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Sea Urchin Spicule Matrix Proteins Form Mesoscale "Smart" Hydrogels That Exhibit Selective Ion Interactions.
Pendola M
,
Davidyants A
,
Jung YS
,
Evans JS
.
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In the sea urchin embryo spicule, there exists a proteome of >200 proteins that are responsible for controlling the mineralization of the spicule and the formation of a fracture-resistant composite. In this report, using recombinant proteins, we identify that two protein components of the spicule, SM30B/C and SM50, are hydrogelators. Because of the presence of intrinsic disorder and aggregation-prone regions, these proteins assemble to form porous mesoscale hydrogel particles in solution. These hydrogel particles change their size, organization, and internal structure in response to pH and ions, particularly Ca(II), which indicates that these behave as ion-responsive or "smart" hydrogels. Using diffusion-ordered spectroscopy NMR, we find that both hydrogels affect the diffusion of water, but only SM50 affects the diffusion of an anionic solute. Thus, the extracellular matrix of the spicule consists of several hydrogelator proteins which are responsive to solution conditions and can control the diffusion of water and solutes, and these proteins will serve as a model system for designing ion-responsive, composite, and smart hydrogels.
Figure 1. Primary sequence and bioinformatics analysis of the mature, processed
SpSM30B/C and SpSM50 proteins (UniProtKB accession numbers P28163 and P11994, respectively).8â11 Predicted regions of intrinsic disorder sequence regions (solid
lines using DISOPRED33 and IUP34 algorithms) and cross-beta strand sequence regions
(dashed lines using AGGRESCAN,35 TANGO,36 and ZIPPER_DB.37).
Negative (red) and positive (blue) charged amino acids are indicated.
Figure 2. Light microscopy
images of rSpSM protein hydrogel particles in
10 mM HEPES, pH 8.0; 10 mM HEPES/30 mM NaCl, pH 8.0; and 8.9 mM NaH2PO4, pH 4.0. Scale bars = 10 microns.
Figure 3. Flow cytometry 2D density plots (FSC vs SSC) of 15 μM
rSpSM30B/C-G
and 1.5 μM rSpSM50 proteins in 10 mM HEPES, pH 8.0; 10 mM HEPES/30
mM NaCl, pH 8.0; and 8.9 mM NaH2PO4, pH 4.0.
FSC refers to a parameter measuring light scattered less than 10°
as a particle passes through the laser beam and is related to the
particle size. SSC is proportional to particle granularity or internal
complexity and is a measurement of mostly refracted and reflected
light that occurs at any interface within the particle where there
is a change in RI. SSC is collected at approximately 90° to the
laser beam by a collection lens and then redirected by a beam splitter
to the appropriate detector.
Figure 4. Light microscopy
and 2D flow cytometry density plots for 15 μM
rSpSM30B/C-G and 1.5 μM rSpSM50 in the presence and absence
of 10 mM CaCl2 in 10 mM HEPES, pH 8.0.
Figure 5. Overlay of 700 MHz 1H 2D DOSY PFG diffusion experiments
of 90% 30 nm filtered H2O/10% 99.9% D2O/133
μM d4-TSP sample in the presence
(blue) and absence (red) of 22 μM rSpSM30B/C-G and rSpSM50,
150 μL volume. The log of the diffusion coefficient is plotted
on the y-axis (F1 domain) and determined from the
contour plot maxima for each sample.
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