Supporting Information Available
In vitro labeling of hydroxyapatite minerals by an engineered protein
Esra Yucaa,b,c, Ayten Yazgan Karatasa, Urartu Ozgur Safak Sekera,c, Mustafa Gungormusc,
Gizem Dinler-Doganaya, Mehmet Sarikayaa,c and Candan Tamerlera,c*
a
Department of Molecular Biology, Genetics and Biotechnology, Istanbul Technical University,
Istanbul, Turkey, b Department of Biology, Yıldız Technical University, Istanbul, Turkey, c
Department of Materials Science and Engineering, University of Washington, Seattle,
Washington, USA
Quartz Crystal Microbalance-Dissipation (QCM-D) Experiments:
The QCM-D measures the change in the frequency and the dissipation following the crystal is
excited under electrical power (Hook et al. 2001; Hook et al. 1998). By comparing the frequency
change at different overtones, one can comment on the elasticity of the film adsorbed on the
sensor surface. Proteins can be considered as polymers with water holding capacities. Upon
adsorption on a solid surface, the proteins are able to capture water and form a viscoelastic film.
Depending on the change in the dissipation of the adsorbed layer, one can make assumptions
about how dense the adsorbed film is. In this section, we provide the dissipation changes
monitored in our studies. The dissipation change for both of the proteins is very small in our case
(Supplementary Figure 1). This may imply that the amount of water held by proteins is very
small upon adsorption. We therefore can assume that a rigid protein film was formed on the
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resonator surface. This is also in agreement with the fact that, GFP is quiet rigid with its beta
sheet dominated barrel like structure.
Supplementary Figure 1. Dissipation change (black) and frequency change (blue) for GFPHABP1 and GFP-HABP2
In calculations, Sauerbrey equation can be used to convert the frequency change (Δf) to the mass
load. Sauerbrey equation assumes that the film adsorbed on the sensor surface is rigid, coupled
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with the sensor and oscillating with the same frequency. By using this approach, mass deposited
on the surface can be calculated using the following equation (Sauerbrey 1959):
Equation 1
where m is the mass deposited, C is the mass sensitivity constant and f is the frequency change
of the sensor and n is the overtone number.
However this equation is not valid and applicable for the soft and elastic films. In our case the
film may not be fully coupled with the resonator (sensor), even though there is only a small
possibility of dissipation throughout adsorption. In this case the change in the deposited mass can
be defined with a correction in the Sauerbery equation as shown in equation 2 (Domack et al.
1997; Fredriksson et al. 1998; Granstaff and Martin 1994; Martin et al. 1991; Voinova et al.
1999, Kankare 2002):
Equation 2
where
is deposited mass on resonator surface,
is the resonance of the given overtone,
is the change in the resonance frequency upon mass deposition on the resonator,
of quartz,
solution,
is thickness of resonator,
is the density
is the density of medium in our case it is protein
density of the protein film adsorbed on to the surface,
solution, is angular frequency of oscillator, and
is the viscosity of the protein
is shear modulus.
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Monitoring time wise mineralization
Supplementary Figure 2: The ability of GFPuv-HABP1 construct to monitor the time wise
mineralization was tested on minerals forming on glass slides. An alkaline phosphatase based
mineralization model (Gungormus et al. 2008) was used for time wise monitoring the
biomineralization. Before the examination by optical microscopy, scanning electron microscopy
(SEM) and fluorescent microscopy, a calcium phosphate layer was formed on glass cover slides.
At the end of each time point (0.5, 2, 8 and 24 hours), the glass slides were taken out and the
mineral covered area on glass slides was calculated using Metamorph v7.5 image processing
software (Molecular Devices, USA). Optical microscopy results showed that the amount of
mineral on the glass slides has increased in a linear fashion.
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TEM and Electron Diffraction Characterization of the HA minerals
Supplementary Figure 3: TEM micrographs of the synthetic HA used for fluorescent binding
characterization and the HA synthesized on a glass slide to monitor the mineral formation in
vitro. The corresponding selected area electron diffraction (SAED) patterns are shown in the
insets. The synthesized particles were collected by scraping off the mineral from the glass slides
using a plastic toothpick.
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