template. The mutant strands were synthesized with T4 DNA polymerase, and ligated
using T4 DNA ligase. The mutated mixtures were transformed to AMH71-18muts host
strain, and the cells were plated on the LB plates containing the selective antibiotics. The
chimeric plasmids were prepared from the colonies grown on the plates, and re-
transformed to JM109. The JM109 clones were screened by manual sequencing, and the
mutation in each construct was verified (Figure 2.2, and 2.3).
Another primer SN254 (complementary of SN247) was synthesized, and
phosphorylated. SN248, SN254, and top-strand selective oligonucleotide primers were
used to introduce both point mutations into the M180-pQE construct. The procedure was
performed as stated previously. The resulting colonies were screened, and the mutation
was confirmed by manual DNA sequence determination to obtain the mutated M l80
construct (Figure 2.4).
These constructs designed above were named “T-mutant-pQE for the Thr^-Ile
mutant, “P-mutant-pQE” for the Pro 4 1 -Thr mutant, and the “D-mutant-pQE” for the
double point mutations Thr2 1 -Be + Pro 4 1 -Thr construct.
15
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Wildtype T-mutant
Figure 2.2 DNA nucleotide sequence determination identified for the Thr^l to He in
the T-mutant amelogenin construct.
16
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G A T C
AACCC-
-AAACC
Wildtype P-mutant
Figure 2.3 DNA nucleotide sequence determination to identify the Pro 41
to Thr mutation in the P-mutant amelogenin construe.
17
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Wildtyp^e
-mutant
D-mutant Wildtype
Figure 2.4 D N A nucleotide sequence determination identified for the Thr2 * to lie,
p ro ^ l to Thr mutation in the D-mutant amelogenin construct. Sequence
1 and 2, Thr 2 1 to lie mutation, 3 and 4, Pro^l to Thr mutation.
18
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2.3 In vitro Expression, Purification o f Recom binant His-tagged Am elogenin
Proteins
To purify the His-tagged amelogenin proteins the M180-pQE, M180AA-pQE,
M180AB-pQE, T-mutant-pQE, P-mutant-pQE, and D-mutant-pQE constructs were
introduced into the M l5 host strain. A single colony from each construct was inoculated
into 5 ml of LB medium containing 50 ug/ml of ampicillin and 25 ug/ml kanamycin at
37°C with shaking overnight. The next morning the cultures were diluted with 500ml of
LB medium containing ampicillin and kanamycin, and allowed to continue growing at
37°C until an O D ^ of 0.7 reading was reached. Protein expression was induced with
EPTG at 2 mM final concentration for 2 hours. The cultures were harvested by
centrifugation at 3,000 rpm for 15 minutes in a GS3 rotor (Dupont Instruments, Sorvall
RC-5B Refrigerated Superspeed Centrifuge).
For M180-pQE, M180AB-pQE, and the three point mutation constructs, the cell
pellets were resuspended in 10 ml of PBS, sonicated on ice (VWR Scientific Bransom
Sonifier 450) using the following condition: 1 minute burst /minute cooling, duty cycle
20%, output 5. The nonsoluble parts were collected by centrifugation at 14,000 rpm for
15 minutes in a SA-600 rotor. The resulted pellets were dissolved in the buffer A (6M
GuHCl, 0.1M Na-phosphate, 0.01M Tris/HCl, pH8.0). In contrast, MI80DA-pQE
culture pellet was lysed directly with the buffer A since M180DA-pQE is soluble.
19
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The pellet-buffer A mixtures were incubated at room temperature for 2 hours to
redissolved the pellet and cleaned by centrifugation in a SA-600 rotor at 14,000 rpm for
15 minutes at room temperature. The resulting supernatants were mixed with 8 ml of
50% of Ni-NTA resin (Qiagen) previously equilibrated with buffer A, and incubated at
room temperature for 1 hour. The resin-protein mixtures were loaded into 5 ml columns
(Qiagen). The columns were sequentially washed with buffer A, buffer B, buffer C, and
finally buffer D. Buffer B, C, D are composed of the same components: 8 M Urea, 0.1 M
Na-phosphate, 0.01 M Tris/HCl, but each was prepared with a different pH (pH 8 for
buffer A, pH 6.9 for buffer C, pH 5.9 for buffer D). Each washing was undertaken until
the O D -jgo absorption measured for the flow-through was less than 0.01. The bound
proteins were eluted from the column with 10 ml of buffer E (8 M Urea, 0.1 M Na-
phosphate, 0.01 M Tris/HCl, and pH 4.5), fractions of 3 ml from each elution were
collected. A 10 ul sample of each fraction was analyzed by 10% SDS-PAGE gel. The
fractions rich in eluted protein were combined, and dialyzed against urea with decreasing
concentration from 6 M, to 4 M, to 2 M, tol M, and finally into water at 4°C, and
lyophilized (Labconco, Lyphlock 18). The protein purity was checked using samples
electrophoresed in a 12% PAGE gel (Figure 2.5). Western blots were performed with
anti-His, anti-amelogenin antibody, anti-Flag, and anti-HA for detection of purified His-
tagged M180 (Figure 2.6), His-tagged M180AA (Figure 2.7), His-tagged M180AB
(Figure 2.8). The proteins having greater than 95% purity were used for plasmon
resonance spectroscopy analysis as analytes.
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4 5 6
97kDa
69kDa
42kDa
28kDa
18kDa
Figure 2.5 SDS-PAGE gel analysis for purified His-tagged amelogenin proteins
used in plasmon resonance spectroscopy analysis. Lanel, Protein
molecular marker; Lane 2, M180; Lane 3, M180AB; Lane 4, M180AA;
Lane 5, T-mutant; Lane 6, P-mutant; Lane 7, D-mutant.
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1 2 3 4 5 6 7
42kDa
18kDa
14kDa
Anti-amelogenin Anti-His tag
Figure 2.6 Western blot results for M180-pQE protein with different
antibodies. Lane 1, protein molecular marker; Lane 2 and 5,
uninduced cell culture; Lane 3 and 6, induced cell culture;
Lane 4 and 7, purified M l80 protein.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3 4
10
%
m r
42 m
29 kD
18 kD
14 kD
A
Anti-amelogenin Anti-His tag Anti-Flag tag
Figure 2.7 Western blot results for M180AA protein with different antibodies.
Lane 1, molecular marker; Lane 2, 5, and 8, uninduced culture;
Lane 3, 6 and 8, induced culture; Lane 4, 7 and 10, purified protein.
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42kD
29kD
18kD
14kD
Anti-amelogenin Anti-Iiis tag Anti-HA tag
Figure 2.8 Western blot results for M180AB protein with different antibodies. Lane 1,
molecular marker; Lane 2, 5, and 8, uninduced cell culture; Lane 3, 6
and 9, induced cell culture; Lane 4, 7 and 10, purified protein.
24
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2.4 Construction o f E xpressive Plasmids for the Yeast Two Hybrid Assay
M l80, M180AB, and the three amelogenins having various point mutations were
subcloned into pPC97 vector, a yeast expression vector containing the DNA binding
domain. The polymerase chain reactions were performed using “Advantage cDNA
polymerase” (Clontech) to generate the inserts. The forward primer is SN86: 5’-TTC
GGATCC TA TGCCCCTACC ACCT-3’ (amelogenin amino acid residue 1-5 with
BamHl site). The reverse prim er is SN286: 5’-TAGGGAGCTCTTAATCCACTTCT
TCCCG-3’ (amelogenin am ino residuel75-180 with SacI site). The PCR products were
cloned into the TA-cIoning vector to facilitate their recovery and verification. The inserts
in the TA-cloning vector w ere released with BamHl and EcoRV, and cloned to pPC97 at
the Bgl II and blunt-ended S acI sites. These constructs were named M180-pPC97,
M180AB-pPC97, T-mutant-p*PC97, P-mutant -pPC97, and D-mutant-pPC97. The
wildtype amelogenin fused to GAL4 activation domain (M180-pPC86) and M180AA
fused to GAL4 DNA binding domain (M180AA-pPC97) constructs were provided by Dr.
Michael Paine.
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Table 2.3 Primers used to construct GAL 4 DNA binding-domain fused amelogenin
(pPC97) and GAL4 activating domain fused amelogenin (pPC86)
in the yeast two-hybrid assay.
Prim er Name Sequence
SN 86 5’ TTCGGATCCCTATGCCCCTACCACCT 3’
(Amelogenin forward primer with BamHl site)
SN286 5’ TAGGGAGCTCTTAATCCACTTCTTCCCG 3’
(Amelogenin 175 to 180 reverse primer with SacI site)
SN 287 5’ ATTCAGATCTACATGCCAGGGCCCGGC 3’
(M180AA forward primer with Bgl H site)
2.5 Im m unoblot Analysis
The pellets from 100 ul of the uninduced; induced liquid culture, or 50 ng of the
expressed proteins following purification (see above) were dissolved in 10 ul of sodium
dodecyl sulfate (SDS) loading buffer. The samples were boiled at 95°C for 5 minutes,
and loaded on 12% SDS-polyacrylamide gels (PAGE). The gels were electrophoresed at
10mA constant current until the bromophenol blue ran off the gels. The proteins were
transferred to polyvinylidene difluoride membrane (Millipore) using an electromotive
transfer device (Semi-Phor™, Hoeffer Scientific Instruments) at 100 mA constant current
for one hour. The membranes were blocked in TBST solution (10 mM Tris/HCl pH 7.5,
100 mM NaCl, 0.1% Tween 20) containing 10% instant non-fat dry milk overnight. The
appropriate first antibody was diluted in TBST solution with 5% nonfat milk, and used to
blot the membranes at room temperature for 1 hour. These antibodies include mouse
monoclonal anti-His antibody (5 ug/ml final concentration) (Qiagen), rabbit anti-
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amelogenin (Simmer et aL, 1994) (3 ug/ml final concentration), rabbit anti-HA (MBL)
(final concentration 5 ug/ml), and mouse monoclonal anti-Flag (Eastman Kodak
Company) (5 ug/ml final concentration). The biotinylated goat anti-mouse IgG (Gibco
BRL), or biotinylated goat anti-rabbit IgG (Gibco BRL) were used for detection of the
antigen-antibody complex at 1:3000 dilution in TBST buffer containing 5% nonfat milk.
The protein-antibody complex was visualized in the presence of streptavidin-alkaline
phosphate (Gibco BRL) as the conjugate, and nitroblue tetrazolium chloride (NBT,
Sigma), 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) as chromogens.
2.6 Polym erase Chain Reaction
The PCR reactions were carried out in a programmable thermal controlled cycler
(MJ Research, Inc, PTC-100). In a 50 ul reaction volume, the following components
were included: 10 ng of plasmid DNA as template, 10 pmol of each primer, 10 uM of
dNTP, 1 X PCR buffer, 2.5 u of pfu DNA polymerase or “advantage” DNA polymerase.
The PCR reactions were performed with initial denaturation at 94°C for 5 minutes
followed by 32 cycles of 94°C x 1 minute for denaturation, 58°C x 1 minute for
annealing, and 72°C x 1 minute for extension. The final extension was continued for 5
minutes. The PCR products were analyzed on 1% agarose gels electrophoresesed at
100V for 1 hour, stained with ethidium bromide, and a photographic record was prepared.
27
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2.7 DNA Nucleotide Determination
The orders of nucleotides in the cloned inserts were determined with Sequenase
Version 2.0 DNA Sequencing Kit (USB). Appropriate primers were designed as
required. A 4 ug of plasmid DNA was denatured with alkaline, and appropriate primers
were annealed to the denatured templates. New strands were synthesized with S[ 3 5 ] dATP
labeling using T7 DNA polymerase with a suitable concentration of terminators. The
reaction mixtures were separated on a 6% PAGE gel at 60 watt using constant power.
The gels were dried at 80°C under vacuum. Radiographic films (35 x 43 cm) (Kodak)
were placed on the gels, exposed overnight, and developed the next morning.
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CHAPTER 3. AM ELOGENIN INTERACTION PROPERTIES DETECTED BY
THE YEAST TW O-HYBRID ASSAY
3.1 Introduction
It has been previously demonstrated that amelogenins self-assemble to form
nanospheres and this super-molecular assembly is presumed to regulate enamel crystal
formation (Deutsch et al., 1995). Mutation or deletion of the putative self-assembly
domains might affect the molecular interaction affinity, or abolish the interaction, leading
to aberrant nanosphere formation.
In 1989, Field and Song (1989) invented a method to detect protein-protein
interaction in the eucaryotic yeast host, and named it the “yeast two hybrid assay”. This
assay is based on the fact that many eucaryotic transcription activators (including GALA)
consist of two physically separable molecular domains, i.e. a DNA-binding domain, and a
transcription activation domain (Keegan et al., 1986). The DNA binding domain
localizes the transcription factor to the upstream region of a specific gene, while the
activating domain contacts other components of the transcription machinery to initiate
transcription (Ma and Ptashine, 1988). Yeast GAL4 is the typical transcription activator
for genes involved in galactose metabolism. In the presence of galactose, the GAL 4
protein binds to GAL 4-responsive elements within the upstream activating sequence
(UAS) of several genes involved in galactose metabolism and activates transcription.
Field and Song engineered the two functional domains of GALA, cloning the domains
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into two different shuttle vector, allowing two unrelated “query” proteins to be fused to
each domain (Bartek et al.,1993). If the two query proteins interact with each other, the
DNA-binding domain and the activating domain of GAL 4 will be tethered, allowing the
transcription function of intact GALA to be reconstituted. The consequence is that the
down stream report gene, Lac Z, will be turned on. The basic principle of the yeast two
hybrid system is shown in figure 3.1.
Yeast cells can perform most of the post-translational events for a protein encoded
by a transgene. These post-translational events include glycosylation and
phosphorylation that mimic mammalian protein modification. Therefore interactions
detected by the yeast two-hybrid assay are likely to reflect events occurring in
mammalian cells. Fields and Song (1989) first tested the yeast two-hybrid system using
yeast proteins known to interact with one another: SNF1 and SNF4. Munder and Furst
(1992) using this assay successfully demonstrated that the yeast CDC25 guanine
nucleotide exchange factor binds to the catalytically inactive form of Ras protein.
Similarly, Mosteller and his coworker (1994) used the yeast two hybrid assay to identify
functional residues of Ras protein that interact with the yeast exchange factor. Using this
assay, Chen et al. (1991) characterized a small domain in the carboxyl-region of yeast
SIR4 protein that mediates SIR4 proteins self-assembly to form homodimers.
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BD
GAL4-UAS
AD
M180
p-galactosidase
AD
M l 80
BD
P-galactosidase
GAL4-UAS
Figure 3.1 The principle of yeast two-hybrid assay. In this study X stands for
M l80, M180AA, M180AB, T-mutant, P-mutant, D-mutant fused to
DNA-binding domain, i.e. pPC97 constructs. Wildtype amelogenin
M l80 was fused to GAL4 activation domain. The P-galactosidase
activity indicates the strength of a interaction.
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Previously, Paine and Snead (1997) identified two functional domains, an amino-
terminal A-domain and a carboxyl-terminal B-domain contained in amelogenin primary
structure required for amelogenin-amelogenin interaction. My study would undertake to
examine A-domain importance in amelogenin self-assembly process. My strategy is to
use the yeast two-hybrid assay to compare the behavior of a series of recombinant GAL4
amelogenins in amelogenin-amelogenin interaction process. Among these constructs are
amelogenin with intact N-terminal A-domain, amelogenin with deleted A-domain,
amelogenin with A-domain modified by phenocopying amino acid mutations seen in a
human disease affecting enamel, or amelogenin bearing a C-terminal B-domain deletion.
My hypothesis is that deletion or mutation o f the A-domain will alter the ability of
amelogenin to self-assemble, while the less-critical C-terminal deletion might or might
not affect amelogenin assembly.
3.2 M aterial and Methods
3.2.1 Constructs
Wildtype amelogenin (M180), truncated amelogenin (M180AA, M180AB), and
mutated amelogenin (T-mutant, P-mutant, and D-mutant) were subcloned into the pPC97
vector containing GAL 4 DNA binding domain. Only wildtype amelogenin M l80 was
cloned into the pPC86 vector containing GA L 4 DNA activating domain. The detailed
information regarding the subcloning was described in the second chapter of this thesis.
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3.2.2 Co-transform ation o f pPC97 and pPC 86 Constructs into a Yeast Strain
CG1945
1) Preparation of Yeast Competent Cells
C G I945 yeast cells (Feilotter et al., 1994) were streaked from the frozen stock on
to a YPD plate (20 g/L of Difco peptone, 10 g/L of-yeast extract, and 15 g/L of agar), and
incubated at 30°C for two days. A single colony was picked from the plate, and grown in
10 ml of YPD medium at 30°C overnight with shaking to provide a “start culture”. The
next morning the start culture was diluted in 100 ml of YPD medium, and grown at 30°C
with shaking for another two hours. The yeast cells were harvested by centrifugation in a
G3 rotor for 15 minute at 3000 rpm. The resulted pellet was washed in deionized water,
resuspended in 2 ml of TE / LiAc buffer (10 mM Tris/HCl, 1 mM EDTA, 100 mM
Lithium acetate, pH 7.5), stored on ice as competent cells to receive selected DNA
constructs.
2) Co-transformation
Twelve pairs of plasmids were cotransformed into the competent cell (Table 3.1).
The positive control pair is CD25-pPC86 with H-ras-pPC97. The negative control pairs
are: a) M180AB-pPC97 with pPC86 empty vector; b) M180AA-pPC97 with pPC86
empty vector; c) T-mutant-pPC97 with pPC86 empty vector, d) P-mutant-pPC97 with
pPC86 empty vector, e) D-mutant-pPC97 with pPC86 empty vector.
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Table 3.1 Groups of constructs used for co-transformation in yeast two-hybrid assay.
Group Binding dom ain Activation domain Function
(pPC97) (pPC86)
1 H-Ras CDC25 Positive control
2 M180-pPC97 M180-pPC86 Experimental
3 M 180AB-pPC97 Vector only Negative control
4 T-mutant-pPC97 Vector only Negative control
5 P-mutant-pPC97 Vector only Negative control
6 D-mutant-pPC97 Vector only Negative control
7 M180AA pPC97 Vector only Negative control
8 M 180AB-pPC97 M180-pPC86 Experimental
9 T-mutant-pPC97 M180-pPC86 Experimental
10 P-mutant-pPC97 M180-pPC86 Experimental
11 D-mutant-pPC97 M180-pPC86 Experimental
12 M 180AA-pPC97 M180-pPC86 Experimental
The following six groups were used to detect potential protein to protein
interaction: a) M180-pPC97 with M180-pPC86, b) M180AA-pPC97 with M180-pPC86,
c) M 180AB-pPC97 with M180-pPC86, d) T-mutant-pPC97 with M180-pPC86, e) P-
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mutant-pPC97 with M180-pPC86, f) D-mutant-pPC97 with M180-pPC86.
Transformations were achieved using 0.1 ug of DNA for each plasmid pair, 100 ug of
salmon sperm DNA, 100 ul of competent cell, and 600 ul of freshly prepared PEG/LiA
solution (40% polyethylene/100 mM of Li Ac acetate) in an Eppendorf tube. The tubes
were vortexed, and incubated at 30°C for 30 minutes with shaking. After adding 70 ul of
dimethy sulphoxide (DMSO) these cells were heat-shocked at 42°C for 15 minutes. The
cells were collected by a brief centrifugation, resuspended in TE buffer (10 mM
Tris/HCl, 1 mM EDTA, pH 7.5), and plated on Petri dishes containing selection medium
lacking leucine and tryptophan amino acids. Only those cells containing both pPC97 and
pPC86 constructs could grow on the selection plates since the pPC 97 vector contains a
gene complementing the absence of leucine in the selection medium and pPC86 vector
contains a gene complementing the absence of tryptophan in the selection medium. These
plates were maintained in a 30°C incubator upside-down for 5 days.
3.2.3 P-Galactosidase Activity Assay
1) Filter Assay
A single colony from each plate described above was transferred to a filter (VWR,
grade 413), grow on an agar plate containing selection medium. The filter-plate
combination was placed in a 30°C incubator to allow the colonies grow for two days.
The filters were lifted, placed into a pool of liquid nitrogen for 10 seconds, and thawed at
room temperature three times in order to permeabilize the yeast cells. The treated filters
were placed on the top of another filter that was soaked with Z-buffer / X-gal (60 mM
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NajHPO,*, 4 mM NaH2 P 0 4, 10 mM KC1, 1 mM M gS04, 50 mM P-mercaptoethanol, 0.8
uM X-gal, pH 7.0), and incubated at room temperature until the appearance of blue
chromogen in the positive control pairs was observed. The intensity of the chromogen
among the different groups was compared.
2) Liquid Assay
A single colony from each plate was inoculated into 5ml of selective medium at
30°C overnight. The next morning, 2 ml of the culture was placed into 8 ml of YPD
medium, and allowed to grow at 30°C until the ODgoo absorption reached 0.6-0.8
absorption units. The O D ^ absorption for each sample was recorded. The cells from 1.5
ml of each culture were collected in Eppendorf tubes by centrifugation at 14,000 rpm for
30 seconds (Eppendorf). The resulting pellets were washed with Z-buffer twice, and
resuspended in 300 ul of Z-buffer, split into 100 ul aliquots in 1.5 ml of Eppendorf tubes,
and freeze-thawed from liquid-nitrogen and 37°C three times. The chromogen was
developed by addition of 700 ul of Z-buffer+(3-mercaptoethanol (Z-buffer with 40 mM of
(3-mercaptoethanol), 160 ul of ONPG+Z-buffer (4 mg/ml) to each tube. Blanks consisted
of all these materials without yeast cells. Samples were incubated at 30°C until the
chromogen developed. Cell debris was removed with centrifugation, the supernatants
were transferred to cuvettes, and absorption at OD4 2 0 was recorded after subtraction of the
blank. To eliminate variance due to cell number, the OD4 0 0 reading for each sample was
normalized to its ODgo,value determined previously. The experiment was repeated on
seven separate occasion. On each occasion the spectrophotometric measurement was
36
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quantified to the amelogenin wildtype construct interaction, i.e. M180-pPC97 with
M180-pPC86. The mean value was calculated and recorded for each group of co
transformation. The relative P-galactosidase activity of each cotransformation was
plotted. The P-galactosidase activity of wildtype amelogenin interaction with itself was
taken as 100%.
3.3 Results
3.3.1 Filter Assay
No chromogen was deposited for the co-transformation of negative control pairs,
indicating that truncated amelogenin (M180AA-pPC97, M180AB-pPC97) or amelogenin
with amino acid mutations (T-mutant-pPC97, P-mutant-pPC97, and D-mutant-pPC97)
did not interact with the empty vector pPC86. The positive control CDC25 with H-ras
produced a strong chromogen deposition. M180-pPC97/ M180-pPC86, P-mutant-pPC97/
M180-pPC86, T-mutant-pPC97/ M180-pPC86, and M180AB-pPC97/ M180-pPC86 all
produced positive interaction although the D-mutant-pPC97/M180-pPC86 produced a
barely detectable signal. No chromogen was deposited for the M180AA-pPC97/M180-
pPC86 co-transformation, indicating that little or no protein to protein interaction
occurred (Figure 3.2).
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Positive Control*
M180/M180
Negative Control
(M180AB/pPC86) '
Negative Control.
(T-mutant/ pPC86)
Negative Control (
(P-mutant/ pPC86)
Negative Control
(M180AA/pPC86)
M 1 8 0 A B / M 1 8 0
&
T-mutant/M180
*
iP-mutant/M180
Mi
iD-Mutant/M180
Negative Control ^ _ w sb^ s s . M180AA/M180
(D-mutant/pPC86) '
Figure 3.2 Yeast two hybrid filter assay results for interaction o f recombinant
amelogenins. Compared with wild type-wild type amelogenin inter
action, single mutation to A-domain decreases the strength o f inter
action. Removal o f A-domain or introducing two mutation to A-
domain result in failure o f amelogenin interaction.
3 8
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3.3.2 Liquid Assay
Relative P-galactosidase activity of each co-transformation is shown in Figure
3.3. The relative P-galactosidase activity of T-mutant-pPC97/ M180 -pPC86 is 55%, P-
mutant-pPC97/M180-pPC86 is 64%, D-mutant-pPC97/M180-pPC86 is 80%, M180AB- .
pPC97/M180-pPC86 is 73%, MI80AA-pPC97/M180-pPC86 is 8% of wildtype M180-
pPC97/M180-pPC86 co-transformation.
125%-<
100% 4
75% 4 I
<
>
| 50%'
Pi
25% 4
: *:X w X ;X ;
0% -*
Z 8 & 8 8 ? :
/ y
IvIvl vXvl
. v. v. wX*!
m
i
i
m m
W ildtype M 180AA M180AB T-mutant P-mutant D-mutant
Figure 3.3 Related P-galactosidase activity detected by liquid yeast two-hybrid assay
for interaction between altered amelogenin with wildtype amelogenin
M 180. The P-galactosidase activity o f wildtype-wildtype interaction was
taken as 100%.
39
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3.4 C onclusion
Using yeast two-hybrid assay the functional motifs contained in amelogenin
required for self-assembly were previously identified. The present experiment showed
that the N-terminal A-domain was required for amelogenin-amelogenin interaction.
Without the N-terminal A-domain, M180AA-pPC97 construct did not interact with
wildtype amelogenin M180-pPC86 construct: no chromogen developed in the filter assay
and only 8% of the P-galactosidase activity were detected in the liquid assay. The T-
mutant-pPC97, P-mutant-pPC97, D-mutant-pPC97, and M180AB-pPC97 constructs did
interact with wildtype amelogenin M180-pPC86, but with less affinity: chromogen
deposited in the filter assay for these constructs was less than wildtype amelogenin
interaction with itself. The percentage of P-galactosidase activity remaining in these
altered amelogenin constructs compared to that of wildtype amelogenin-amelogenin
interaction is: 55% for T-mutant-pPC97/M180-pPC86, 64% for P-mutant-pPC97/M 180-
pPC86, 80% forD-mutant-pPC97/M180-pPC86, 73% for M180AB-pPC97/M180-
pPC86.
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CHAPTER 4. CHANGED INTERACTION BEHAVIOR OF AM ELOGENIN
DETECTED BY PLASMON RESONANCE SPECTROSCOPY
4.1 Introduction
Traditional approaches to the study of molecular interaction such as crossliking,
co-immunoprecipitation, and co-fractionation by chromatography have long been known
to be not applicable to amelogenin since the physic-chemical properties of amelogenin or
the inorganic phase of in vivo enamel obscure interpretation. Development of an
alternative system, plasmon resonance spectroscopy which is commercially available sold
as Biacore (Biomolecular interaction analysis core) may allow me to obtain kinetic data
(Fisher and Fivash, 1994; Szabo et al., 1995) for amelogenin self-assembly.
Plasmon resonance spectroscopy is based on an optical phenomenon occurring at
an interface between two transparent media with different refractive indices such as water
and glass. Light coming from the side with higher refractive index is partially reflected
and partially refracted. Above a certain incident angle, no light is refracted across the
interface, and total internal reflection occurs. Although the light is totally reflected, an
electromagnetic field component termed an evanescent wave penetrates a short distance
into the medium with lower refractive index. If the light is monochromatic and polarized,
and the interface between the media is coated with a thin layer of metal, the intensity of
reflective light will be dramatically reduced at a specific incident angle, so a sharp
41
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shadow will be created. This phenomenon is called surface plasmon resonance, i.e. SPR.
The incident angle at which the shadow occurs is termed the SPR angle.
Several physical properties affect the SPR angle. One is the refractive index of
the medium that the evanescent wave penetrates on the non-illuminated side of the
surface. The sensor chip provides a gold interface between the glass support and the
surface matrix. The refractive index of surface matrix is affected by the surface
concentration of solute. In an experiment with plasmon resonance spectroscopy, one
molecule is immobilized onto the surface of a sensor chip as the “ligand”, and another
molecule flows over the surface in free solution as the “analyte”. If the analyte
selectively binds to the ligand, the concentration of solute on the surface matrix changes
which results in a change in refractive index of the surface matrix causing an SPR angle
to be shifted (Figure 4.1). A plot of the SPR angle shift with time is called a sensogram.
The unit for SPR signal measurement is scaled in response units, i.e. RU.
Plasmon resonance technology could provide detailed information on the binding
mechanisms, and rate constants associated with macromolecular interaction. It has been
used to: a) identify ligand-receptor interaction (Olazaki et al.,1995; Myszka et al., 1996),
b) characterize interactions involved in signal transduction (Nmelson et al.,1994;
Balasubramanian et al., 1995), c) characterize engineered antibody (Glaser, 1993;
Karlsson et al., 1993), d) characterize protein-DNA binding (Helmann and Haseth, 1999),
and in aspects of virus research (Bondesson et al.,1993).
42
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Reflected Light Intensity SPR Angle (RU)
Time
SPR Angle
Polarized Ligh
Detection Unit
Reflected Light
Prism
ka
How Cell
— > q p RecomBmant Amelogenins imlbm
Figure 4.1 The principle of plasmon resonance spectroscopy. In this study, M179
was immobilized on a CM5 sensor chip as a ligand, recombinant
amelogenins flow through the chip as analytes. When s analyte binds
to the ligand, the SPR angle will shift due to the changed efractive
index of the surface layer. Plotting the SPR angle against time creates
a sensorgram w hich displays the interaction happening on the chip.
43
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Two major advantages arise from this technology in my study of amelogenin self-
assembly process: a) it is not necessary to label amelogenin, and b) detection of
amelogenin-amelogenin interactions in real time. Amelogenin wildtype M l79 (e.g. full
length of amelogenin without first methionine and His-tag, purified by HPLC, and
provided by Dr. Alan Fincham) was immobilized onto the CMS chip as ligand. The
following altered, His-tagged amelogenin proteins were used as analytes: a) wildtype
amelogenin (M180), b) the A-domain deleted amelogenin (M180AA), c) the B-domain
deleted amelogenion (M180AB), d) T-mutant, e) P-mutant, f) D-mutant. The
sensorgrams created by the protein to protein interactions were recorded, and compared
among these engineered amelogenin proteins. In this assay, it is possible for
amelogenin analytes to first partially assemble in solution and to then interact with the
amelogenin ligand on the chip. Based on the result of the yeast two hybrid assay, I
predict that the N-terminal A-domain of amelogenin plays a critical role in the assembly
process, and any changes involved in the N-terminal A-domain will alter the interaction
behavior.
4.2 M aterial and Methods
4.2.1 E quipm ent and Reagents
Biacore 2000 system, a sensor chip CM5 (BR-1005-50), BIA evaluation software,
HBS buffer (10 mM Hepes with 0.15 M NaCl, 3.4 mM EDTA and 0.005% P20, pH 7.4),
amine-coupling kit (BR-1000-50) were obtained from Biacore AB company.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.2.2 Im m obilization o f M179 to a CM5 Chip
There are four flow-cells in a CM5 chip. Among these four-cells, three were
used for the assay. Flow cell 1 was used as a control surface; flow cell 2 and 3 were used
as test surfaces. The carboxyl-groups on the surface were activated with an injection of a
solution containing 0.2 M N-ethyl-N’-(3-diethylamino-propyl) carbodiimide (EDC), and
0.05 M N-hydroxysuccinimide (NHS). M179 was diluted in 10 mM acetic acid, pH 4.5
at concentration of 100 ug/ml, and injected over flow cell 2 and flow cell 3 until 800 RU
of protein was coupled. The immobilization was completed by a 7-minute injection of 1
M ethanolamine hydrochloride to block any remaining activated carboxyl-groups.
4.2.3 Kinetic Study
HBS was used as running buffer at a flow rate of 20 ul/min. The six different
concentrations of His-tagged recombinant amelogenin proteins were diluted from stock
solution in 10 mM acetic acid, pH 4.8 with HBS buffer, and were injected at 20 ul/minute
at 25°C with 2 minutes contact time, followed by 2 minutes dissociation period. The
sensor chip was regenerated with 1 M MgCl2 after each run. All procedures were
performed using an automated program provided by Biacore AB company.
4.2.4 Data Analysis
Data transformation and overlay plots for all experimental interactions were
prepared with BIAevaluation software 3.0 (Biacore, AB). In order to correct for
refractive index changes and nonspecific binding, the binding responses generated from
45
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flow cell 1 were substrated from the responses generated in flow cell 2 and flow cell 3.
The association rate constants (ka) were determined from the linear portion of
sensorgrams during the early association phase. The dissociation rate constants were
calculated from the linear portion of the sensorgram approximately 10 seconds after
completion of the injection of samples. The data was fitted to a basic kinetic 1:1 binding
model provided in the BIA evaluation software (Biacore AB)
4.3 Results
4.3.1 M utated or Truncated Amelogenin Proteins Bind M179 with Less Affinity
The real-time interaction of wildtype with wildtype amelogenin (Ml 80 /M l79),
mutant amelogenin with wildtype amelogenin (T-mutant/M179, P-mutant/M179, D-
mutant/M179), truncated amelogenin with wildtype amelogenin (M180AA/M179,
M180AB/M179) were determined using plasmon resonance spectroscopy. Figure 4.2
shows a representative overlay of a sensorgram generated for each set of interaction at a
concentration of 0.4 uM. At the same concentration, M180/M179 interaction produced
the highest binding response, while the M180AA/M179 yielded the smallest binding RU
value. The order of binding RU, from strongest to weakest, is M l 80/M 179 > T-mutant/
M179 > P-mutant/M179 > M180AB/M179 >D-mutant/M179 >M180AA/M179.
46
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1 8 0 -
1 6 0 -
140 ■
1 2 0 -
1 0 0 4
o
«
e
o
a
e
I: M I80
II. T-mutant
2 0 -
IH. P-mutant
IV. M180AB
V. D-mutant
VI. M180AA
IV
VI
• 2 0 '
-150
-t—
-100 -50 50 100 150 200 250 300
T u n e
Figure 4.2 Overlaid M l 79-binding sensorgrams by M l 80, M180AA, M180AB,
T-mutant, P-mutant, D-mutant at 0.4 jiM o f concentration.
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4.3.2 Amelogenins with A lteration in the A-domain Are Less Able to Self-assem bly
Figure 4.3 through figure 4.8 provide the overlaid sensorgrams of kinetic study as
well as the binding model fitting results for wildtype amelogenin (M l80); mutated
amelogenin (T-mutant, P-mutant, D-mutant); truncated amelogenin (M180AA, M180AB)
interaction with wildtype amelogenin M l79 at six different concentrations. When fitted
to the basic kinetic 1 to 1 binding model, only the interaction of M180AA with M179, D-
mutant with M179 fitted very well (% 2 is 1.19 for M180AA/M179, 7.07 for D-mutant/
M179). In comparison, interaction between M180 with M179 (% 2: 105), T-mutant with
M179 (% 2: 51), P-mutant with M179 (% 2: 19.6) and M180AB with M179 (% 2: 43.8) did
not fit the 1 to 1 binding model, indicating that amelogenin without the A-domain or with
introduction of two amino acids changed to the A-domain greatly reduced the ability to
undergo pre-assembly in solution.
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M180 wtih M179
2 5 0 T
200 -
1 5 0 -
100 -
5 0 -
0 -
-5 0
3 0 0 2 5 0 15 0 200 100 5 0 -5 0 -1 5 0 -100
Time
Fitting Result for 1 to 1 Binding Model
Time
Figure 4.3 An overlaid sensorgram for M179-binding kinetics of M180 at 15,7.5,
3.75, 1.88,0.94, 0.47 uM, as well fitting results for 1 to 1 binding model.
The x 2 is 105.
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IUI180AA wtih M179
4 5 -
4 0 -
3 5 -
3 0 -
«
£ 2 5 -
I 20-
01
* 1 5 -
10 -
5 - -
0-
250 30 0 15 0 200 100 5 0 -5 0 -1 5 0 -100
Time s
r u Fitting R esu lt for 1 to 1 Binding Model
4 5 -
3 5 -
E 25 -
5 -
250 300 100 150 200 50 -150 0 -100 -50
Time s
Figure 4.4 An overlaid sensorgram for M 179-binding kinetics o f M180AA at 10,5,
2.5,1.25,0.63,0.3*7111*1, as well fitting results for 1 to I binding model.
T h e s i s 1.19.
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Response
RU
M180 AB with M179
9 0 T
8 0 -
70 -
60 -
50 -
40 -
30 -
2 0 -
10-
0 -
-10
300 250 150 200 100 50 -50 0 -100 -150
Time
RU
Fitting Result for 1 to 1 Binding Model
90 -r
8 0 -
7 0 -
60 -
5 0 -
c
8.
m
& 3 0 -
2 0 -
1 0 -
0-
-10
300 250
200 100 150
-100 -50 -150
s
Time
Figure 4.5
An overlaid sensorgram for M 179 binding kinetics o f M180AB at 0.7,
0 .3 5 ,0 .1 8 ,0 .0 9 ,0 .0 4 5 ,0 .0 2 3 uM, as w ell fitting results for 1 to 1 binding
model. The y? is 43.8. .
5 1
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T-mutant with M179
RU
1 4 0 - r
1 2 0 -
1 0 0 -
8 0 -
o
c
8.
6 0 -
•>
t c
4 0 -
2 0 -
0 -
-20
Fitting Result for 1 to 1 Binding Model
— t—
100
— i—
1 5 0
-1 5 0
— t—
-100
- t —
-5 0
— I ----->
5 0
Time
200 2 5 0 3 0 0
Figure 4.6 An overlaid sensorgram for Ml 79-binding kinetics of T-mutant at 6, 3,
1.5, 0.75, 0.375, 0.19 uM, as well fitting results for 1 to 1 binding model.
The%2 is 51.
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Response Response
r u P-mutant with M179
160
140
120 -
100 -
8 0 -
6 0 -
4 0 -
20 -
0 -
-20
-1 5 0 - 1 0 0 -5 0 0 5 0 1 0 0 1 5 0 2 0 0 ’ 2 5 0 3 0 0
Time s
RU Fitting Result for 1 to 1 Binding Model
1 6 0 t
1 4 0 -
120 -
100 -
8 0 -
4 0 -
2 0 -
0 -
-20
2 5 0 3 0 0 -100 5 0 100 200 -1 5 0 -5 0 0 1 5 0
Time
Figure 4.7 An overlaid sensorgram for M179-binding kinetics of P-mutant at 6, 3,
1.5, 0.75, 0.375, 0.19 uM, as well fitting results for 1 to 1 binding model.
The % 2 is 19.6.
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RU
70
0
«
c
o
a.
i n
0
0 1
6 2 -
5 4 -
4 6
3 8
3 0 -
2 2 -
14 -
6
-2
-10
D-mutant w ith M179
— t—
-5 0
— t---------------'
5 0
Time
-1 5 0 -100 100 15 0 200 2 5 0 3 0 0
RU
7 0 t
6 2
5 4 -
4 6 -
„ 3 8 -
•ft
| 304
• f t
S 2
14 -
6 -
- 2 -
-10
Fitting Result for 1 to 1 Binding Model
— t -
50
Time
-1 5 0 -100 -5 0 100 15 0 200 2 5 0 3 0 0
Figure 4.8 An overlaid sensorgram of M179-binding kinetics o f D-mutant at 6, 3, 1.5,
0.75, 0.375, 0.19 uM, as well fitting results for 1 to 1 binding model. The
X 2 is 7.07.
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4.3.3 M utated Am elogenin Interact with W ildtype Amelogenin with Aberrant
Rate Constant and Affinity
Table 4.1 shows a summary of kinetic parameters for the interaction between
mutant, truncated, and wildtype amelogenins with wildtype amelogenin M179. The
interaction of M180 with M179 exhibits the affinity, greater than any point mutant with
M179, and M180AA with M179. The approximate ka for M180 with M179 is 28.3 x 104
M 'IS'1 ; 4.24 xlO4 forT-mutant with M179; 3.01xl04 for P-mutant with
M179; 1.02 x 104 M ^S'1 for D-mutant with M179; and 2.40 x 104 MrlSA for M180AA
with M179. Interestingly, the interaction of M180AB with M179 showed highest affinity
at 41.1 x 104M'IS'1 .
The dissociation rate constants for M180 with M179 interaction was calculated as
12.3 x 10'3S'1 , a value which is higher than T-mutant with M179 (8.41 x 10'3 S'1 ); P-
mutant with M179 (8.38 x 10'3 S'1 ); D-mutant-pQE with M179 (6.15 x 10"3 S*1 ); and
M180AA with M179 (4.51 xlO*3 S'1 ). When the B-domain deleted amelogenin (M180AB)
was allowed to interact with wildtype amelogenin M179 a dissociation rate constant of
12.2 x 10'3 S' V as measured, a value that was almost the same as that for wildtype
amelogenin (e.g. M180 with M179) interaction.
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Table 4.1 Summary of rate constants of kinetic assay for engineered amelogenin
proteins interaction with M179.
k a
m V (xlO4 )
k d
s'1 (x 10'3 )
M 180/M 179 28.5 12.3
T-mutant / M179 4.24 8.41
P-mutant / M179 3.01 8.38
D-mutant / M179 1.02 6.05
M l 80 A A / M180 2.04 4.51
M180AB / M179 41.1 12.2
4.3 Conclusion
Plasmon resonance spectroscopy showed that the N-terminal A-domain is
important to the amelogenin self-assembly process. Deletion of N-terminal A-domain
(e.g. M180AA) or introduction of two amino-acid point mutations to the N-terminal A-
domain, resulted in alteration to the interaction from heterogeneous binding style, to 1 to
1 binding style, indicating that the analytes did not partially assembly in solution before
binding to M179 on the chip. Single amino acid mutation in the A-domain or the B-
domain deletion produced less binding RU response in the corresponding amelogenin
interaction. Deletion of the entire A-domain or introduction of double amino acid
mutations to the A-domain showed the lowest binding RU response. Compared with
wildtype amelogenin M180, T-mutant, P-mutant, D-mutant, and M180AA interacted with
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wildtype amelogenin M179 with reduced binding affinity. Interaction of M180 AB with
M179 showed an increased binding affinity. These data indicate that compared to the
wildtype amelogenin-amelogenin interaction, the mutated amelogenin or the truncated
amelogenin, interact with wildtype amelogenin M l79 in a manner evidenced by an
aberrant rate constant. The mutation of a single amino acid in the A-domain such as the
T-mutant or the P-mutant reduced the binding affinity. However mutation of both amino
acids in the A-domain, or deletion o f the whole A-domain (residues 1-42) dramatically
reduced the binding affinity, leading to the absence of amelogenin self-assembly.
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CH APTERS. DISCUSSION
A hallmark of calcified tissues is that various extracellular matrix proteins attract
and organize calcium and phosphate ions into a structural mineral phase of carbonate
apatite (Boskey, 1996). Collagen based hard tissues are characterized by organic matrix
consisting mainly of type I collagen frame. Teeth, on other hand, contain mainly
amelogenin during secretory stage, and no collagen (Robinson and Weatherell, 1981).
Amelogenin in developing dental enamel is secreted continuously by ameloblasts, and
self assembles into nanosphere (Moradian-Oldak et al., 1994). Nanosphere along with
mineral crystallites are believed to be the basic building blocks of enamel (Fincham et al.,
1994).
Compared with bone and dentin, where the structural framework of collagen is
retained, the enamel extracellular matrix proteins are processed and degraded completely
in matured enamel (Fincham et. al., 1999). Amelogenin nanosphere formation does not
involve covalent bonds or covalent cross-linking reactions, but rather, all the
consequence of interactions among hydrophobic side chain of amino acid residuals such
as Phe, Ala, Val, Leu, and lie in amelogenin. Amelogenin amino acid sequence is highly
conserved through evolution showing a moderately hydrophobic amino terminal motif
and a hydrophilic carboxyl-terminal sequence (Snead et al., 1995; Simmer and
Snead,1995).
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Following the initial description o f amelogenin proteins in the early 1960’s,
scientists have attempted to gain information about its secondary and tertiary structure.
Amelogenin CD spectrum performed by Goto et al. (1993) suggests that the amelogenin
N-terminus contains (3-sheet structure, while the central region and C-terminal region
exhibits a random coil conformation. Scientists (Termine and Torchia, 1980) later on
found that amelogenin protein largely fails to retain stable secondary or tertiary structure.
Instead, amelogenin self-assembles to form supermolecular nanospheres (Fincham et al.,
1994, 1995). W ith tools of molecular biology, scientists have concentrated on the
molecular basis for amelogenin self-assembly. Previously Paine and his colleague (1997)
using the yeast two-hybrid system identified two motifs contained in the amelogenin
primary structure. These motifs were shown to participate in self-assembly process.
Their data shows that amelogenin self-assembly depends on the N-terminal 42 amino
acids interacting either directly or indirectly with 17 (157-173) amino acids on the C-
terminus.
My goal was to better characterize the participation of these motifs during
amelogenin self-assembly into nanospheres. A series of recombinant amelogenin were
engineered, and used to study their behavior in the self-assembly process using yeast two
hybrid assay and plasmon resonance spectroscopy. Essentially similar results were
obtained from either of these assays.
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5.1 Removal o f A-dom ain Abolishes Protein-protein Interaction, Resulting in
Failure o f Am elogenin Self-assembly
When the first 42 amino acids are deleted from full length amelogenin, the
resulting amelogenin M180AA could not interacted with full length amelogenin M l80 as
judged by the yeast two-hybrid assay. The failure to interact with full length M l80
suggests that the N-terminal domain play a critical role in protein-protein interactions. In
the plasmon resonance spectroscopy assay, analyte M180AA interacted with wildtype
amelogenin M l79 ligand in a one to one binding pattern, demonstrating no amelogenin
polymer formed in the solution.
My data for M180AA is corroborated by the observations obtained with dynamic
light scattering (DLS) and atomic force microscopy (AFM) imaging reported by Dr.
Moradian-oldak et al. (2000). Moradian-Oldak and colleagues showed that M180AA
produced a majority of nanosphere particles with a hydrodynamic radium of
approximately 2.67 nm, indicating that most of the protein are unassembled, and exist as
monomer. I conclude that the A-domain is required for amelogenin self-assembly since
in the absence of the A-domain the self-assembly process is abolished.
5.2 M utation o f A-dom ain Resulted in Aberrant Assembly
Aberrant assembly behavior also resulted from introducing amino acid point
mutation into the amelogenin A-domain. When wildtype amelogenin interacted with
either T-mutant amelogenin (T-mutant-pPC97/ M180-pPC86) or P-mutant amelogenin
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(P-mutant-pPC97/M180-pPC86), relative P-galactosidase activity was lower than that of
wildtype-wildtype amelogenin interaction (M180-pPC97/M180/pPC86) detected by
either filter assay or liquid assay (55% of P-galactosidase activity for T-mutant, 64% for
P-mutant) in the yeast two hybrid assay. In the plasmon resonance spectroscopy assay,
T-mutant, as well P-mutant interact with M l79 with reduced ka compared with that of
M l 80/M 170 interaction, indicating that the binding affinity is lower than wildtype
amelogenins.
When both amino acid point mutations are present in the A-domain, the resulting
D-mutant-pPC97 amelogenin failed to interact with M180-pPC86 detected by filter
assay, however, 80% P-galactosidase activity was detected in liquid assay for D-mutant-
pPC97/M180-pPC86 in liquid assay in yeast two-hybrid system. The contradictory
results from the yeast two-hybrid assay need more work. In the assay based upon
plasmon resonance spectroscopy, the lowest binding RU was produced by the interaction
of D-mutant with M l79 when compared with other group interaction at the same
concentration. Kinetic data showed that the D-mutant interacts with M l79 with the
smallest ka and the binding curves fit the 1:1 model very well, indicating that the double
mutant synergizes both single amino acid mutation effects, thus abolishing the self-
assembly process.
These same proteins were used by Moradian-Oldak et al. (2000) to analyze
nanosphere formation by dynamic light scattering and atomic force microscopy. They
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found that T-mutant, P-mutant, and D-mutant did assembly, but the nanosphere formed
by those mutants exhibited larger radii and greater polydispersity, indicating these
mutations interfere amelogenin self-assembly and result in altered nanosphere formation.
Previous investigation (Goto et al., 1993) suggests that the amelogenin N-
terminus form a P-sheet structure. It is well known that proline favors P-tums in
proteins. Changing amino acid residue 41 from proline to threonine would destroy the P~
tum, deforming the P-sheet of the N-terminal domain, thus inhibiting proper amelogenin
assembly. The consequence to protein folding resulted from the 21st amino acid being
mutated from threonine to isoleucine is difficult to understanding. However a solution is
suggested when the amino acid sequence around the 21st amino acid was examined.
There is a proline next to the threonine. Since isoleucine has longer side chain than
threonine, substitution of threonine with isoleucine may block the 22nd proline and
prevent the p-tum from occurring.
5.3 B-dom ain Deletion Increases the Affinity of Am elogenin Interaction
Removal of B-domain (M180AB) resulted in less P-galactosidase activity
determined by yeast two-hybrid assay (73% of wildtype). Plasmon resonance
spectroscopy data showed that the interaction of M180AB with wildtype amelogenin
M179 produced reduced binding RU compared with wildtype-wildtype amelogenin
interaction at the same concentration. However the binding affinity of M180AB to
wildtype amelogenin M179 was increased, about 1.5 times of wildtype amelogenin
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according to ka value. What is the explanation for this apparently contradictory data? In
a model for enamel biomineralization, Fincham et al. (1995) and Paine (1997) have
suggested that the removal of the hydrophilic carboxy-terminus will promote
hydrophobic interaction. It has previously been postulated that the amelogenin
hydrophilic carboxy-terminal is exposed on the surface of the nanosphere, which makes
them soluble in an aqueous solution, and also prevent the fusion of neighboring
nanospheres (Fincham et al., 1994; Paine and Snead, 1997). With these suggestions in
mind the increased ka for M180AB interaction with wildtype amelogenin is postulatable.
The increased binding affinity might allow M180AB to pre-assemble to an almost
saturated level, so that the pre-assembled complex could not bind M180-pPC86 in the
yeast two-hybrid assay, or the M179 monomer immobilized on sensor chip in plasmon
resonance spectroscopy assay.
His-tagged M180AB protein was analyzed by Moradian-Oldak et al. (2000) using
dynamic light scattering (DLS) and atomic force microscopy (AFM). DLS and AFM
image data showed that the size of nanosphere formed by M180AB increased with time,
indicating that without the B-domain the nanosphere is not stable, and readily to coalesce.
Taken together, these data suggest the B-domain might play a role in nanosphere
stabilization.
However, the exact role played by the C-terminal motif in amelogenin self-
assembly process still remains unclear since there is no reported mutation occurring in
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the C-terminus happened in nature. More work is needed to characterize the contribution
made by the B-domain in amelogenin self-assembly process.
5.4 Summary and conclusion
The data from this study suggests that: 1) removal of A-domain abolishe the
amelogenin self-assembly process; 2) in the A-domain the 21st and 41st amino acids be
important for functional assembly. Mutation in either of these two amino acids will
reduce assembly affinity, resulting in altered nanosphere formation; and 3) deletion of the
B-domain increases the affinity of amelogenin interaction.
The cumulative results from the data created by my investigation coupled to
interpretation of the literature indicate that the N-terminal domain is required for
amelogenin self-assembly, the C-terminal domain does not participate in self-assembly,
but instead stabilizes the nanosphere, thus preventing abnormal assembly.
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R E F E R E N C E S
Balasubramanian, S., Chemov-Rogan, T., Davis, A.M., Shitehom, E., Tate, E., Bell, M.,
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Bartek, P.L., Chen, C.T., Stemglanz, R., and Field, S. (1993). Using the two-hybrid
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Oxford, pp 153-179.
Bondesson. K., Frostell-Karlsson, A., Fagerstab, L., and Magnusson, G. (1993). Lactose
repressor-operator DNA interactions: Kinetic analysis by a surface plasmon
resonance biosensor. Anal. Biochem. 214, 245-251.
Boskey, A.L. (1996). Matrix protein and mineralization: An Overview. Conn. Tissue
Res. 35, 357-363.
Chen, C.T., Stemglanz, R., and Fields, S. (1991). The two-hybrid system: A method to
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Asset Metadata

Creator Lei, Yaping (author)

Core Title Amelogenin domains in a self-assembly process

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Tokes, Zoltan A. (committee chair), Kasahara, Noriyuki (committee member), Snead, Malcolm (committee member), Stellwagen, Robert H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-35548

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