advances.sciencemag.org/cgi/content/full/3/8/e1700765/DC1

Supplementary Materials for

Control of nacre biomineralization by Pif80 in pearl oyster

So Yeong Bahn, Byung Hoon Jo, Yoo Seong Choi, Hyung Joon Cha

Published 2 August 2017, Sci. Adv. 3, e1700765 (2017)

DOI: 10.1126/sciadv.1700765

This PDF file includes:

fig. S1. PTM analyses of native Pif80.

fig. S2. Turbidimetric measurement of Ca2+-induced coacervation of rPif80 in the presence of 4 mM CaCl2.

fig. S3. SDS-PAGE analysis with Stains-All staining of rPif80.

fig. S4. Turbidimetric measurement of Ca2+-rPif80 coacervates according to additional NaCl.

fig. S5. Optical micrograph images (top) and Raman spectra (bottom) of mineralized CaCO3.

fig. S6. Cryo-scanning TEM image and EDS mapping analyses of rPif80-CLP.

fig. S7. Dot blotting with Coomassie staining after CaCO3-binding analysis of rPif80.

fig. S8. Structural analyses of a cross-sectioned plate mineral induced by rPif80 at a concentration of 50 μg/ml.

fig. S9. Morphology and polymorph analyses of grown minerals in the presence of protein impurities.

fig. S1. PTM analyses of native Pif80. (A) Glycoprotein staining analysis. Following

SDS-PAGE, the gels were stained with Coomassie (left) and periodic acid-Schiff

(PAS; right) staining reagents. (B) Phosphoprotein staining analysis. The gels after

SDS-PAGE were stained with Coomassie (left) and Pro-Q Diamond (right) staining

reagents. The control proteins were used according to the manufacturer’s instructions.

The red arrows indicate Pif80 from AIM. The white and black arrowheads indicate

positive and negative control proteins of Pro-Q Diamond staining, respectively, in

PM. Lanes: M, protein molecular weight marker; AIM, acid-insoluble and SDS-

soluble organic matrix of nacre; PC, positive control (horseradish peroxidase) for PAS

staining; NC, negative control (soybean trypsin inhibitor) for PAS staining; PM,

PeppermintStick phosphoprotein molecular weight standard.

fig. S2. Turbidimetric measurement of Ca2+-induced coacervation of rPif80 in

the presence of 4 mM CaCl2. (A) Turbidity of the coacervates in a moderately basic

pH. The pH was modulated using a 20 mM Tris buffer. (B) Turbidity of the

coacervates according to rPif80 concentration. (C) Turbidity of the coacervates

according to NaCl concentrations up to 200 mM. The coacervation with NaCl was

performed in a 20 mM Tris buffer (pH 8). The turbidimetry was performed in

triplicate.

fig. S3. SDS-PAGE analysis with Stains-All staining of rPif80. BSA was used as a

negative control. Lanes: M, protein molecular weight marker; P, purified rPif80;

BSA, bovine serum albumin.

fig. S4. Turbidimetric measurement of Ca2+-rPif80 coacervates according to

additional NaCl. The dissolution of the coacervates was performed by adding NaCl

with a range of concentrations up to 500 mM into the pre-formed coacervate solution.

The turbidimetry was performed in triplicate.

fig. S5. Optical micrograph images (top) and Raman spectra (bottom) of

mineralized CaCO3. CaCO3 precipitates obtained (A) in the absence of an additive

and (B) in the presence of lysozyme-HA coacervate. Peaks indicated by C and Si in

the Raman spectra correspond to calcite and silicon, respectively.

fig. S6. Cryo-scanning TEM image and EDS mapping analyses of rPif80-CLP.

Mapping was performed at the site surrounded by the red rectangle in the cryo-

scanning TEM image.

fig. S7. Dot blotting with Coomassie staining after CaCO3-binding analysis of

rPif80. The first and second lanes are the results of the calcite- and aragonite-binding

experiments, respectively. After incubation with calcite, rPif80 was recovered in each

fraction, including the FT, the washes, and the dissolved calcite. However, rPif80 was

not recovered in the FT and the washes after incubation with aragonite, and only

eluted after the dissolution of aragonite. These results indicate the more specific

binding of rPif80 to aragonite compared to the binding to calcite. Lanes: FT, flow

through fraction; W1, wash fraction with 10 mM Tris (pH 8); W2, wash fraction with

10 mM Tris (pH 8) supplemented with 0.1 M NaCl; W3, wash fraction with 10 mM

Tris (pH 8) supplemented with 0.5 M NaCl; E, dissolved CaCO3 with 4 M acetic acid.

fig. S8. Structural analyses of a cross-sectioned plate mineral induced by rPif80

at a concentration of 50 µg/ml. (A) TEM image of a cross-section of the plate

mineral of Fig. 3D prepared by FIB. (B) Selected area electron diffraction pattern of a

cross-sectioned mineral, indicating aragonite. (C) HR-TEM image of the inner

structure (left) with related fast Fourier transform patterns (right) of the sites,

indicated as red rectangles.

fig. S9. Morphology and polymorph analyses of grown minerals in the presence

of protein impurities. (A to C) SEM images of β-chitin surface after crystallization

at 20 C for 48 h in the presence of the host cell protein impurities at a concentration

of (A) 0 µg/mL, (B) 5 µg/mL, and (C) 50 µg/mL. (D) Raman spectra of grown

minerals in (A) and (B). The Raman spectrum of calcite powder is presented for

comparison.