1. In Vitro Self Assembly of Human Amelogenin at Oil-Water Interface
P W-82
Olga Martinez-Avila1, *, Shenping Wu2, Yifan Cheng2, Xiaodong He1 , Stefan Habelitz1
1
Department of Preventive and Restorative Dental Sciences, School of Dentistry, 2Department of Biochemistry & Biophysics, University of California, San Francisco
Results
Results
The role of Calcium and Phosphate in Amelogenin self assembly
Amelogenin rH174 metastable emulsions phase separation
1. Amelogenin assembly at the water-oil interface require Calcium
AFM and TEM images of Amelogenin assembly after 7 days of incubation at pH 4.5 in the oil-water system
1-2 days
2-3 hours
(a)
&
(b)
nanoribbons
Amelogenin nanoribbons are formed from reverse micelles that facilitated the interaction between
the hydrophobic tails of the protein molecules and prevent the formation of amelogenin
nanospheres.
A model for ribbon extension proposes the addition of short segments or dimers to the ends of the
ribbon to achieve growth. The formation of self-aligning and uniaxially elongating amelogenin
structures triggered by the presence of calcium and phosphate may change the current paradigm
of protein-controlled mineralization in enamel.
(c)Amelogenin assembly in
the absence of calcium into
nanospheres
Amelogenin Reverse Micelle and assembly tracking: AFM & DLS
Reverse Micelles
Reverse Micelles
Nanoribbons
Nanospheres
500 nm
hydrophilic
C-terminus
1 µm
AFM images of initial ribbon like structures observed
within first minutes after emulsion preparation (a)
time = 0 and nanoribbons (b) at time = 7 days
Hydrophobic tail
d
e
(e) EDTA (0.5 mM) adition to the solution (b) followed
by 48 hours incubation revealed a disintegration of the
ribbons and formation of nanospheres.
Ribbons show consistent width of 16.7 ± 1 nm as
determined by TEM
Objective
1:4
In a typical preparation, calcium solution, containing rH174, CaCl2, and oil phase, and phosphate
solution, containing equal concentrations of rH174, KH2PO4 and oil phase were vortexed
separately at 900 rpm for 60 seconds. Then, the phosphate solution was added to the calcium
solution and the mixture was vortexed again for 60 seconds along with adjusting pH with Bistris/HCl or KOH/HCl as necessary.
Calcium and phosphate concentration maintain the degree of saturation (DS) for hydroxyapatite in
the metastable range. The final concentrations were 33.1 mM CaCl2 and 20.9 mM KH2PO4 at pH
4.5 (DS = 3.6) and pH 5.5 (DS = 11-12)
TEM analysis of amelogenin assemblies formed in a water control system and in an
oil-water system (2/1 oil/water) at incubation times of up to 7 days
1 Day
7 Days
Water control
Stabilized Amelogenin emulsions were prepared with a mixture of ethyl acetate/ octanol equal to
3/7, oil-water ratio equal to 1/1, 1/2 or 4/1 and acidic pHs. Emulsification is indicated by a creamy
white or turbid protein/water/oil mixtures that formed after vortexing.
Amelogenin assembly at water-oil interface vs water
2 hours
Water-oil
Preparation of amelogenin stabilized emulsion
•
A range of Ca/P ratios, appropriate degree of saturation and elevated concentrations of
amelogenin are required for the formation of these fibrillar structures.
•
We propose a model by which amelogenin assembly into nanoribbons may guide and control
apatite crystal growth
(d) Analysis of the fibrillar structures
versus amelogenin powder using microRaman spectroscopy showed
similarities, but also indicated a shift
towards ß-turn prone structure.
Methods
Human recombinant full-length amelogenin protein, rH174 at concentrations between 0.4-3.7
mg/ml was dissolved in calcium and phosphate solutions at acidic conditions and mixed with an oil
phase (octanol/ethyl acetate) to form metastable amelogenin water-oil emulsions. Incubation of
amelogenin water-oil system for up to 7 days was followed by in situ pH increase to induce apatite
formation. The effects on protein self-assembly and crystal formation as a function of calcium and
phosphate concentration, protein concentration, pH, water-oil ratio and incubation time were
studied. The gel-matrix was analyzed using Atomic force microscopy, Transmission and Scanning
electron microscopy, Energy Dispersive X-ray analysis and Dynamic Light Scattering
7 Days
The control samples revealed
the
presence
of
the
characteristic
amelogenin
nanosphere of about 25 nm in
diameter.
The
spheres
occasionally associated with
each
other
and
formed
elongated chain-like structures
after 2 or 7 days.
Nanoribbons have an average diameter of 16.7nm which does not vary with pH, ion
concentracion or time, unlike nanospheres formed by amelogenin
Amphiphilic Amelogenin assembles into organized parallel arrays of nanoribbons in water-oil
system
•
(c) The more randomly distributed
ribbons were also observed by AFM but
only in areas were large amounts of the
protein could be immobilized to the
glass surface
2. Increasing oil/water ratio from 1/1 to 4/1 in the
preparation decreases stability of emulsion and
produce longer nanoribbons
1:2
We have produced assembly of amelogenin rH174 other than nanospheres using a water-oil
system
(b) Elemental analysis by EDX
confirmed the presence of small
amounts of Calcium and Phosphate
ions in these structure.
Lengths ranged from 500 nm to 1.5 mm
1:1
To take advantage of the bipolar nature of amelogenin and produce amelogenin assemblies
other than nanospheres by using an oil-water system commonly used for assembly of surfactants
•
(a) Large amounts of amelogenin
ribbons were observed by SEM
Height was measured as 3.1 ± 0.6 nm from AFM
images and supported the nanostructure as ribbons
How to facilitate Amelogenin assembly and ribbon elongation?
1. In situ pH increase or DS to facilitate HAP
formation along with amelogening assembly
Conclusions
•
Chemical composition of amelogenin nanoribbons
500 nm
Hydrophilic head
c
b
(d) Acidic treatment at pH 2 of AFM (a) specimen for
one minute resulted in small ribbons or nanospheres
aligned along the initial nanoribbon.
Nanoribbons
PLPPHPGHPGYINFSYEVLTPLKWYQSIRPPYPSYGYEPMGGWLHHQIIPVLSQQHPPTHTLQPH
HHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQHHQPNLPPPAQQPYQPQPVQPQPHQPMQPQPP
VHPMQPLPPQPPLPPMFPMOPLPPMLPDLTLEAWPSTDKTKREEVD
hydrophobic
c
b
Amphiphilic Amelogenin
N-terminus
Amelogenin
Averaging > 500 equally sized
ribbon
segments
further
revealed the evidence of a
dark central line that indicate
higher electron density which
could be associated with the
presence of calcium and/or
phosphate ions which are
both
required
for
selfassembly into ribbons.
AFM and TEM analysis of amelogenin assemblies formed in a water control system versus oil-water system (4/1
oil/water) at incubation times of up to 4 days followed by in situ pH increase. Control sample showed the presence
of nanospheres. After 4 days of incubation, only short and isolated ribbons formed. After raising the pH, the
number of nanoribbons increased dramatically and appeared organized in bundles of co-aligned filaments
Model: Crystal Growth in Enamel Controlled
by Amelogenin Nanoribbons
PO43Amelogenin emulsion
Amelogenin Reverse Micelle
PO43-
Ca2+
PO43Ca2+
hydrophobic parts interact
electrostatic forces of hydrophilic part
prevent non-specific protein agglomeration
500 nm
500 nm
250 nm
1µm
When aqueous amelogenin
solutions were mixed into an oil
phase, ribbon-like structures
appeared within hours. Later
time points showed higher
concentrations of ribbons which
were oriented in a parallel
fashion.
2 µm
Micelles sink down to the
water/oil interface and merge &
release protein onto the
interface
Oil
------Water
Grows to several micrometer length
References
Supported by NIH/NIDCR RO1- DE017529 and R01-DE017529S2
Water control
Day 4
Day 4 followed by in sity pH raise up to 5.6
2-3 nm
Time 0
2. TEM shows the precence of a dark central line running along the long axis of each nanoribbon
1)
Self-assembly of amelogenin proteins plays a key role in the biomineralization of developing
enamel by regulating the growth and organization of nanofibrous apatite crystals. It is accepted
that amelogenin proteins, the main constituent of the developing enamel matrix, form nanospheres
of 20 to 40nm in vitro, but the amphiphilic nature of the of the bipolar full-length protein provide with
characteristics that might allow assembly into supramolecular structures of high order. This study
tested if the use of metastable oil-water emulsions can induce the formation of supramolecular
assemblies of amelogenin.
(00
Introduction
17 nm
amelogenin dimer
